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Metal chelating and oxidative behaviour of maillard reaction products extracted from model and food systems Wijewickreme, Arosha Nilmini 1996

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M E T A L C H E L A T I N G A N D O X I D A T I V E B E H A V I O U R O F M A I L L A R D R E A C T I O N P R O D U C T S E X T R A C T E D F R O M M O D E L A N D F O O D S Y S T E M S by A R O S H A M L M T N I W T J E W I C K R E M E B . S c , University o f Peradeniya, Sr i Lanka , 1986 M . S o , The University o f Bri t ish Columbia, Canada, 1990 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Department o f F o o d Science W e accept this thesis as confonrdng to the required standard T F l E T j N l V E K S I T Y O F ' B R I T I S H C O L U M B I A December 1996 © Arosha Ni lmin i Wijewickreme, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying, of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. •" Department of The University of British Columbia Vancouver, Canada ' f t Date fee % '(m DE-6 (2788) 11 ABSTRACT T h e elementary composi t ion, metal chelating, oxidat ive behaviour and related genotox ic , and cy to tox ic activit ies o f different Ma i l l a rd reaction products ( M R P s ) , der ived f rom both model and coffee systems were studied in vitro in the presence, or absence, o f polyvalent metal ions. Non-d ia lysab le model M R P s were synthesized by heating £)-Glucose (Glu) or Z)-Fructose (Fru) wi th Z - L y s i n e (Lys) under variable t ime-temperature, init ial p H and ini t ial water act iv i ty (a w ) combinat ions. M R P s were also extracted f rom instant, brewed, and bo i led roasted g round coffee powders to represent a f ood system. E lementary composi t ion o f Fru-Lys MRPs constantly exhibited a higher percentage o f n i t rogen and a relatively smaller number o f C u 2 + b inding sites compared to simi lar ly der ived Glu-Lys MRPs. B o t h M R P types exhibited only antioxidant activit ies in a metal free l inole ic acid emuls ion, but a combinat ion o f distinct anti- /prooxidant act ivi ty was observed in C u 2 + supplemented l ip id emulsions w i th oxygen electrode. The two Glu-Lys MRPs der ived from synthesis experimental condit ions 3 and 13 (i.e., t ime, temp., p H , a w : 119 m in , 127 °C, 6.14, 0.74 ; 71 min , 129 °C, 8 .41, 0.78) and the two Fru-Lys MRPs der ived f rom synthesis experimental condi t ions 5 and 11 (107 min , 157 °C, 8.51, 0.57 ; 43 min, 159 °C, 8.57, 0.62) were found to produce the highest M R P yields. The products were subsequently used to evaluate the eff icacy to retard l ip id ox idat ion in a model cook ie dough system. Wh i l e Glu-Lys MRPs exhibi ted antioxidant act iv i ty in the cook ie dough model , Fru-Lys MRPs showed prooxidant act iv i ty regardless o f the presence o f C u 2 + . Fract ionat ion o f M R P s derived f rom specif ic Glu-Lys (Expt . N o s 3 and 13) and Fru-Lys (Expt . N o s 5 and 11) reaction condit ions using C u 2 + chelat ing chromatography resulted in two pr incipal metal chelating components having molecular weights o f 5.7 k D and Ill 12.4 k D , respectively. Glu-Lys MRPs (5 k D ) decreased (p>0.05) F e 2 + induced n ick ing o f P M 2 bacter iophage D N A , relative to the n ick ing caused by both metals alone or metals associated w i th Fru-Lys MRPs (5 k D ) . B o t h M R P sources induced a higher percentage o f D N A n ick ing in the presence o f F e 2 + relative to the n ick ing caused by M R P s alone. The extent o f D N A breakage by polyvalent metals and M R P reactants was p H dependent and corresponded to the reducing potent ial o f M R P s in modulat ing the Fenton reaction. Cy to tox ic i t y evaluations using C 3 H 1 0 T 1 / 2 mouse embryo f ibroblast cel ls demonstrated that M R P s cou ld decrease cytotoxic i ty, regardless o f C u 2 + concentrat ion o r preincubat ion condi t ions used. Individual ly, M R P s and F e 3 + enhanced cytotox ic i ty dur ing direct appl icat ion; a similar effect was not noted wi th F e 2 + . These f indings indicated that metal chelat ing and reducing activi t ies are important factors in determining model M R P s eff icacies in modulat ing metal catalyzed cytotoxic i ty . Cof fee derived M R P s (5-6 k D ) possessed a relat ively higher percentage o f carbon but exhibited l o w reducing and higher C u 2 + chelating activi ty compared to mode l M R P s . A s a result, coffee M R P s effectively reduced metal catalyzed in vitro cytotoxic i ty . The complex i ty o f different M R P s due largely to variat ions in react ion condi t ions was shown to inf luence both antioxidant and prooxidant characterist ics o f specif ic M R P s . These results are important in determining the funct ional and safety propert ies o f M R P s . IV TABLE OF CONTENTS S E C T I O N P A G E T I T L E P A G E , I A B S T R A C T II T A B L E O F C O N T E N T S I V L I S T O F T A B L E S X I L I S T O F F I G U R E S XI I I L I S T O F S Y M B O L S A N D A B B R E V I A T I O N S X X A C K N O W L E D G E M E N T X X I I 1.0 I N T R O D U C T I O N 1 2.0 L I T E R A T U R E R E V I E W 2 2.1 Introduct ion and H is to r i ca l B a c k g r o u n d 2 2.2 S ign i f i cance o f the M R i n O u r F o o d 3 2.2.1 Conven t iona l Sources o f M R P s 4 2.2.2 Contemporary U s e s o f M R P s i n Foods 7 2.3 C h e m i c a l Pa thways o f M R 9 2.3.1 In i t ia l Stage o f the M R 11 2.3.2 Intermediate Stage o f the M R 16 2.3.3 F i n a l Stage o f the M R 28 2.4 Parameters In f luenc ing the M R 29 2.5 K ine t i cs o f the M R 34 2.6 Prevent ion o f the M R 36 2.7 Phys i cochem ica l Propert ies o f M R P s 36 2.7.1 An t i ox ida t i ve A c t i v i t y 37 2.7.2 M i n e r a l Interactions 39 2.7.3 An t imu tagen ic i t y 44 2.8 Nut r i t i ona l Consequences o f M R 45 2.8.1 Dest ruc t ion and L o s s o f Essent ia l A m i n o A c i d s 46 2.8.2 Pro te in D iges t ib i l i t y 47 2.9 Phys io l og i ca l Consequences o f M R 47 2.9.1 Inf luence o f M R o n V i t a m i n Content 47 V 2.9.2 Ef fec t o f M R o n D iges t i ve E n z y m e s 48 2.10 T o x i c o l o g i c a l Aspec ts o f M R 48 2.10.1 Mutagen ic i t y 48 2.10.2 In vivo G l y c a t i o n o f Prote ins 49 2.10.3 T o x i c C o m p o u n d s F o r m e d through the M R 50 2.11 Thes is Deve lopment 50 2.11.1 Hypothes is 50 2.11.2 A i m s 51 2.11.3 Object ives 51 3.0 S T U D Y I: M E T A L C H E L A T I N G A N D A N T I - / P R O - O X I D A N T A C T I V I T Y O F GLUCOSE-LYSINE A N D FRUCTOSE-LYSINE M O D E L M A I L L A R D R E A C T I O N P R O D U C T M I X T U R E S 52 3.1 Introduct ion 52 3.2 Hypo thes is 55 3.3 Object ives 55 3.4 Mater ia ls 56 3.4.1 Produc t ion o f M o d e l M a i l l a r d Reac t i on Product M i x t u r e s 56 3.4.2 Measurement o f C o p p e r B i n d i n g A c t i v i t y o f M o d e l M R P M ix tu res and Frac t ionat ion o f M o d e l M R P M i x t u r e s b y Che la t i on Chromatography 56 3.4.3 Assessment o f An t i ox i dan t A c t i v i t y o f M o d e l M R P M i x t u r e s i n a L i p i d M o d e l Sys tem U s i n g an O x y g e n E lec t rode and Th iobarb i tu r ic A c i d ( T B A ) M e t h o d 57 3.4.4 Assessment o f T o x i c i t y o f M o d e l M R P M ix tu res i n a D N A Sys tem 57 3.5 Me thods 58 3.5.1 Produc t ion o f M o d e l M R P M ix tu res 58 3.5.2 Cor rec t ion for The rma l L a g s 59 3.5.3 Spectral Character is t ics o f M o d e l M R P M i x t u r e s 63 3.5.4 M e t a l Che la t ing A f f i n i t y o f M o d e l M R P M i x t u r e s 63 3.5.4.1 B i n d i n g A c t i v i t y o f Coppe r Ions to M R P M i x t u r e s 63 3.5.4.2 Fract ionat ion o f M o d e l M R P M ix tu res b y C o p p e r Che la t i on Chromatography 64 3.5.5 E lementary C o m p o s i t i o n and M o l e c u l a r We igh ts o f M o d e l M R P M ix tu res and M R P M i x t u r e Componen ts Fract ionated by Che la t i on Chromatography J65 3.5.6 Assessment o f An t i ox i dan t A c t i v i t y o f M R P M i x t u r e s 66 3.5.6.1 O x y g e n C o n s u m p t i o n Measurements 66 3.5.6.2 Measurement o f Th iobarb i tu r ic A c i d Reac t i ve Substances ( T B A R S ) 66 3.5.6.3 Assessment o f D N A N i c k i n g A c t i v i t y o f m o d e l v i M R P M ix tu res 67 3.5.7 Stat ist ical A n a l y s i s 72 3.6 Resu l ts 72 3.6.1 Y i e l d o f G l u c o s e - L y s i n e (Glu-Lys) and F ruc tose -Lys ine (Fru-Lys) M R P M ix tu res 72 3.6.2 Spectra l Character is t ics o f M R P M i x t u r e s 76 3.6.2.1 A b s o r p t i o n Spectra 76 3.6.2.2 Abso rp t i on at 420 n m 76 3.6.2.3 Hunter L a b L, a and b V a l u e s 79 3.6.3 M e t a l Che la t i ng A f f i n i t y o f M o d e l M R P M ix tu res 79 3.6.3.1 The Coppe r B i n d i n g A c t i v i t y o f M R P M ix tu res 79 3.6.3.2 Frac t ionat ion o f M o d e l M R P M ix tu res by Che la t i on Chromatography 83 3.6.4 E lemen ta l A n a l y s i s o f C rude M R P M i x t u r e s and M R P M i x t u r e Componen ts 85 3.6.5 Assessment o f An t i ox idan t A c t i v i t y o f M R P M ix tu res 90 3.6.5.1 Assessment o f An t i ox idan t A c t i v i t y by T B A M e t h o d 90 3.6.5.2 Assessment o f An t i ox idan t A c t i v i t y by O x y g e n C o n s u m p t i o n Measurement 94 3.6.5.3 In vitro D N A N i c k i n g Studies 100 3.7 D i s c u s s i o n 106 3.7.1 E f fec t o f Reac t i on Cond i t i ons o n the Intensity o f B r o w n i n g between Glu-Lys and Fru-Lys M o d e l React ions 106 3.7.2 Character izat ion o f M e t a l Che la t i ng A f f i n i t y o f M o d e l M R P M ix tu res 109 3.7.2.1 M e t a l Che la t ing A f f i n i t y o f C rude M R P M ix tu res 109 3.7.2.2 Separat ion o f M o d e l M R P M ix tu res by Che la t ing Chromatography 109 3.7.3 M a s s Spect roscopy 110 3.7.4 E lemen ta l A n a l y s i s o f C rude M R P M i x t u r e s and the Fract ionated M R P Componen ts I l l 3.7.5 An t i ox idan t / P roox idan t A c t i v i t y o f M o d e l M R P M ix tu res . . . . 112 3.7.5.1 Measurement o f An t i ox i dan t A c t i v i t y by O x y g e n C o n s u m p t i o n Measurements 112 3.7.5.2 An t i ox idan t A c t i v i t y o f M R P M ix tu res as Measu red by the T B A M e t h o d 113 3.7.5.3 Geno tox i c i t y o f M o d e l M R P M i x t u r e s 115 3.8 C o n c l u s i o n 117 S T U D Y II: A S S E S S M E N T O F A N T I - / P R O - O X I D A N T A C T I V I T Y O F GLU-LYS A N D FRU-LYS M O D E L M A I L L A R D R E A C T I O N P R O D U C T M I X T U R E S I N A L I N O L E I C A C I D E M U L S I O N S Y S T E M A N D A M O D E L C O O K I E D O U G H F O O D S Y S T E M 118 4.1 Introduct ion 118 4.2 Hypo thes is 121 4.3 Objec t ives 121 4.4 Mate r ia l s 122 4.5 M e t h o d s ; 122 4.5.1 Preparat ion o f M o d e l M R P M i x t u r e s 122 4.5.2 Assessment o f L i p i d Ox ida t i on i n a L i n o l e i c A c i d E m u l s i o n Sys tem i n the A b s e n c e and Presence o f C o p p e r 122 4.5.3 Preparat ion o f C o o k i e D o u g h s 123 4.5.4 Assessment o f L i p i d Ox ida t i on i n C o o k i e D o u g h s 123 4.5.5 C o l o u r Measurement o f C o o k i e D o u g h s 125 4.5.6 Assessment o f R e d u c i n g A c t i v i t y o f M o d e l M R P M i x t u r e s . . . . 125 4.6 Resu l ts 125 4.6.1 M o d e l L i n o l e i c A c i d E m u l s i o n Sys tem 125 4.6.1.1 Assessment o f L i p i d Ox ida t i on i n the A b s e n c e o f Coppe r Ions 125 4.6.1.2 Assessment o f L i p i d Ox ida t i on i n the Presence o f Coppe r Ions 127 4.6.2 M o d e l C o o k i e D o u g h Exper imen t 127 4.6.2.1 Intensity o f D o u g h C o l o u r 127 4.6.2.2 Changes i n L i p i d Ox ida t i on i n Con t ro l C o o k i e D o u g h s 129 4.6.2.3 E f fec t o f A d d e d M R P M ix tu res and a - tocophero l on the Rate o f L i p i d Ox ida t i on Occu r r i ng i n C o o k i e D o u g h s wi thout A d d e d C o p p e r 129 4.6.2.4 E f fec t o f M R P M i x t u r e s and cc-Toc i n Retard ing L i p i d Ox ida t i on Occu r r i ng i n C o o k i e D o u g h s w i t h A d d e d C u p r i c ions 137 4.6.2.5 Eva lua t i on o f the Poss ib le Synergest ic / An tagon is t i c Ef fec t o f a -Toe and M R P M ix tu res i n Retard ing L i p i d Ox ida t i on 143 4.6.3 R e d u c i n g A c t i v i t y o f M o d e l M R P M i x t u r e s 143 4.7 D i s c u s s i o n 147 4.7.1 Assessment o f L i p i d Ox ida t i on i n M o d e l Systems Con ta in ing M R P M i x t u r e s and cc-Toc wi thout A d d e d C o p p e r 147 4.7.2 A n t i - / Proox idant A c t i v i t y o f M o d e l M R P M i x t u r e s and a - T o c i n Coppe r Supp lemented C o o k i e D o u g h s 149 4.8 Conc lus ions 151 S T U D Y III: E F F E C T O F GLU-LYS A N D FRU-LYS M O D E L M A I L L A R D R E A C T I O N P R O D U C T M I X T U R E S O N M E T A L I N D U C E D D N A N I C K I N G 152 5.1 Introduct ion 152 5.2 Hypothes is 155 5.3 Objec t ives 155 5.4 Mater ia ls 156 5.5 Me thods 156 5.5.1 Preparat ion o f M o d e l M R P M ix tu res 156 5.5.2 G e l f i l t rat ion Chromatography 156 5.5.3 Preparat ion o f Bacter iophage P M 2 D N A 157 5.5.4 Assessment o f D N A N i c k i n g Caused by M o d e l M R P M i x t u r e s and Po lyva len t M e t a l Ions at D i f ferent p H Cond i t i ons 157 5.5.5 Studies (a - c ) o n the E f fec t o f M o d e l M R P M i x t u r e s o n M e t a l Dependent D N A N i c k i n g 158 5.5.6 E f fec t o f A s c o r b i c ac id , Phy t i c ac id , and E D T A on M e t a l Ca ta l yzed D N A N i c k i n g 159 5.6 Resul ts 159 5.6.1 D N A N i c k i n g Caused by Po lyva len t M e t a l Ions and M R P M ix tu res at D i f ferent p H V a l u e s 159 5.6.2 D N A N i c k i n g i n the Presence o f M R P M i x t u r e s Together w i t h F e 3 + or C u 2 + ions 162 5.6.3 D N A N i c k i n g i n the Presence o f M R P M i x t u r e s Together w i t h F e 2 + Ions 168 5.6.3.1 Study Conduc ted w i t h a M R P Concent ra t ion o f 0 . 0 0 0 1 % w/v ) 168 5.6.3.2 Study Conduc ted w i t h a M R P Concent ra t ion o f 0 . 0 0 1 % (w/v) 172 5.6.4 Assessment o f M e t a l Ca ta l yzed D N A N i c k i n g i n the Presence o f Phy t i c A c i d , A s c o r b i c A c i d , and E D T A 176 5.7 D i scuss ion 181 5.7.1 M e t a l C a t a l y z e d H y d r o x y l R a d i c a l Fo rma t i on 181 5.7.2 Proox idan t A c t i v i t y o f M R P M ix tu res i n a M o d e l D N A Sys tem 183 5.7.3 M e t a l Ca ta l yzed D N A N i c k i n g in the Presence o f M R P M i x t u r e s 183 5.7.4 A n t i / P roox idan t A c t i v i t y o f A s c o r b i c A c i d , E D T A , and Phy t i c A c i d i n the Presence o f M e t a l Ions 187 5.8 C o n c l u s i o n 188 i x 6.0 S T U D Y I V : M O D U L A T I O N O F P O L Y V A L E N T M E T A L I O N I N D U C E D C Y T O T O X I C I T Y B Y M O D E L M R P M I X T U R E S 191 6.1 Introduct ion 191 6.2 Hypothes is 192 6.3 Object ives 192 6.4 Mater ia ls 193 6.5 Me thods 193 6.5.1 Produc t ion o f M R P M i x t u r e s 193 6.5.2 Assessment o f Cy to tox i c i t y o f M R P M i x t u r e s and M e t a l Ions 193 6.5.3 Assessment o f the M R P M ix tu res A b i l i t y to M i t i ga te M e t a l Induced C y t o x i c i t y 194 6.6 Resu l ts 194 6.6.1 C u 2 + , F e 2 + , and F e 3 + Ca ta l yzed Cy to tox i c i t y 194 6.6.2 Cy to tox i c i t y o f M R P M i x t u r e s 196 6.6.3 Assessment o f M e t a l Induced C y t o x i c i t y i n the Presence o f M o d e l M R P M i x t u r e s 196 6.7 D i s c u s s i o n 205 6.7.1 M R P Induced Cy to tox i c i t y 205 6.7.2 Cy to tox i c i t y i n the Presence o f M e t a l Ions and M R P M ix tu res 207 6.8 C o n c l u s i o n 208 7.0 S T U D Y V : M O D U L A T I O N O F M E T A L I N D U C E D D N A N I C K I N G A N D C Y T O T O X I C I T Y B Y C O F F E E M A I L L A R D R E A C T I O N P R O D U C T S 210 7.1 Introduct ion 210 7.2 Hypothes is 212 7.3 Objec t ives 212 7.4 Mater ia ls 213 7.5 Me thods 213 7.5.1 Isolat ion o f Co f fee M R P s 213 7.5.2 G e l F i l t ra t ion and E lemen ta l A n a l y s i s o f Co f fee M R P s 213 7.5.3 Coppe r B i n d i n g A c t i v i t y o f Co f fee M R P s 214 7.5.4 D N A N i c k i n g Studies and T issue Cul tu re Exper imen ts 214 7.6 Resu l ts 214 7.6.1 P A R T I - C h e m i c a l Character is t ics o f Co f fee M R P s 214 7.6.2 P A R T II - D N A N i c k i n g A s s a y s Conduc ted w i t h Co f fee M R P s 218 7.6.2.1 D o s e Response D N A N i c k i n g Ef fec t o f Co f fee M R P s and F e 2 + Ions 218 7.6.2.2 D N A N i c k i n g Ef fec t o f Co f fee M R P s i n the Presence o f F e 2 + 218 X 7.6.3 P A R T III - C o l o n y F o r m i n g E f f i c i e n c y o f C 3 H 1 0 T 1 / 2 M o u s e E m b r y o F ibrob las t C e l l s i n the Presence o f Po lyva len t M e t a l Ions and Co f fee M R P s 223 7.6.3.1 C o l o n i z a t i o n E f f i c i e n c y o f F ibrob las t C e l l s i n the Presence o f F e 2 + and Co f fee M R P s 223 7.6.3.2 C o l o n i z a t i o n E f f i c i e n c y o f F ibrob las t C e l l s i n the Presence o f F e 3 + and Co f fee M R P s 229 7.6.3.3 C o l o n i z a t i o n E f f i c i e n c y o f F ibrob las t C e l l s i n the Presence o f C u 2 + and Co f fee M R P s 232 7.7 D i s c u s s i o n 232 7.7.1 C h e m i c a l Character is t ics o f Fract ionated Cof fee M R P s 232 7.7.2 D N A N i c k i n g Patterns Observed w i t h Cof fee M R P s i n the Presence o f Po lyva len t M e t a l Ions 235 7.7.3 In Vitro C o l o n i z a t i o n E f f i c i e n c y o f F ibrob las t C e l l s i n the Presence o f Po lyva len t M e t a l Ions 236 7.8 C o n c l u s i o n 238 8.0 G E N E R A L C O N C L U S I O N 240 9.0 L I S T O F R E F E R E N C E S 244 10.0 A P P E N D I X 267 x i LIST OF TABLES T A B L E P A G E 2.1 A c t i v a t i o n energy ( E J parameters for the non-enzymat ic b rown ing react ion ( A ) and pote in qual i ty loss (B ) due to M a i l l a r d react ion i n dif ferent foods 35 3.1 In i t ia l and f ina l exper imenta l condi t ions [reaction t ime, oven temperature, in i t ia l water act iv i ty (a^), in i t ia l p H ] used and obtained i n the preparat ion o f Glu-Lys and Fru-Lys non-d ia lysab le M R P mixtures and the y i e l d gained at the end o f the react ion 60 3.2 Equ iva len t react ion t imes ( U ) * ca lcu lated for two act ivat ion energy ( E J va lues for dif ferent M R P mix ture synthesis exper iments. . . . 61 3.3 B o u n d copper ( p M C u / m g M R P ) , d issoc ia t ion constants ( K d ) and number o f copper b ind ing sites (n) i n crude Glu-Lys and Fru-Lys MRP mix tures 81 3.4 The d issoc ia t ion constants ( K d ) and number o f copper b ind ing sites (n) i n crude and fract ionated Glu-Lys and Fru-Lys MRP mixtures 86 3.5 Resu l ts o f e lemental analys is and emp i r i ca l fo rmulae o f crude Glu-Lys and Fru-Lys MRP mix tures 91 3.6 The elementary compos i t i on and emp i r i ca l fo rmulae o f crude M R P mix tures and M R P mixtures fract ionated b y copper chelat ing chromatography 92 4.1 Exper imen ta l des ign used i n the preparat ion o f cook ie doughs 124 4.2 Assessment o f l i p i d ox ida t ion i n a l i no le ic ac id emu ls ion w i t h added M R P mix tures i n the absence and presence o f copper 126 4.3 C o l o u r intensi ty o f cook ie doughs measured us ing Hunter L a b t r i -s t imulus co lor imeter 128 5.1 Percentage o f superco i led D N A remain ing after incubat ing P M 2 D N A w i t h C u 2 + and M R P mixtures at p H 7.5 165 Xll 5.2 Percentage o f superco i led D N A remain ing after incubat ion o f P M 2 D N A w i t h F e 3 + and M R P mixtures at p H 7.5 167 7.1 Coppe r che la t ing act iv i ty o f b rewed (B r ) , b o i l e d ( B o ) , and instant (I) cof fee M R P p igments 215 7.2 E lementary compos i t i on o f coffee M R P p igments 216 LIST OF FIGURES F I G U R E P A G E 2.1 R e v i e w o f M a i l l a r d react ion pathway 10 2.2 In i t ia l stage o f the M a i l l a r d react ion 12 2.3 Condensat ion o f ca rbony l compounds w i t h am ino compounds 12 2.4 ( A ) Fo rmat ion o f sugar f ragmentary products. (B ) T w o poss ib le pathways [(I) and (II)] o f free rad ica l fo rmat ion 14 2.5 Proposed react ion scheme for generat ion o f chemi luminescence ( C L ) i n M R 17 2.6 Structures o f three most predominant ly found deoxyosones 19 2.7 T w o poss ib le pathways o f A m a d o r i compound degradat ion. Pathway (I) via 1,2 eno l i za t ion and Pathway (II) via 2,3 eno l iza t ion 19 2.8 Deoxyosone degradat ion via Strecker degradat ion Pa thway III 21 2.9 Strecker degradat ion pathway I V via t ransaminat ion o f the S c h i f f b a s e 21 2.10 F i f t h pathway o f A m a d o r i rearrangement product degradat ion 21 2.11 Degradat ion products o f 1 -deoxyosone 23 2.12 Degradat ion products o f 3 -deoxyosones 25 2.13 Degradat ion products o f 4 -deoxyosones 26 2.14 Some o f the N - , S - , (I) and O - (II) heterocyc l ic compounds fo rmed dur ing the M a i l l a r d react ion 27 3.1 Appearance o f P M 2 phage band in ces ium ch lor ide f o l l o w i n g gradient centr i fugat ion 70 x i v 3.2a Y i e l d o f crude non-d ia lysab le M R P mixtures versus the in i t ia l water act iv i ty ( a ^ ( A ) , and in i t ia l p H va lue (B ) 74 3.2b Y i e l d o f crude non-d ia lysab le M R P mixtures versus the ca lcu lated equivalent react ion t imes (U ) 75 3.3 Spectra l patterns o f crude Glu-Lys and Fru-Lys MRP mix tures 77 3.4 Abso rbance readings o f crude Glu-Lys and Fru-Lys MRP mix tures at 420 n m versus the y i e l d o f non-d ia lysab le M R P mixtures 78 3.5 Hunter L a b 'L', ' a 'and 'b' va lues against the y i e l d o f crude Fru-Lys ( A ) and Glu-Lys (B ) MRP mix tures 80 3.6 A m o u n t o f copper bound to crude M R P mixtures versus their n o n -d ia lysab le y i e l d 82 3.7 Percentage o f M R P mixtures recovered i n components f ract ionated b y che la t ion chromatography 84 3.8a M A L D I - mass spectra o f component number 1 o f ( A ) Glu-Lys M R P mix ture 3 and (B) Fru-Lys MRP m ix ture 5 fract ionated b y che la t ion chromatography 87 3.8b M A L D I - mass spectra o f component number 5 o f ( A ) Glu-Lys M R P mix ture 3 and (B ) Fru-Lys MRP m ix ture 5 f ract ionated by che la t ion chromatography 88 3.8c M A L D I - mass spectra o f f ract ionated component number 1 around 12,000 Da l t on mo lecu la r we ight range 89 3.9 Percent ant ioxidant act iv i ty o f crude Glu-Lys and Fru-Lys M R P mix tures as measured by the T B A method 93 3.10 Percent oxygen consumpt ion b y non-d ia lysab le M R P mix tures i n a mode l l i no le ic ac id emu ls i on ( M L E ) system 95 3.11 Protect ive index (PI) va lues ca lcu lated for crude Fru-Lys and Glu-Lys M R P mixtures synthesized b y each M R P synthesis exper iment 96 3.12 Re la t ionsh ip between the protect ive index (PI) va lues and the y i e l d o f crude non-d ia lysab le M R P mix tures 98 XV 3.13 Re la t ionsh ip between protect ive index (PI) va lues and the copper b ind ing act iv i ty o f crude M R P mixtures 99 3.14 Concent ra t ion dependent D N A n i c k i n g observed for two M R P mixtures 101 3.15 Percentage o f supercoi led D N A remain ing after incubat ing P M 2 phage D N A for 1 hour at 37 °C w i t h dif ferent concentrat ions o f crude M R P mixtures der ived f r om dif ferent synthesis exper iments 102 3.16 Percentage o f n i cked c i rcu lar D N A remain ing after incubat ing P M 2 phage D N A for 1 hour at 37 °C w i t h dif ferent concentrat ions o f crude M R P mixtures der ived f r om dif ferent synthesis exper iments 103 3.17 Percentage o f degraded D N A remain ing as a smear after incubat ing P M 2 phage D N A for 1 hour at 37 °C w i t h dif ferent concentrat ions o f crude M R P mixtures der ived f r o m dif ferent synthesis exper iments 104 4.1 Tempora l pattern o f malona ldehyde fo rmat ion i n cont ro l cook ie doughs w i t h and wi thout supplemented copper ions 130 4.2a Tempora l pattern o f malona ldehyde fo rmat ion i n cook ie doughs w i t h added Glu-Lys MRP mix ture 3 i n the absence o f supplemented copper 131 4.2b Tempora l pattern o f malona ldehyde format ion i n cook ie doughs w i t h added Glu-Lys MRP mix ture i i i n the absence o f supplemented copper 132 4.3a Tempora l pattern o f ma lona ldehyde fo rmat ion i n cook ie doughs w i t h added Fru-Lys MRP mix ture 5 i n the absence o f supplemented copper 133 4.3b Tempora l pattern o f ma lona ldehyde fo rmat ion i n cook ie doughs w i t h added Fru-Lys MRP mix ture 11 i n the absence o f supplemented copper 134 4.4 Tempora l pattern o f malona ldehyde fo rmat ion i n cook ie doughs w i t h added a - T o c o p h e r o l i n the absence o f supplemented copper 136 XVI 4.5a Tempora l pattern o f ma lona ldehyde format ion i n cook ie doughs w i t h added Glu-Lys MRP m ix ture 3 i n the presence o f supplemented copper 138 4.5b Tempora l pattern o f ma lona ldehyde format ion i n cook ie doughs w i t h added Glu-Lys MRP m ix ture 13 i n the presence o f supplemented copper 139 4.6a Tempora l pattern o f ma lona ldehyde format ion i n cook ie doughs w i t h added Fru-Lys MRP mix ture 5 i n the absence o f supplemented copper 140 4.6b Tempora l pattern o f ma lona ldehyde format ion i n cook ie doughs w i t h added Fru-Lys MRP mix ture i i i n the absence o f supplemented copper 141 4.7 Tempora l pattern o f ma lona ldehyde format ion i n cook ie doughs w i t h added oc-Tocopherol i n the presence o f supplemented copper 142 4.8 Tempora l pattern o f ma lona ldehyde fo rmat ion i n cook ie doughs w i t h added c t -Tocophero l together w i t h Glu-Lys MRP mix tures or Fru-Lys MRP mix tures i n the absence o f supplemented copper 144 4.9 Tempora l pattern o f ma lona ldehyde format ion i n cook ie doughs w i t h added a - T o c o p h e r o l together w i t h Glu-Lys MRP mix tures or Fru-Lys MRP mix tures i n the presence o f supplemented copper. . . . 145 4.10 R e d u c i n g act iv i ty o f mode l M R P mix tures and ascorb ic ac id 146 5.1 D N A n i c k i n g caused b y dif ferent meta l ions at p H 7.5 160 5.2 F e induced D N A n i c k i n g at four dif ferent p H values 161 5.3a D o s e dependent D N A n i ck i ng i n the presence o f Glu-Lys MRP mixture 3 ( A ) and Glu-Lys MRP m ix tu re 13 (B ) at four dif ferent p H values 163 5.3b D o s e dependent D N A n i ck i ng i n the presence o f Fru-Lys MRP mixture 5 ( A ) and Fru-Lys MRP m ix ture 11 (B ) at four dif ferent p H va lues 164 XVII 5.4a Ef fec t o f 0 . 0 0 0 1 % (w/v) Glu-Lys and Fru-Lys MRP mix tures on F e 2 + ca ta lyzed D N A n i c k i n g at p H 7.5 ( A ) and p H 4.0 ( B ) 169 5.4b Ef fec t o f 0 . 0 0 0 1 % (w/v) Glu-Lys and Fru-Lys MRP mix tures on F e 2 + cata lyzed D N A n i c k i n g at p H 3.2 ( A ) and p H 2.6 (B) . . . 170 5.5a E f fec t o f 0 . 0 0 1 % (w/v) Glu-Lys and Fru-Lys MRP mix tures o n F e 2 + ca ta lyzed D N A n i c k i n g at p H 7.5 ( A ) and p H 4.0 (B ) 173 5.5b Ef fec t o f 0 . 0 0 1 % (w/v) Glu-Lys and Fru-Lys MRP mix tures o n F e 2 + cata lyzed D N A n i c k i n g at p H 3.2 ( A ) and p H 2.6 ( B ) 174 5.6 D N A n i ck i ng patterns observed after incubat ing P M 2 phage D N A w i t h two concentrat ions o f Glu-Lys MRP m ix ture 3 ( A ) and Fru-Lys MRP m ix tu re 5 (B ) at p H 7.5 w i t h F e 2 + ions 175 5.7 Percent superco i led D N A remain ing after incubat ion o f D N A w i t h three chelat ing agents at several concentrat ion leve ls 177 5.8 Percentage o f superco i led D N A remain ing after incubat ion o f D N A together w i t h ( A ) C u 2 + (10 p M ) and ascorb ic ac id (10 p M ) , (B ) F e 3 + (10 p M ) and ascorb ic ac id (10 p M ) , (C ) F e 2 + (10 p M ) and ascorbic ac id (10 p M ) 178 5.9 Percentage o f superco i led D N A remain ing after incubat ion o f D N A together w i t h ( A ) C u 2 + (10 p M ) and E D T A (10 p M ) , (B ) F e 3 + (10 p M ) and E D T A (10 p M ) , (C ) F e 2 + (10 p M ) and E D T A (10 p M ) . 1 7 9 5.10 Percentage o f superco i led D N A remain ing after incubat ion o f D N A together w i t h ( A ) C u 2 + (10 p M ) and phy t i c ac id (10 p M ) , (B ) F e 3 + (10 p M ) and phy t i c ac id (10 p M ) , F e 2 + (10 p M ) and phyt ic ac id (10 p M ) 180 5.11 D N A n i c k i n g patterns observed after incubat ing P M 2 phage D N A w i t h ascorbic ac id , E D T A , and phyt ic ac id i n the presence o f F e 2 + , F e 3 + and C u 2 + ions at p H 7.5 182 6.1 D o s e response cy to tox ic effect o f three meta l ions. . 195 6.2 Co lon i za t i on e f f i c iency o f mouse embryo f ibroblast ce l ls i n the presence o f m o d e l M R P mixtures 197 XVlll 6.3a M o u s e embryo f ibroblast ce l l co lon iza t ion e f f i c iency i n the presence o f M R P mixtures (0.001 % , w /v ) w i th C u ions at 0 .1 , 10, and 50 p M levels. . . . 198 6.3b M o u s e embryo f ibroblast ce l l co lon iza t ion e f f ic iency i n the presence o f M R P mixtures ( 0 .01%, w /v ) w i t h C u ions at 0 .1 , 10, and 50 p M levels. . . . . . 2 0 0 6.4a M o u s e embryo f ibroblast ce l l co lon iza t ion e f f ic iency i n the presence o f M R P mixtures ( 0 . 0 0 1 % , w / v ) w i t h F e 3 + ions at 0 .1 , 10, and 50 p M levels 201 6.4b M o u s e embryo f ibroblast ce l l co lon iza t ion e f f i c iency i n the presence o f M R P mixtures (0.01 % , w / v ) w i t h F e 3 + ions at 0 .1 , 10, and 50 p M leve ls . . . ; , . . .203 6 .5a M o u s e embryo f ibroblast c e l l co lon i za t i on e f f i c iency i n the presence o f M R P mixtures (0.001 % , w /v ) w i th F e 2 + ions at 0 .1 , 10, and 50 p M levels 204 6.5b M o u s e embryo f ibroblast ce l l co lon iza t ion e f f ic iency i n the presence o f M R P mixtures (0 .01%, w /v ) w i t h F e 2 + ions at 0 .1 , 10, and 50 p M levels. . . . . . . . . . . 206 7.1 R e d u c i n g act iv i ty o f cof fee M R P p igments and ascorb ic ac id 217 7.2a D o s e response o f coffee M R P concentrat ions on D N A n i c k i n g act iv i ty . ( A ) = p H 7 . 5 , (B ) = p H 4 . 0 219 7.2b D o s e response o f coffee M R P concentrat ions on D N A n i c k i n g act iv i ty . ( A ) = p H 3.2, ( B ) = p H 2.6 220 7.3a Ef fec t o f cof fee M R P (0.001 % , w /v ) on F e 2 + ca ta lyzed D N A n i c k i n g act iv i ty . ( A ) = p H 7 .5 , ( B ) = p H 4.0 221 7.3b Ef fec t o f coffee M R P (0.001 % , w /v ) on F e 2 + ca ta lyzed D N A n i c k i n g act iv i ty . ( A ) = p H 3 . 2 , (B ) = 2.6 222 7.4a Ef fec t o f coffee M R P (0.01 % , w / v ) on F e 2 + cata lyzed D N A n i c k i n g act iv i ty . ( A ) = p H 7 . 5 , ( B ) = p H 4 . 0 224 x i x 7.4b E f fec t o f coffee (0.01 % , w / v ) o n F e 2 + a ca ta lyzed D N A n i c k i n g act iv i ty . ( A ) = p H 3.2, ( B ) = p H 2.6 225 7.5 D o s e response co lon iza t ion e f f i c iency o f C 3 H 1 0 T 1 / 2 ce l ls i n the presence o f three coffee M R P s 226 7.6a C o l o n i z a t i o n e f f i c iency o f C 3 H 1 0 T 1 / 2 ce l ls i n the presence o f cof fee M R P (0 .001%, w /v ) and F e 2 + 1 227 7.6b C o l o n i z a t i o n e f f ic iency o f C 3 H 1 0 T 1 / 2 ce l ls i n the presence o f cof fee M R P (0 .01%, w /v ) and F e 2 + 228 7.7a C o l o n i z a t i o n e f f ic iency o f C 3 H 1 0 T 1 / 2 ce l ls i n the presence o f cof fee M R P (0 .001%, w /v ) and F e 3 + : 230 7.7b C o l o n i z a t i o n e f f ic iency o f C 3 H 1 0 T 1 / 2 ce l ls i n the presence o f cof fee M R P (0 .01%, w /v ) and F e 3 + 231 7.8a C o l o n i z a t i o n e f f ic iency o f C 3 H 1 0 T 1 / 2 ce l ls i n the presence o f coffee M R P (0 .001%, w /v ) and C u 2 + 233 7.8b C o l o n i z a t i o n e f f i c iency o f C 3 H 1 0 T 1 / 2 ce l ls i n the presence o f coffee M R P (0 .01%, w /v ) and C u 2 + . . . . . . .234 10.1 T i m e temperature curves o f Glu-Lys.and Fru-Lys MRP synthesis exper iment numbers 5 ( A ) and 14 (B ) 267 LIST OF SYMBOLS AND ABBREVIATIONS Symbols: C Degrees Celsius °F Degrees Farenheit K Kelvin t Heating time tb Unmodified heating time Z Temperature change required to change the decimal reduction time by a factor of 10 E a Activation Energy L Litre mL millilitre uL microlitre m M millimole/Litre n M nanomole/Litre nm nanometer g gram mg milligram ug microgram kD Kilodalton K j Dissociation Constant mmol millimole p. mol micromole Abbreviations: AGES Advanced glycated end products A N O V A Analysis of variance A R P Amadori rearrangement product B(a)P Benzo-a-Pyrene B H A Butylatedhydroxyanisole BHT Butylatedhydroxytoluene B S A Bovine serum albumin C L Chemiluminescent D M E Dulbecco's Modified Eagles medium D N A Deoxyribonucleic acid DPHH Dipicrylhydrazyl E D T A Ethylenediaminetetraaceticacid Fig. Figure Figs. Figures Olu-P-I 2-amino-6-methylpyrido [ 1,2-a: 3', 2'-d] imidazole H M F Hydroxymethylfurfural 5-HMF 5-Hydroxymethylfurfural H 2 0 2 H y d r o g e n perox ide I Q 2 -am ino -3 -me thy l im idazo -4 ,5 -qu ino l i ne M A L D I - M S M a t r i x assisted laser desorpt ion ion iza t ion- mass spectroscopy M D A Ma lona ldehyde M e I Q x 2 -am ino -3 ,8 -d ime thy l im idazo [4 ,5 - f ] qu inoxa l i ne M N N G N-methy l -N ' -n i t ro -N-n i t roguan id ine M W C O M o l e c u l a r we ight cu to f f M R M a i l l a r d react ion M R P s M a i l l a r d react ion products M R P M a i l l a r d react ion product M S G M o n o sod ium glutamate N A D P H N i co t i nam ide adenine d i -nuc leot ide N B T N i t rob lue te t razonium 0 2 *~ Superox ide ions * O H H y d r o x y rad ica l P G P r o p y l gal late P M S Phenaz ine methosulphate T A E T r i s acetate E D T A 2 - T B A 2-O- th iobarb i tu r i cac id T B A R S Th iobarb i tu r i cac id react ive substances T B H Q Ter t -buty l hydroqu inone T D T Therma l death t ime T P N Tota l parenteral nutr i t ion T rp -P - I 3 - a m i n o - l , 4 - d i m e t h y l - 5 H - p y r i d o [ 4 , 3 - b ] indo le U V U l t rav io le t V i s V i s i b l e XXII ACKNOWLEDGEMENTS I a m greatly indebted to m y thesis superv isor , D r . D . D . K i t t s o f Depar tment o f F o o d Sc ience , for h is guidance, encouragement, and enthusiast ic interest throughout this research project. H i s pro fess iona l attitude w i l l surely have a great in f luence on m y future as a research scientist. I w o u l d also l i ke to thank D r . T . D . Durance and D r . B . Skura , Depar tment o f F o o d Sc ience , for their he lp fu l c r i t i c i sm and suggest ions as w e l l as c la r i f y ing d iscuss ions. M y sincere appreciat ion is also extended to D r . S . S . Tsang , Depar tment o f E p i d e m i o l o g y , Cancer Research Institute, B . C . , for p rov id ing access to h is laboratory fac i l i t ies and h is va luab le adv ice . M A L D I - M S evaluat ion o f M a i l l a r d compounds w o u l d not have been poss ib le wi thout the generous support extended b y D r . G . E igendor f , Depar tment o f Chemis t r y , Un ive rs i t y o f B . C . Gra te fu l thanks are due to D r . B . E . M a r c h , Department o f A n i m a l Sc ience , for rev iew ing this thesis at very short not ice and her va luab le comments . A n a l y t i c a l guidance p rov ided by D r . C . Perera , Department o f Mathemat i cs , S i m o n Fraser Un i ve rs i t y dur ing statist ical data analys is and technica l assistance of fered by M r . S. Y e e , M s . V . Skura , and M s . D . S m i t h are deeply appreciated. Suppor t and assistance p rov ided b y the Un i ve rs i t y o f B r i t i sh C o l u m b i a and Natu ra l Sc ience and Eng ineer ing Research C o u n c i l o f C a n a d a are grateful ly acknowledged . F i n a l l y , very specia l thanks go to m y f ive year o l d son , A y e s h , and m y husband, D h a r m a , for their encouragement, pat ience, and understanding throughout the var ious stages o f m y study and to m y parents fo r their w a r m w e l l w ishes. 1 1.0 Introduction The non-enzymatic browning that occurs between reducing sugars and amino groups in foods is widely known as the Maillard browning reaction (MR). The reaction conditions that govern the yield of M R include: pH, aw, reaction time, reaction temperature, type of reactants, and molar ratio of the reactants. There remains, however, little information concerning the influence of reaction conditions on the physico-chemical and biological effects of different MRPs. MRPs have been demonstrated to possess antioxidant activities when applied to food systems, as a result of characteristic chelating activity, reducing power, or affinity to scavenge free radicals. Since antioxidant activity is an important attribute in food processing and preservation, and that MR may occur in thermally processed foods by inter-compartmental interactions, it is important to characterize the products of this reaction not only from a chemical stand point but also in relation to its oxidative behaviour (antioxidant and prooxidant activities). Moreover, the most recent findings in this area have widened to include food safety aspects of MRPs since in vivo glycation of proteins, enzymes, pigments, as well as D N A with reducing sugars may result in potential genotoxic and cytotoxic activity. This thesis will address various factors influencing both the yield, chemical composition, as well as the bioactive potential of MRPs extracted from both model and a food system. Due to the ubiquitous nature of metal ions and their involvement in oxidation reactions, the role of MRPs in food quality and safety may be manifested by the characteristic metal chelating and reducing activities of MRPs. Thus, the purpose of these studies was to determine the efficacy of defined MRPs to modulate DNA nicking and cytotoxicity in the presence of polyvalent metal ions and to evaluate the underlying mechanisms of action for observed antioxidant and prooxidant prooxidant activity. 2 2.0 Literature Review 2.1 Introduction and Historical Background The Mai l la rd reaction ( M R ) is a non-enzymatic browning reaction caused by the condensation o f an amino group with a reducing compound. L i n g (1908), in England, first postulated that the colour changes derived during a brewing process stemmed f rom reactions occurr ing between sugars and proteins. However , it was Lou is Camil le Mai l lard, who, in 1912, performed the first experiment, in which 1 part o f glycine combined with 4 parts o f glucose was heated in water (Mai l lard, 1912). In that experiment, after 10 minutes o f heating, the l iquid turned a yel low colour fo l lowed by a rapid progression to deep orange before turning a deep brown colour. Format ion o f CO2 was noted and the presence o f oxygen, nitrogen, hydrogen, or a vacuum did not affect the reaction. The brown compound formed f rom this reaction, termed melanoidin, was reported to be water soluble in the early stages o f the reaction and insoluble at the later stages with continued heating. The insoluble melanoidin obtained was composed o f 58 .85% C , 4 .92% H , 4 .35% N , and 31 .88% O ; thus a formula o f C i 6 H i 5 N 0 6 was derived. Mai l lard (1916) repeated the same reaction wi th sarcosine, alanine, valine, leucine, tyrosine, and glutamic acid and found that alanine was the most active amino acid. The different amino acids in this reaction when placed in the descending order o f relative reactivity were found to fol low: alanine, valine, glycine, glutamic acid, leucine, and sarcosine. Similarly, when glycine was reacted with a variety o f saccharides, xylose and arabinose reacted instantaneously; whi le fructose, galactose, glucose, and mannose reacted fairly rapidly and lactose and maltose reacted slowly. Ma i l l a rd (1916) observed that sucrose, a non-reducing sugar, gave no browning upon short heating w i th glycine, but the browning reaction started after 3 hours o f heating. Mai l la rd postulated that the M R might lead to profound analytical errors in chemical biology, and further hypothesized that the 2.0 Literature Review 5 physiology and medicine. Maillard even theorized that this condensation reaction would be of relevance to human health in relation to diabetes. In essence, most of Maillard's predictions have proven to be correct. In recent years, the M R has become a focal point of research by scientists in different disciplines, including Food Science, Nutrition, Geological Science, Agricultural Science, and Medicine. In the past decade, the M R has been the subject of six international symposia (Ericksson, 1981; Waller and Feather, 1983; Fujimaki et al, 1986; Baynes and Monnier, 1988; Finot et al, 1990), including the latest (Labuza et al., 1993) which was dedicated to the physiological and biological aspects of this reaction. The importance of the M R stems from the fact that it can be initiated under mild conditions and therefore becomes an important reaction to consider in many processed food systems. 2.2 S ign i f i cance o f the MR i n Our F o o d Heat treatment of foods rich in reducing sugars and free amino acids results in the production of MRPs. Various technological modes of heat treatment applied to foods, to name a few, include baking, frying, broiling, and stewing. These procedures enabled humans to widen their choice of foods immensely since a greater variety of foods become palatable only after a heat treatment. For example, browning reactions are an integral part in the production of caramel, coffee, chocolate, bread, and maple syrup. In addition, M R products (MRPs) are often added to foods and beverages (e.g. soft drinks, beers, sake) as ammonia caramel or ammonia sulphite caramel (O'Brien and Morrissey, 1989) to impart desirable colours, aromas and flavours. Fermented oriental foods, notably soy sauce and miso (bean paste), which are composed of amino acids, peptides, proteins, and sugars, also contain MRPs (Hashiba et al., 1981). Therefore, understanding the reaction conditions for generating MRPs may provide an important way of improving the control of this reaction in different processed food systems. 2.0 Literature Review 4 2.2.1 C o n v e n t i o n a l Sources o f M R P s a) Bread M a n y aspects o f bread flavour have been investigated by various scientists during the last few decades. Bake r et al. (1953) emphasized that fermentation fol lowed by the formation o f a b rown crust was essential for the development o f full bread flavour and odour. A fully fermented dough baked by internal electrical resistance heating, without the formation o f crust yielded a mi ld bread f lavour and odour (Baker and M i z e , 1939). Folkes and Gramshaw (1977) and Mulders et al. (1976) investigated the structures o f volatile compounds in baked bread crusts and reported that most o f them were produced as a result o f Mai l lard and Mail lard-type reactions. Thus, daily consumption o f bread contributes a certain amount o f M R P s to the human body. b) Dehydrated vegetables and fruits Non-enzymat ic browning is a major route for deterioration o f fruits and vegetables during dehydration and in storage (Braverman, 1963), with the Strecker degradation pathway in the M R being o f particular importance in this undesirable browning (Sull ivan et al, 1974). The use o f sulphur dioxide in potatoes containing a l ow concentration o f reducing sugars is required for the production o f dehydrated potatoes with l ow levels o f Strecker aldehydes (Sull ivan, 1981). Therefore, breeding o f potato plant varieties to obtain a low sugar content is an active area o f research based on minimizing the occurrence o f M R . c) Processed meat and meat products A great deal o f effort has gone into the determination o f the compounds present in the volatile fractions o f cooked meat either in roasted or broiled form. Despite the large number o f quantitative data on the chemical composit ion o f volatiles derived from cooked meat, no one has been able to duplicate meat aroma by the combination o f pure chemical(s) identified in meat aromas (Danehy and 2.0 Literature Review 5 Wolnak, 1983). The very first attempt to produce meaty aromas useful in foods via M R was published by K ie l y et al. (1960). These scientists reported that the heating o f reducing sugars wi th cysteine and cystine resulted in meaty odours. C o l d water extracts o f minced and lean meat have also been shown to contain a variety o f amino acids, glycoproteins, lactic acid, and glucose (Hornstein and Crowe, 1960; W o o d , 1961; M a c y et al, 1964a; b). Dur ing the ageing process o f meat carcasses, glycogen undergoes glycolysis to lactic acid and the autolysis o f proteins and nucleic acids take place inside the muscle tissue. Melanoid in formation in cooked meat products was studied by Obretenov et al. (1993) by characterizing the melanoidin components in meat patties using spectrophotometry and gel filtration chromatography. A s such, it is evident that boil ing, broil ing, and roasting o f meat can facilitate the interaction o f these reducing sugars with amino groups leading to the M R . d) Soy foods It is wel l known that some brewed products such as soy sauce, miso, wine and Sake (Japanese rice wine) darken during storage when in contact wi th atmospheric oxygen. M a n y scientists have reported that the colour formation in soy foods is due to M R . Soy sauce is prepared by digesting mixtures o f soy beans and wheat wi th enzymes produced by Ko j i molds fol lowed by fermentation by yeasts in the presence o f 17-18% N a C l . Hashiba et al. (1981) found six Amador i compounds in soy sauce, miso, white wine, and Sake, and explained the important role o f Amador i compounds in oxidative browning. Since soy sauce contains large amounts o f amino acids, peptides, and sugars, and is brewed with the process o f 6-12 months o f ageing, intermediate browning products accumulated within this food system ultimately result in the presence o f brown pigments. e) Chocolate and cocoa Chocolate, one o f the worlds most popular flavours, is produced through the M R . T w o entirely separate stages: (a) the fermentation o f the cacao beans and (b) the roasting o f the fermented beans are 2.0 Literature Review 6 essential for the development o f chocolate flavour (Danehy and Wolnak, 1983). Unfermented cacao beans, wh ich contain sucrose, have little flavour. Dur ing the fermentation step, sucrose is converted to glucose and fructose, and the concentration o f free amino acids is increased by three to four fold (Danehy and Wolnak, 1983). This sets the stage for the development o f the chocolate aroma which is produced during subsequent roasting o f fermented cacao beans by the interaction o f free amino acids wi th reducing sugars through Strecker degradation reaction (Rohan and Stewart, 1965). M o r e than 350 cocoa volatiles have been identified and the routes for the formation o f many o f these compounds have been elucidated (Hoskin and Dimick, 1984; Danehy, 1985). E v e n though chocolate flavour is produced naturally through the M R during the roasting operation in the manufacture o f chocolate, there is no evidence that any o f the cocoa and chocolate manufacturers have adapted the Mai l lard technology to their manufacturing processes. O n the other hand, product ion o f cocoa substitutes or extenders (bulking agents with added flavour and colour) and chocolate flavours depend largely on the Mai l la rd technologies. A few o f the extenders, as mentioned by Danehy and Wo lnak (1983), are carob® (producing a nutrient f lavouring agent), V iob in cocoa replacer®, and Cocomost®. f) Coffee and tea Coffee bean processing, which involves unit operations such as drying, roasting, mil l ing, and extraction wi th hot water, causes the amino-carbonyl reaction and polymerization o f phenols leading to the formation o f melanoidin. In some studies, coffee has appeared to be mutagenic in the absence o f S-9 mix (Nagao et al, 1979). Glyoxal , methyl glyoxal, and di-acetyl have been reported to be the mutagenic compounds present in coffee (Nagao et al, 1979). However , in the presence o f S-9 mix, coffee compounds have been reported to be non-mutagenic (Aesbacher et al, 1980). 2.0 Literature Review 7 2.2.2 C o n t e m p o r a r y Uses of M R P s in Foods a) Production of flavours and aromas The most important aspect o f the M R in food preparation is its ability to transform a great variety o f food and raw materials into palatable food products with pleasant aromas and flavours. A l though M R is not the only means o f imparting flavour and colour to create acceptable food systems, it plays a key role in cook ing traditions all over the wor ld. M R flavours are found in many heated foods. However , in some food processes, M R is also characterized as causing discolouration and off-flavour production. A classic example is the production o f aged or stale flavour during the storage o f dairy products (Mauron, 1981). Parks (1967) reported that the stale flavours in dairy products were associated wi th l ipid deterioration reactions and as wel l as the occurence o f M R . In most traditional food processing operations, such as frying, roasting, and baking, the development o f colour and flavour formation is controlled by long term practical experience. However , in new technologies such as, microwave, infra-red, and extrusion cooking technologies a new basis for the control o f product quality has to be developed (Lingert, 1990). In microwave-processing, microwaves do not result in increased surface temperature o f food. Therefore, drying o f the surface to produce water activities favourable for the M R does not occur. A s a result, food products have to be adapted for microwave heating by treating the surface wi th pre-mixes containing reducing sugars and amino compounds in order to improve colour and flavour formation during heating. Alternatively, using a coating that works as a moisture barrier that keeps a l o w moisture content at the surface o f food wi l l also produce colour. A third method involves the use o f artificial M R based flavours (Lingert, 1990). Similarly, considerable research activity has been initiated to develop Mail lard-type colours and flavours for infrared-processed and extrusion-processed foods. 2.0 Literature Review 8 Product ion o f artificial meat flavours can be considered as another area o f research that has exploited the characteristics o f M R . Danehy and Wolnak (1983) reported a list o f companies producing artificial meat flavours using M R . Some o f the companies that were listed by Danehy and Wo lnak (1983) include: K n o x Ingredient Technology (KIT) , Ed long Corporat ion (E lk G rove Vi l lage, IL ) , W m . M . Be l l C o . (Melrose Park, IL) , Borden Industrial F o o d products (Northbrook, IL) , Hydroca l , S. A . (Geneva, Switzerland), A lex Fries and Bros. , Inc. (Cincinnati, O H ) , Fr i tzsche-Dodge & Olcott , Inc. ( N Y ) , and Haarmann & Reimer (Springfield, N J ) . c) Production of colour The colour and flavour o f caramel constitutes an important quality characteristic o f many foods and beverages since caramel is primarily employed as an added component o f many food systems for its colour attributes (Greenshields and Macgi l l ivery, 1972). Caramel can be produced by a variety o f chemical processes and it is also considered as a natural constituent o f many carbohydrate r ich foods which have been baked, fried, or roasted. The most common, and longest known, technique o f caramalization involves the production o f a dark coloured product by heating sugars above their melting points in the absence o f amino acids or proteins. Caramel can also be produced by the M R . F o r example, ammonia caramel that is used in many soft drinks is produced by heating sugar with added ammonia. The added ammonia immediately reacts with the aldehyde group in the sugar to form an aldehyde ammonia complex; this reaction is then fol lowed by rearrangement, condensation, and polymerization reactions culminating in caramel. Generally, caramel colour contains approximately 5 0 % digestible carbohydrate, 2 5 % indigestible carbohydrate and 2 5 % melanoidins. Therefore, it is worthwhile to note that consumption o f soft drinks wi th added ammonia caramel expose human beings to a certain amount o f melanoidins. 2.0 Literature Review 9 c) Anti-microbial activity It has been demonstrated that M R P s obtained by reacting amino acids and sugars are inhibitory to bacterial growth (Einarsson et al, 1983). These compounds act at three different target sites in the cell : i) cellular membranes (causing changes in cell integrity); ii) genetic material (causing genotoxicity); iii) enzyme systems (causing suppression) and thereby exhibiting antimicrobial activity (Eckland, 1980). The inhibitory effects o f many antibacterial compounds have also been attributed to their ability to chelate metal ions, thus making them unavailable to microorganisms. F o r example, the presence o f M R P s inhibit the uptake o f oxygen by chelating iron, a co-factor in many enzyme reactions related to oxygen metabolism and thus impart antibacterial activity (Einarsson and Er i ckson , 1990). The antibacterial activity o f arginine-xylose M R P s has been reported to be similar to or higher than that o f sorbic acid under certain experimental conditions (Einarsson and Er ickson, 1990). In addition, Einarsson (1990) reported that M R P s exhibit a stimulating effect on microbial growth up to a certain concentration level, and above that level they act as growth inhibitors. 2.3 C h e m i c a l P a t h w a y s o f M R The description o f the early stages o f the M R precursor formation has been wel l cited by many food chemists. A m o n g the many chemical reviews on M R (Danehy and Pigman, 1951; Hodge , 1953; H o d g e and Rist , 1953; El l is , 1959; Reynolds, 1963; 1965; Feeny etal, 1975; M a u r o n , 1981), the M R scheme (Fig. 2.1) originally reported by Hodge (1953; 1967) is specially notable. Accord ing to Hodges ' s scheme, M R has been identified to occur in three different stages: (1) an initial stage (reactions A and B ) involving the formation o f glycosyl-amino products fo l lowed by an Amador i rearrangement; (2) an intermediate stage (reactions C , D , and E ) involving dehydration and fragmentation o f sugars, amino acid degradation, and others; and (3) a final stage (reactions F and G ) 2.0 Literature Review aldose sugar + amino compound N-substkuted glycosylamine + H20 Amadori rearrangementf-B/ (Q 1 - amino - 1 - deoxy -2- ketose (1,2 enolform) Amadori compound -3H20 Schiffbase of HMF or furfural -2H20 (Q Reductones -amino compound + ^ 0 -2H HMF or furfural +2H dehydro reductones (D) \ + amino acid (E) Strecker degradation Fission products (acetol, pyruvaldehyde, diacetyl, etc) co2 aldehyde (G) + amino compound Aldimines (G) (F) with or without amino compound (F) (F) (F) Aldols and N-free polymers + amino compd amino compd. (G) Aldimines Aldimines or ketimines I + amino compd (G) — » ' MELANOIDINS e ^ Z Z (G) F i g . 2 .1 : R e v i e w o f Ma i l l a rd react ion pathway (Hodge, 1953). 2.0 Literature Review 11 involving aldol condensation, polymerization, and the formation o f heterocyclic nitrogen compounds and coloured products. 2.3.1 In i t ia l Stage o f the M R The initial stage o f the M R is presented in Fig.2.2. The very first step o f the reaction is the simple condensation between the carbonyl group (aldehyde form o f the reducing sugar) and the free amino group giving rise to a N-substituted glycosylamino compound fol lowed by the reversible formation o f the Schi f f base derivatives. This condensation reaction is initiated by an attack o f a nucleophil ic amino nitrogen wi th an unshared electron pair on the carbonyl carbon. This reaction usually requires an acid catalyst. Protonat ion o f the carbonyl group enhances its reactivity to the nucleophil ic reagent while protonation o f the nitrogen o f the amino group inhibits the attack on the carbonyl carbon. The favourable combination for these two reactants is shown in F ig . 2.3. Schif f base is an intermediate step in the M R since it rapidly cyclizes to a N - substituted glycosyl amine. U p to this step, the reaction is reversible because the glycosylamine can be hydrolyzed to its parent compounds in aqueous solutions. The reaction o f an aldose with an amino group results in formation o f aldosylamines and the reaction o f a ketose wi th an amino group results in the formation o f ketosylamines. Because o f the l o w stability o f aldosylamines and ketosylamines, these intermediates are readily converted into Amador i compounds (1-amino- l -deoxy-2-ketose) and Heyns products (1-amino-l-deoxy-2-aldose), respectively. These Heyns and Amador i rearrangement reactions are catalyzed by weak acidic conditions. Since amino acids serve as their own acid catalysts, the reaction is rapid even in the absence o f added acids (Namik i , 1988). It is also worthwhi le to note that the neutral or alkaline p H conditions that promote the browning reaction are not conducive to the initial stage o f the M R which is favoured by acidic conditions. This issue has been addressed by Namik i (1988) as presented in a later section in this chapter. 2.0 Literature Review 12 HOO RNH RN RNH RNH | +RNH2 I -H^O | | | I (CHOH). <s> CHOH <=> CH o H<^ 1 <z> <^H2 CH&H (CHOH)m (CHOH)„ (CHOH)n., O C=0 CHJ)H CHfiH HC^ 1 (H(^OH)n CHfiH CH^H Aldose in Addition Schiff base N-substituted N-substituted aldehyde form compound glycosylamine 1-amino-l-deoxy-2-ketose Amadori compound F i g . 2.2: Initial stage o f the Ma i l l a rd reaction (Hodge, 1967). F i g . 2.3: Condensat ion o f carbonyl compounds v/ith amino compounds. ( N a m i k i , 1988) 2.0 Literature Review 13 Free Radical Character of MRPs Format ion o f free radicals at an early stage o f M R have been reported by several scientists, namely, M i t suda et al. (1965), Nam ik i et al (1973), and Namik i and Hayashi (1981; 1983). Fair ly stable free radicals have also been observed in alkaline solutions o f reducing sugars (Lagercrantz, 1964) and in amino-carbonyl reactions o f ninhydrin with amino acids (Yuferov et al, 1970). Those studies suggest that M R may also involve a free radical process or produce some form o f free radical compounds through a similar process that occurs in the above mentioned compounds. A free radical formation mechanism through sugar fragmentation as proposed by Namik i (1988) is presented in F ig . 2.4. Structures and the Process of Free Radical Formation T w o possible pathways (Fig. 2.4 B) for the formation o f M R free radical products as suggested by N a m i k i and Hayashi (1981; 1983) and Hayashi and Namik i (1980; 1986) include: (1) free radicals develop rapidly prior to the formation o f Amador i product which then begin to decrease while the Amador i product continues to increase and 3-deoxyosone is produced thereafter; (2) free radicals form from a glycosylamino compound alone while no free radicals develop from the Amador i compound or from amino acid or sugar. The pathways, proposed by N a m i k i (1988), for the formation o f free radicals involves sugar fragmentation which give rise to a very reactive two carbon enaminol compound (glycoaldehyde) with extremely high reactivity in both free radical formation and browning. Furthermore, Nam ik i (1988) suggested that this reaction sequence is a different chemical pathway in the degradation o f reducing sugars which take place without the formation o f Amador i compounds. The above corroborates the previous work by Hodge (1953) who noticed similar sugar fragmentation in M R and the active role o f the low molecular weight carbonyl compounds in browning although a reverse aldol mechanism was proposed to explain this reaction. Quantitative measurements indicated that product ion o f the two-carbon sugar fragmentation product increased after the production o f 2.0 Literature Review A) CHO R' sugar CH=NR CHOH + RNH2 CHOH I • | -CHOH - H20 CHOH R' Schiff base HC=NR f I ( HC-OH HC-OH NHrR R' reverse aldol reaction H HC-NR II HC-OH 2-C fragmentary product CHO I /?' \ HC-NR II HC-OH condensation u > H2C-NR R N R Browning HC=0 glycoaldehyde dialkyl-dihydro pyrazine dialkyl pyrazinc radical dialkyl pyrazinium compound H HC-NR k HC=NR w HC=NR -HjO HC=NR + H20 HC=0 II ^ I ^ 1 ^ 1 * I HC-OH H2C-OH oxidation / f t>0 +RNH2 HC=NR -2RNH, i/C=0 glycolaldehyde glyoxal glyoxal glyoxal alkylimine mono-alkylimine di-alkyiimine F i g . 2.4: A ) Format ion o f sugar fragmentary products. B ) T w o possible pathways [(I) and (II)] o f free radical formation. ( N a m i k i , 1988) 2.0 Literature Review 15 glycosyl amino compounds which was fol lowed by free radical formation and subsequent browning. In addition, formation o f a four-carbon product has been identified to occur almost in parallel wi th the two-carbon product (Namik i and Hayashi, 1983). Al though formation o f fragmentary products at an early stage o f the M R was obtained mainly in glucose-alkylamine systems (Namik i , 1988) reverse aldol conversions have also been shown to be possible with ketoses and amino-ketoses (Led l , 1990). F o r m a t i o n o f S u g a r F r a g m e n t a r y Produc ts H o d g e (1953; 1967) proposed a mechanism for formation o f sugar fragmentary products (methylglyoxal, diacetyl) f rom Amador i products through 2,3 enolization and deamination, although no clear evidence was presented on this process at the time. Hayase and K a t o (1986) reported that a large number o f l ow molecular weight fragments was produced very rapidly at p H 11.4 wi th no heterocyclic compounds while the products formed at p H 4.0 were mainly heterocyclic. Similarly, Hayashi and N a m i k i (1986) quantified C -2 and C-3 carbonyl products in a glucose n-alanine reaction mixture and reported that production o f these compounds was greatly influenced by p H o f the medium. Alkal ine p H values were reported to greatly influence C -2 and C-3 products whi le no products were formed at acidic p H values. Neutral p H values were reported to produce only trace amounts o f C - 2 and C-3 products. The structures o f C -2 and C-3 fragmentation products have been assumed to be glycoaldehyde, glyceraldehyde, methylglyoxal, etc., or associated imine derivatives (Namik i , 1988). The relative browning activity o f l ow molecular weight sugars and carbonyl compounds wi th respect to glucose and glucose n-alanine mixtures were reported to be several fold higher than that o f glucose. Therefore, it clearly demonstrates that the browning observed at an early stage o f the M R at higher p H values may result f rom sugar fragmentation pathways. This fact explains the reasoning for increased Mai l la rd browning under alkaline p H values although the initial stages in the M R are favoured at an acidic p H . 2.0 Literature Review 16 Chemiluminescent Character of MRPs Certain organic compounds slowly oxidize in air and produce extra weak chemiluminescent character (Kurosaki et al, 1989). Bordalen (1984), first reported that the amino carbonyl reaction results in chemiluminescence ( C L ) . F ive years later, Kurosak i et al (1989) reported results o f an extensive study designed to elucidate the mechanism o f C L during M R P formation. It was determined that C L is p H dependent wi th a linear relationship between C L intensity and hydrogen ion concentration. In addition, when sugars used in their study were arranged according to specific effects o f browning, aldopentoses (ribose, arabinose, xylose) produced more browning than aldohexoses (glucose, galactose), which in turn produced greater browning than ketohexoses (fructose). Similarly, the relative effectiveness on C L also fol lowed the same order except for Z)-glucose, by exhibiting a direct relationship between C L and extent o f browning reaction. It was further suggested that C L in amino carbonyl reaction originates from free radicals present in melanoidin or associated intermediate compounds. The reaction pathways o f C L production as reported by Kurosak i et al. (1989) are given in F ig . 2.5. 2.3.2 Intermediate Stage of the MR Amador i and Heyns compounds are the initiators o f the intermediate step in M R . These compounds have been detected in heated, dried, and stored foods as wel l as inside the human body (Mauron , 1981; McPherson et al, 1988). Since Amador i and Heyns products play a very important role on physical, nutritive, and physiological properties o f proteins, Amador i product formation in both food and biological systems have been investigated in a number o f studies (Adr ian, 1974; Mauron , 1981; Mester et al, 1981; Monn ier and Cerami, 1983; Baynes et al, 1986 and Erbersdobler, 1986). 2.0 Literature Review 1. RNH RNH CH2 Heat „ HC-2. RNH 3. RNH HC^OO' L i R' 4. K/V// L?0» J. 5. /yv// too// L J. i u I J J HCC I J U " * J HCX i RNH \H2 u I. RNH CH2 L RNH CH2 c=o I HC* +' ' O ; I. RNH RNH //loo - J» J . J RNH HCOO-L I -• Polymers RNH \oOH L HCC  J. 'o, RNH I / / C J /JAW CH2 RNH CH2 > I + c=o I CL R 7. '02 -* 3o2 CL F i g . 2 .5 : P roposed react ion scheme for generat ion o f chemiluminescence ( C L ) in (Ku rosak i et a l . , 1989). 2.0 Literature Review 18 A m a d o r i R e a r r a n g e m e n t P roduc ts ( A R P s ) Amador i products are weak in browning reactivity even in the presence o f amino compounds. Therefore, the main process o f colour and flavour product formation through Amador i products involve degradation reactions associated with formation o f deoxyosones and Strecker degradation (Mauron , 1981). In the p H range between 4 and 7, Amador i compounds and Heyns products are predominantly degraded to deoxyosones 1,3, and 4, as shown in F ig . 2.6 (Beck et al, 1988; Huber et al., 1988), and several pathways o f degradation have been proposed in this regard. T w o major pathways o f A R P degradation and deoxyosone production which ultimately produce colour and f lavour products are shown in F ig . 2.7. Pathway (I) occurs via 1,2-enolization, fo l lowed by elimination o f the hydroxy group at C-3 and deamination at C - l thus yielding 3-deoxyhexasone, a reactive carbonyl product and a hydroxy-methyl furfural ( H M F ) . Pathway (II) occurs via 2,3-enolization o f the A R P , fo l lowed by elimination o f the amino group from C - l which gives rise to a 1-deoxydicarbonyl intermediate. This compound further reacts to produce methylglyoxal, diacetyl, and other intermediates (Hodge, 1967). Accord ing to the above scheme, pathway (I) is favoured under acidic conditions and pathway (II) is favoured under alkaline conditions. Deoxyosones are important intermediate products o f the M R since ring formation, enolization, dehydration, condensation, and C - C fission reactions o f these degradation products o f Amador i compounds lead to a multiplicity o f M R P s . However , Hashiba (1976, 1978) reported oxidative coloration o f Amador i products in soy sauce without undergoing conversion to deoxyosones. Depending on the reaction conditions (e.g., type o f sugars, amines, reaction times, temperatures, water activity, pH) , the concentrations o f M R intermediates such as, Amador i and Heyns products or deoxyosones vary in yield. Consequently, the compositions o f M R P s differ, wh ich in turn result in 2.0 Literature Review Amadori compound CH, HC = 0 IhC-Oll C=0 £ = 0 6 = 0 J=o <Lfc c=o HC-OR HC^-OR J/6 HC-OH uJ-OH id-OH II :C -Oil H2C-OH II:C-OH 1-deoxyosone 3-deoxyosones 4-deoxyosones F i g . 2.6: Structures o f three most predominent ly found deoxyosones. Low pH (i) H2C^ CrO I Amadori CH(OH) CMPD. CH C (OH) H(OH) ( N ) High pH HC-N< C-OII ICOH c=o 1=0 J \ HON*< 1 OH HC=0 loHC=0 UCH(OH) CH(OH) I CH(OH) CH(OH) CH CH(OH) CH(OH) L + amine 'H(OH) (3-deoxy osone) intermediate reductones f CHtJ20-CH0 ^ CH1-CO-CO-CH3 HOCHiCO-CO-CHi \ / etc. F i g . 2 .7: T w o possible pathways o f A m a d o r i compound degradation. Pa thway I via 1,2 enol izat ion and Pathway II via 2,3 enol isat ion. ( N a m i k i , 1988) 2.0 Literature Review 20 variabilities in both sensory (e.g., flavour, colour, and aroma), as wel l as non-sensory (e.g., antioxidative properties, mutagenic activities) attributes. S t recke r Deg rada t i on P roduc ts Strecker degradation is the third pathway in the formation o f brown coloured compounds f rom Amador i rearrangement products. This series o f reactions involves the oxidative degradation o f arnino acids by oc-dicarbonyl compounds produced from the breakdown products o f Amador i compounds via pathways (I) and (II) (Fig. 2.7). In the Strecker degradation (Fig. 2.8), a-amino acids react wi th cc-dicarbonyl compounds to form Schiff bases which in turn enolize to form amino acid derivatives that are easily decarboxylated. The new Schiff base with one carbon atom less is then easily split hydrolytically into the amine and the aldehyde which in turn correspond to the original amino acid wi th one less carbon atom. The main a-dicarbonyl compounds resulting from this reaction are glyoxal, methyl glyoxal , diacetyl, and also probably 3-deoxyosones. The net result o f the reaction is a transamination which could be an important reaction for the incorporation o f nitrogen into melanoidins. M o s t o f the CO2 released during the M R is produced during the Strecker degradation step (Stadtman etal, 1952; Wo l f r om and Rooney, 1953). In addition to these three pathways, a fourth pathway was proposed by Hol terman (1966), which completely bypasses the Amador i rearrangement (Fig. 2.9). This reaction starts at the Schif f base and involves the migration o f the C = N double bond and subsequent hydrolysis wi th water. In this reaction, amino acid is converted to a oxo-acid and sugar is converted to a non-reducing amino sugar. The oxo-acid reacts further with an intact amino acid resulting in decarboxylation and liberation o f an aldehyde by the Strecker degradation. A fifth pathway (Fig. 2.10), proposed by Bur ton and M c W h e e n y (1964) involves the subsequent regeneration o f the amino acid from the Amador i compound while the sugar moiety is dehydrated to 5-hydroxy-methyl-furfural ( 5 - H M F ) . 2.0 Literature Review Deoxyosones + RNH2 F i g . 2.8: Deoxyosone degradation via Strecker degradation Pathway III ( N a m i k i , 1988). R R HOOC-C-N-C-U UOOC-C"N-C-H. H^'-C-J/. u I | " +HjO I R I • I • I + UOOC-OO J| I Oxo acid UjOU CUfiH CJliOH Schiff base Amino sugar F i g . 2 .9 : 1990). Strecker degradation Pathway I V via transamination o f the Sch i f f base (Led l , O H OH QH N OH H 2 C . -NH I CH2 I COOH OH OH OH) OH H 2 C - -NH — I CH2 I COOH OH OH h CH2 Amadori compound HC HOCH2 CH CHO OH HCz= CH HOCH2 O CHO CHO I c=o I CH II T CHOH I CH2OH CHO I c = o I CH2 CHOH I CHOH I CH2OH F i g . 2 .10: F i f th pathway o f Amado r i rearrangement product degradat ion (Led i , 1990). 2.0 Literature Review 22 D e g r a d a t i o n o f Deoxyosones a) Degradation of 1-deoxyosones A whole series o f characteristic M R P s are formed f rom 1-deoxyosones. Some compounds extracted by Led l (1990) are shown in F ig . 2.11. Cycl izat ion and water elimination f rom 1-deoxyosone lead to the production o f hydroxyfuranones (e.g., 1A to i Q . Products ID and IE shown in F ig . 2.11, are the main coloured compounds when pentoses are heated with primary and secondary amines, respectively. Format ion o f a six ring compound is also possible wi th hexoses (IF and 1G). El iminat ion o f water f rom IF leads to formation o f n-pyranone 1H which is very unstable, and thus quickly rearranged to form an ester 11. Compound 1G is found in all types o f heated food, thus proving the occurrence o f M R in foods. Isomerization o f 1-deoxyosones results in the formation o f a n-diketo compound IJ and other fragmentary products. El iminat ion o f the hydroxy group in the n-posit ion creates the acetylformine 1M which rearranges in the presence o f secondary amines producing a product named amino hexose reductone IL. There are many other degradation compounds formed from 1-deoxyosones which are not mentioned herein. b) Degradation of 3-deoxyosone Deoxyosones are the precursors o f most o f the heterocyclic and carbonyl compounds o f M R . Add i t ion o f a hydroxy group at position 4 o f 3-deoxyosone, fol lowed by enolization and isomerization leads to formation o f metasaccharinic acid lactone 3A (Sengel et al, 1989). Another compound, pyranone 3C, is formed when the hydroxy group o f the carbon atom 5 adds to the aldehyde function and water is eliminated. Compound 3D is formed from 3C and H M F . Jurch and Tatum (1970) reported that compounds 3E (pyrrole) and 3F (pyridinium) are formed through the water elimination and isomerization o f 3-deoxyosone. Pyrrole 3E dimerize very easily to compound 3G and 3H. 3-deoxyosone is also involved in the formation o f several intermediate compounds through condensation 2.0 Literature Review F ig . 2.11. Degradat ion products o f 1- deoxyosone. (Led l , 1990) 2.0 Literature Review 24 wi th Amador i compounds (compound 31). The reaction pathways o f the above are given in F i g . 2.12. Water elimination o f 31 results in the formation o f formyl pyrrole 3J (Farmer et al, 1988), or alternatively the retero-aldol reaction converts 3J to compound 3IT(Njoroge etal, 1987). Substitution o f hydroxy groups in compounds 3J and 3K with ether and methyl ether groups results in formation o f compounds 3L and 3M, respectively. Treatment o f reaction mixtures o f prolamine wi th glucose and sodium periodate have revealed further pyrrole (30,3P, 3Q, and 3R) formation (Led l , 1990). React ion o f secondary amines (eg. proline) wi th 3-deoxyosone led to product ion o f many different r ing structures which are not discussed herein. A good review o f this complex pathway is given by L e d l (1990). c) Degradation of 4-deoxyosones The hydroxyacetyl furane 4A (Fig. 2.13) is believed to be a primary decomposit ion product o f 4-deoxyosone (Ledl , 1990). In the presence o f primary amines, formation o f product 4A is completely inhibited and the products o f 4B and 4C are obtained (Pachmayr et al, 1985). F o r m a t i o n o f He te rocyc l i c C o m p o u n d s Greater numbers o f heterocyclic compounds are derived during the intermediate and final stages in the M R . These compounds include N - , S- , and O - heterocyclic structures. A m o n g the N - heterocyclic structures are pyrazine compounds which have received the most attention. Pyrazines are typical products o f advanced M R , and have been reported in many heated food systems. Pyrazines (Fig. 2.14 T) can be formed through condensation o f Strecker degradation products such as aminoketones (Mauron , 1981). Other pathways were proposed by Koehler and Odel l (1970). Pyrroles, pyrrolidines, and pyridines (Fig. 2.14 I) represent another group o f compounds that belong to the class N - heterocyclics. They are formed by reaction o f furfurals and their homologues 2.0 Literature Review 25 -CH-CO-NHJ:H-CO-NH-CH n^-nn^n- H2N-CH-COOH f] Tl CH2 CH2 HOH2C^\^CH2 CH2 CH2 ^1 CH2 Q H - CH2 HOH2C^N^CHO IT XT JTJL 3F(I) 1MR 1 CH2 —O — H20^K^CHO I 3H F i g . 2 .12: Degradat ion products o f 3-deoxyosones. (Ledl , 1990) 2.0 Literature Review 26 + wc=o t CH2 HC-OH HC-OH H2C-H H H r. fl — HOH2C— C _<">>^1 _ U OH OH OH OH HC-OH HO 'HO + NalO 4 OH H^-OH H2C-OH 3K HO OH IJ—L HC-OH HC-OH H)c-OH H2C-OH 3M OHC CHO I H O OHC X 1 N I 3R •HO OHC XX. CHO N CHO 30 OC ^~CHO I 3P F ig . 2.12: Degradat ion products o f 3-deoxyosones. H2C-OH o HC-OH H2C-OH 44 x = 0 4B X = N-4C F ig . 2 .13: Degradat ion products o f 4 - deoxyosones. ( L e d l 3 1990) 2.0 Literature Review I I) Pyrroline 2-acetyl-l,4,5,6 tetrahydro pyridine Maltol Dehydro furanone Iso maltol Dehydro pyrone Amino reductone Cyclopentenolone F i g 2.14: Some o f the N - , S - , (I) and O - (DT) heterocycl ic compounds formed dur ing the M a i l l a r d react ion (Mau ron , 1981). 2.0 Literature Review 28 wi th a-arnino acids. R i zz i (1974) proposed a mechanism which explains the formation o f pyrroles from corresponding furans. Oxasolines have also been isolated from heated foods and have been assumed to result from M R (R izz i , 1969). Thiazoles constitute another group o f compounds present in foods in wh ich M R has occurred. These compounds possess both N - and S- in their ring structures and are formed through the Mai l la rd reactions involving sulphur amino acids. A number o f thiazoles have been isolated from foods which have undergone Mai l la rd- type reactions (Mauron, 1981). O-heterocycles are the compounds responsible for the aroma o f caramel and are formed as a result o f sugar pyrolysis and also as a result o f the M R . T w o primary structures o f known O-heterocycles are the maltol and isomaltol wi th few other minor compounds o f this type including dehydrofuranones, dehydropyrones, aminoreductones, and cyclopentenolones (Fig.2.14 TJ) (Mauron, 1981). 2.3.3 F i n a l Stage o f the M R The final stage o f the M R produces brown pigments, often referred to as melanoidins. However , the knowledge accumulated so far, concerning the formation and structures o f these high molecular weight compounds is limited. It is believed that melanoidin is formed as a result o f polymerization o f many highly reactive compounds formed during the intermediate steps in the M R (Reynolds, 1965). Melanoid ins have different solubilities. Some are readily soluble in water whi le others are slightly soluble in water, or completely insoluble in water. Moreover , soluble pigments have been found to be dialysable as wel l as non-dialysable (Mauron, 1981). Ka to and Tsuchida (1981) and Cammerer and K r o h (1995) proposed a possible repeating unit for melanoidin structure. N a m i k i (1988) emphasized that regardless o f how small the quantity o f a product isolated from a reaction mixture, it may be important in melanoidin formation. It was also noted that the more reactive 2.0 Literature Review 29 the product, the more difficult it is to isolate and identify, thus conveying the complexity and ongoing dynamics o f this reaction under different conditions. 2.4 Pa rame te rs In f luenc ing the M R The course o f the M R is strongly influenced by the reaction conditions (Mauron , 1981). A number o f factors influencing M R are discussed below. (a) Nature and molar ratio of reactants It is considered that low molecular weight compounds are more reactive than high molecular weight compounds. A s such, aldopentoses are generally more reactive than aldohexoses (Spark, 1969), and monosaccharides are more reactive than d i - or ol igo- saccharides. A ldoses have been reported to be more reactive than ketoses (O' Br ien and Morr issey, 1989). L e w i s and L e a (1959) reported that sugars when placed in descending order o f reactivity have the fo l lowing sequence: (i) xylose; (ii) arabinose; (iii) glucose; (iv) lactose; (v) maltose; and (vi) fructose. A s a result, Finot et al. (1981) showed that replacement o f lactose by fructose in milk samples resulted in a decreased lysine destruction in the order o f 0-2%. Furthermore lysine destruction was decreased up to 5 0 % when such samples were spray dried. The ratio o f reducing sugar to amino acid has been suggested as an important factor in determining the rate o f M R and has important implications both f rom a food formulation standpoint and in vivo glycation o f proteins (Baisier and Labuza, 1992). Generally, an excess o f reducing sugar to that o f an amino compound accelerates M R (O 'Br ien and Morr issey, 1989). L e a and Hannan (1951) observed a maximum browning rate at a 3:1 ratio in a glucose : casein system. Warmbier et al. (1976) reported that the rate o f browning increased as the ratio o f glucose : glycine increased from 1:0.5 to 3:1 in an intermediate moisture model system. Moreover , studies performed by Wo l f r om et al. (1974) indicated colour development in Z)-glucose-glycine mixtures, containing 6 5 % water and stored at 65°C, was 2.0 Literature Review 30 markedly dependent on the relative proportion o f the amino acid to sugar ratio. These results indicated that colour production was relatively modest at a Z)-glucose-glycine ratio o f 10:1 to 2:1, but that the development o f colour rapidly accelerated at a ratio o f 1:1 to 5:1. The degree o f browning, therefore, appears to be maximum when sugar is present in excess. A n optimal molar ratio o f 2:3 has been reported for a methionine : glucose system (Dworschak and Ors i , 1977). However , an opposite trend on the sugar-amino acid molar ratio on browning was reported by Warmebier et al. (1976). The reason for the opposite trend observed above remains unclear; it perhaps may have stemmed from the lower water activity (0.52) used by these scientists compared to other work done in solution (Kannane and Labuza , 1989). The chemical nature o f the amino acid also affects the rate o f M R formation. A m i n o acids with s-and end terminal amino groups are the most reactive in M R . F o r example, the amino acid lysine which posesses two amino groups on the molecule, is more reactive than other amino acids wh ich have no available amino side chains. Therefore, the concentration o f lysine in a protein is closely related to the degree o f browning that takes place in a particular food fol lowing storage and processing. It is also known that amino acids affect the p H o f the system and, therefore, have an influence on the degree o f browning. Ashoo r and Zent (1984) reported that amino acids and amides may be classified into three groups according to their Mai l la rd reactivity when heated in the presence o f common reducing sugars: v iz. (i) highest reactive group including; lysine, glycine, tryptophane, and tyrosine; (ii) an intermediately reactive group including; proline, leucine, iso-leucine, alanine, hydroxy proline, phenylalanine, methionine, valine and the amides glutamine and asparagine; (iii) the least reactive group including; histidine, threonine, aspartic acid, arginine, glutamic acid and cysteine. This classification is not in agreement wi th the results reported by Massaro and Labuza (1990) who observed that lysine produced 2.0 Literature Review 31 the least browning when compared to tryptophan and cysteine. In another study, F ry and Stegink (1982) found that proline and amino acids with hydrophobic side chains reacted more slowly than amino acids without these side chains. These conflicting results may be explained by the p H dependence o f different amino acids in the M R . A s such, it may be expected that the amino acids that react similarly at a certain p H range may not necessarily respond the same way and hence wi th the same relative efficacy under other p H ranges. W o l f r o m et al. (1974) designed a comprehensive study to determine the effect o f amino acid type on the extent o f browning in a D-glucose-amino acid mixture. The study involved using amino acids that were commonly found in orange juice and other foods. The results o f those studies indicated that both L-arginine, and aminobutyric acid, for example, produced the most intense colour, fo l lowed by glycine, aJariine, serine, and /--proline. Aspartic acid, Z-glutamic acid, and L-glutamine showed a behaviour similar to that o f glycine. (b) Moisture content of the food M R in foods and model systems occur over a wide range o f water activities. Water activity exerts a primary influence by controll ing the liquid phase viscosity by dissolution, concentration, or by dilution o f the reactants (O'Br ien and Morr issey, 1989). M a x i m u m browning occurs in most foods with a water activity in the range o f 0.3 to 0.7 (Labuza et al., 1970). The reaction rate increases exponentially wi th increasing moisture content up to a maximum in the intermediate moisture range due to increased mobil i ty o f reactants. A further increase in water activity decreases the reaction rate due to a dilution effect. However , the position o f maximum reaction kinetics depends on the physicochemical properties o f the respective food system (Supplee, 1926). F o r example, an amorphous food system absorbs more moisture than a crystalline food. Eichner and Kare l (1972) studied the isolated effect o f water content and that o f water activity similarly on the browning rate. They reported that a decrease 2.0 Literature Review 3 2 in water content wou ld increase the rate o f browning except in those systems where water mobil i ty was limited by high viscosity. In essence, the extent o f browning was determined to be influenced by the amount o f available water and by the state o f water binding in the system. (c) Temperature and duration of heating M a n y scientists have observed that the rate o f browning increases wi th the reaction temperature and the reaction time. Cuzzon i etal. (1988) showed that the reaction temperature, absorbance at 420 nm and 280 nm, mutagenicity, and the level o f furfurals were all correlated at temperatures between 120-140 °C. Baxter (1995) reported that the reaction rate increases also wi th an increase in p H , temperature, and reducing sugar content. The importance o f reaction temperature and duration o f heating was further illustrated by Hurrel l and Carpenter (1974) who demonstrated the similarity in the amount o f e-aminolysine groups reacted in an albumin-glucose system in l o w temperature, long duration experiments (30 days at 37 °C) wi th those o f high temperature, short duration experiments (15 minutes at 121 °C). (d) Effect of pH Numerous research projects have been undertaken to investigate the effect o f p H on browning reactions in a variety o f foods (Underwood et al, 1959; K a t o et al, 1969; W o l f r o m et al, 1974; Ashoo r and Zent, 1984). Ashoor and Zent (1984) reported that M R increases as the p H o f the system is increased up to a maximum o f 10, and that the reaction rate then declines above a p H value o f 10. A l though the reason for a decrease in the rate o f browning at p H values above 10 is not known, two mechanisms have been proposed to explain the effect o f p H on the increased rate o f browning for those p H values below 10. These mechanisms can be summarized as fol lows: i) mechanism I: The availability o f an unprotonated amino group is a prerequisite for the occurrence o f M R . Thus, an increase in the reaction medium p H which ultimately drives the amino group from a protonated form to 2.0 Literature Review 33 an unprotonated form increases the rate o f browning. The protonated and unprotonated forms o f the amino group are shown by the fol lowing : - N H 3 + ( p H > 7) > - N H 2 + F T (Eq. 2.1) Accord ing to equation 2.1, at p H values higher than the pKa o f the amino acid, the amino groups do not possess a positive charge thus an increase in the extent o f browning occurs; ii) mechanism JJ: The amount o f acyclic form, or the reducing form, o f sugar also affects the rate o f brovvning and is p H dependent. W i th an increase in medium p H , the concentration o f acyclic form o f reducing sugar increases, which in turn increase the rate o f M R (Kannane and Labuza, 1985). In addition to the two above mentioned mechanisms, sugar fragmentation also plays an important role in M R kinetics. Higher p H values at the beginning o f the reaction increase the degree o f sugar fragmentation leading to increased reaction rates due to the greater reactivity o f smaller compounds. M o r e detailed information on the effect o f p H on M R can be found in an extensive review on the control o f browning reaction in foods conducted by Labuza and Schmidl (1986). (e) Other miscellaneous factors affecting the rate of MR Certain metal ions are known to affect M R kinetics. Copper and iron salts appear to accelerate the M R while tin and manganese salts appear to inhibit the reaction (Bohart and Carson, 1955; K a t o et al, 1981). Iron has been suggested to catalyze the browning o f soy sauce and also has been identified to be a constituent o f the melanoidin chromophore (Hashiba et al, 1981). T o a certain extent, the effect o f metal ions on browning is known to result f rom the p H changes occurring in the medium due to the presence o f metal salts. Another set o f compounds that affect M R are phosphates and sulphites. Whi le phosphates promote the M R and chromophore development (Reynolds, 1959), sulphur dioxide and sulphites act as inhibitors o f the M R (Taylor and Bush , 1986; Modderman, 1986; Wedz icha and Kapu to , 1987). 2.0 Literature Review 34 2.5 K i n e t i c s o f the M R T w o approaches with respect to M R kinetic studies have been proposed in the literature. The first approach focuses on the rate o f browning, and the other relates to the rate o f loss o f sugar and amino acids. General kinetic models for M R have been proposed by various research groups (Baisier and Labuza , 1992; Vern in et al., 1992; Yaylayan and Forage, 1992; Be l l , 1996). M o s t o f these models have been based on a general scheme in which the reducing sugar reacts wi th the amine to produce a Schif f base that undergoes the Amador i rearrangement to produce the Amador i product. Extensive reviews on the kinetics o f M R (Baisier and Labuza, 1992) and that o f Amador i rearrangement products (Yaylayan and Huyghues-Despointes, 1994) have been reported. Bais ier and Labuza (1992) reported that although the overall kinetics o f M R are more complex than the individual loss o f sugar or amino acid, the initial stage o f the reaction fo l lows pseudo-first order kinetics. Af ter the initial first order period, the loss o f reactants tapers of f into a phase wi th little reactant disappearance (no loss period) which can be explained by means o f steady state kinetics (Massaro and Labuza, 1990). The first report on the absence o f reactant loss during initial M R phase was published by W o l f e / al. (1977). Massaro and Labuza (1990) were the first two scientists to show a first-order period fol lowed by steady state kinetics. M o s t o f the other published reports in the literature do not seem to be based on enough data to quantitatively establish the occurrence o f a steady state kinetic phase. A kinetic study on glucose and lysine loss during M R was conducted by Massaro and Labuza (1990), and their report indicates that these reactions individually fo l low first order kinetics initially and second-order kinetics overall, as predicted mechanistically. Some o f the published activation energy (E a ) values calculated for M R , and sugar or amino acid losses during M R , as summarized by Labuza and Baisier (1992) are presented in Table 2.1. 2.0 Literature Review Tab le 2 .1 : Ac t i va t ion energy ( E a ) parameters for the non-enzymat ic b rown ing react ion ( A ) and protein qual i ty loss (B ) due to Ma i l l a rd react ion in different foods. ( A ) F o o d Temperature (°C) a w E a (kcal /mole) D r y cabbage 37 0.60 28 D r y cabbage 37 0.01 40 D r i e d potatoes 40-80 0.98 25 D r i ed potatoes 40-80 0.15 37 Casein/g lucose model system 25-45 0.52-0.60 33 Glucose-g lyc ine 60-100 1.00 16-22 Processed cheese 5-40 0.98 24 A p p l e ju ice 37-130 0.99 27 F igs 20-30 0.60-0.80 20 Rais ins 20-30 0.60-0.80 24 ( B ) : F o o d Temperature (°C) a w E a (kcal /mole) Soybean meal 100-120 <0.1 30 D r y cod 105-115 0.5 24 B o v i n e serum albumin 85-145 0.8 24 Soy-g lucose model 80-130 0.3-0.8 35 Case in-g lucose model 0-70 0.7 29 R i c e meal 115-184 >0.9 12.5 Pasta 40-90 0.5 13 Casein-g lucose 25-45 0.41 23 (Labuza and Bais ier , 1992) 2.0 Literature Review 36 2.6 Prevention of the MR In contrast to the extensive research work that has been conducted to understand the M R , a l imited number o f studies have been conducted to investigate means o f retarding occurrence o f M R in food systems. Such methods are discussed in this section. Sulfhydryl compounds Sulphur amino acids and sulphur rich proteins have been reported to prevent both enzymatic and non-enzymatic browning reactions (Molnar-Per l and Friedman, 1990a; b; Fr iedman and Bautista, 1995). Prevention o f the Mai l lard browning reaction either by trapping the initial reactants, or by preventing the activation o f intermediary compounds to biologically active compounds through the use o f sulphur amino acids has been examined (Friedman, 1996). Deglycation A s described by Gerhardinger et al. (1995), deglycation o f Amador i compounds by a bacterial enzyme, fructosyl-N-alkyl oxidase, is considered as an alternate means o f preventing the occurrence o f M R . However , the use o f these enzymes to reverse the browning reaction in foods as wel l as in vivo is still under investigation. Acetylation of amino groups Modi f ica t ion o f amino group o f lysine to amide groups with the use o f transglutaminase has also been investigated (Friedman and Finot, 1990). In this approach the objective was to replace lysine wh ich initiates browning, wi th y-glutamyl lysine which does not initiate browning. 2.7 Physicochemical Properties of MRPs In contrast to the understanding over the past two decades where polymeric M R P s were assumed to be chemically and biologically inert substances, recent research has revealed that polymeric M R P s possess metal chelating, antioxidant, and antimutagenic physicochemical properties. 2.0 Literature Review 37 2.7.1 A n t iox idat ive A c t i v i t y L i p i d deterioration is a common reaction in many foods, and in such cases it is desirable for the food to possess some antioxidative activity to prevent oxidation o f l ipid components. The most widely uti l ized antioxidants to date are the synthetic phenolic compounds, such as butylated-hydroxy anisole ( B H A ) , butylated-hydroxy toluene ( B H T ) , tert-butyl hydroquinone ( T B H Q ) , and propyl gallate (PG) , to name a few. Recently increased attention has been given towards using non-synthetic antioxidants in food systems. It is wel l known that M R influences the oxidative stability o f food products (Griff i th and Johnson, 1957). M o s t o f the studies on antioxidative activity o f M R P s have been peformed wi th model M R P s . Accord ing to Namik i (1988), the presence o f an amino compound is essential to induce antioxidative activity by M R in a food system. This is evident f rom the fact that neither sugars nor pyrolytic products (caramel) alone show antioxidant activity while there are many studies that have suggested antioxidant activities for secondary and tertiary linear and cycl ic amines, amino acids, and peptides (Namik i , 1988). Moreover , antioxidant activity o f amino compounds reacting without sugar has been found to be far weaker than those induced by reacting amino compounds wi th sugars. Grif f i th and Johnson (1957) reported that addition o f 2 .5% glucose to cookie dough, at the expense o f an equal amount o f sucrose, resulted in increased browning during baking, and a greater stability against oxidative rancidity. Anderson et al. (1963) observed an improvement in storage stability o f wheat, corn, and oats after heat treatment. The effect o f browning reaction products on the stability o f fats contained in biscuits and cookies as reported by Yamaguchi et al. (1964), Yamaguchi and K o y a m a (1967), and the ability o f glycine-glucose reaction products to inhibit the development o f rancidity in corn oi l (Malek i , 1973) further supports the above observations. 2.0 Literature Review 38 Eichner (1975), studied the M R in dehydrated foods and in dehydrated model systems. They reported that l ipid oxidation was inhibited in a heat treated (80°C; 5 min) glucose-lysine-avicel model system containing sodium linoleate, even under storage at room temperature. Evans et al. (1958) reported that the antioxidative effect o f melanoidin was associated wi th the presence o f reductones since pure reductones prepared f rom hexoses and secondary amines possessed antioxidative properties. Yamaguchi and Fuj imaki (1974a; b) fractionated M R P s obtained by heating 2 M glycine and 2 M xylose at 100 °C for 2 hours, by gel nitration and thin layer chromatography and found a strong antioxidative melanoidin fraction with a molecular weight o f about 4500 daltons. K i r igaya et al. (1968; 1969) studied the antioxidative effect o f several amino acids and sugars and found that alanine, serine, threonine, histidine, and arginine formed potent antioxidants when reacted wi th sugars. K i r igaya et al. (1971) also observed that the antioxidative activity o f M R P s o f a lower molecular weight range was stronger than the M R P s with a higher molecular weight range. Moreover , the antioxidative effect o f M R P s f rom xylose-glycine was reported to be comparable to that o f B H A , higher than that o f tocopherols, but lower than that o f B H T (Yamaguchi and Fuj imaki , 1974a and b). Ch iu et al. (1991) described the formation o f antioxidant componds during the M R o f tryptophan wi th fructose or glucose. Tanaka et al. (1992) reported that the glucose-tryptophan M R P s exerted an antioxidative effect in linoleic acid and performed as effective antioxidants for sardine lipids. Those same researchers also observed that the extent o f browning determined by measuring absorbance at 420 nm, or fluorescence determined by measuring excitation at 452 nm and emission at 530 nm, and reducing power as measured at 700 nm, were all correlated with antioxidative activity. Nakamura et al. (1992) observed a remarkable enhancement in antioxidative activity o f ovalbumin when it was covalently bound to dextran or galactomanan through a controlled M R . Bedinghaus and Ockerman 2.0 Literature Review 39 (1995) reported that reducing sugars and free amino acids generated effective antioxidants in cooked ground pork patties. In addition to the above, the antioxidative activity o f M R P s in vivo was also studied by Chuyen et al. (1990). The M R P s formed by heating amino acids such as histidine, arginine, lysine, glycine, and proteins, such as casein, and soy protein, with glucose exhibited some antioxidative activity in vivo. These findings demonstrate the effectiveness o f M R P s in prevention o f l ipid peroxidation in foods as wel l as possibly in vivo. Since the M R is very common in food processing and storage, especially in protein r ich foods that are subjected to heat treatment, it appears that food processors over the years have benefitted f rom the antioxidative capabilities o f M R P s even though such activities were not scientifically known at the time. 2.7.2 M i n e r a l Interact ions A l though the presence o f metal ions has long been known to influence the M R (Patton, 1955), much effort has been devoted towards investigating the effect o f M R on protein quality, f lavour, and aroma, than its effect on metal chelation. Recently, however, the influence o f M R P s on mineral metabolism has been investigated in vitro as wel l as in vivo. In vivo studies carried out with MRPs Stegink et al. (1975) reported that M R P s could adversely affect mineral metabolism in humans. A sudden increase in urinary zinc excretion in rats was reported when animals were given intravenously heat treated amino acids or hydrolysed proteins with glucose. Similarly, Andr ieux et al. (1980) observed that a diet containing 3 % M R P s caused a significant decrease in the retention o f calcium, phosphorus, magnesium, and copper in rats. Increased urinary excretion o f copper, i ron and zinc was noticed in patients receiving total parenteral alimentation nutrition ( T P N ) (Freeman et al., 1975). The sugar-amine compounds present in those T P N diets were considered responsible for increased mineral 2.0 Literature Review 4U excretion. Moreover , Johnson et al. (1983) reported that the consumption o f toasted brown corn products affected absorption o f zinc, iron, and copper in human subjects. Those scientists observed a decrease in long term zinc retention in subjects consuming thermally processed corn flakes. The distribution o f z inc in urine o f the subjects in that study was also changed since some o f the urinary zinc was bound by high molecular weight compounds. Since zinc is one o f the trace elements essential to many enzymatic reactions, the study o f M R P s affinity to bind this metal ion is important. In contrast, Stegink et al. (1981) suggested that the M R P s producing these metal interactions were not absorbed from the intestine in quantities necessary to exhibit the above mentioned observations. A l though there is evidence that at least some products o f the M R are absorbed from the digestive tract, there has been little w o r k on whether or not browning in ordinary diets may affect metal absorption o r excretion. A t p H 5, corn flakes bound 1.76 and 1.55 times as much zinc and copper, respectively, as did unheated corn grits. However , no significant differences were reported in iron retention over a 21 day study period (Johnson et al., 1983). The mechanism and the site o f action o f M R P s on calcium and magnesium absorption in the gut were shown to be different from the effect produced by poorly digestible carbohydrates such as lactose (Andr ieux and Sacquet, 1984). O'Br ien and Morr issey (1989) reported the effect o f M R P s on calcium absorption wi th respect to its effect on lactose. In that study, it was noticed that whi le M R P s inhibited the duodenal and ileal transport o f calcium, lactose appeared to enhance intestinal uptake o f calcium. The inhibitory effect o f M R P s on small intestinal calcium transport have been suggested to constitute the action o f either a chelator or an inhibitor o f enterocyte metabolism (O'Br ien and Morr issey, 1989). In vitro studies performed with MR I ron deficiency in humans is prevalent in populations where coffee is commonly consumed ( M u n o z et al, 1988). Chelat ion o f i ron by coffee constituents has been assumed to be the reason for the above 2.0 Literature Review 41 observation. Meta l chelation by coffee has been mainly attributed to phytic acid, thiol, and phenolic compounds. However , the method o f processing coffee beans also results in the amino-carbonyl reaction and polymerization o f polyphenols leading to melanoidin formation. The research group ( H o m m a et al, 1986) that reported the complexation o f metal ions in vivo by instant coffee M R P s indicated that not only the above mentioned compounds in coffee, but also the M R P s found in coffee, may furthermore contribute to iron deficiency anemia in humans. In another in vivo study conducted by Gregor and Emery (1987), where rats were fed with decaffeinated coffee, a decrease in total liver i ron and an increase in total liver copper indicated a negative effect o f feeding coffee extracts on dietary iron absorption. The concentration o f tibia zinc was also elevated in animals whi le bone strength was reduced. It is wel l known that iron accelerates M R (Hashiba et al, 1977) and also increases the colour intensity when added to melanoidin, or to a mixture o f amino acid and glucose (Hashiba, 1985). Iron not only catalyses the M R but it also acts as a chromophore in pigment formation since subsequent addition o f E D T A decreases the colour intensity o f the reaction. Hashiba et al (1977) also suggested that hydroxypyridone and hydroxypyranone residues present in melanoidin polymer combine wi th iron to produce colour. However , the possible role o f metal chelation by melanoidins present in coffee is not yet known. Rendleman (1987) reported that coffee brew and milk-free toasted bread had no measurable calcium binding activity. The weak calcium binding activity o f coffee brew and milk-free bread was explained as a structural difference existing between pigments formed in foods, to those formed by direct heating o f reducing sugars with amino acids (model M R P s ) . Since the number o f acidic donor groups in a food pigment is smaller than in a model system, melanoidins formed through the interaction o f proteins or peptides with reducing sugars in a food system possess less charge than those formed through the interaction o f amino acids with reducing sugars. A s a result, food melanoidins possess a 2.0 Literature Review 42 relatively lower charge density and may bind calcium to a lesser degree compared to melanoidins produced in model-systems. Rendleman (1987), during an investigation on the influence o f copper on browning in aqueous glucose-glycine systems, found that cupric ions were very strongly bound by an insoluble melanoidin produced in the glucose-glycine reaction. Wi th only a slight excess presence o f cupric ions, the extent o f binding at p H 5 very closely approached the maximum binding capacity o f the melanoidin. Furthermore, for each cupric ion bound, two hydrogen ions were released by the melanoidin. The C u : N ratio in the melanoidin was 1:4; and the nitrogen content o f the complex was 5.2%. Terasawa et al. (1991) studied the copper chelating activity o f a glycine-glucose model system using a copper chelating Sepharose 6 B column. Non-dialysable soluble melanoidins produced during this reaction were fractionated into seven components all having metal chelating activity and a molecular weight above 10 k D . T h e role o f metals i n react ion w i t h oxygen Transit ion metals play a significant role in reacting with oxygen since they have the access to a variety o f valence and spin states which al low them to undergo facile changes in oxidation states. Simply by accepting or donating a single electron, transition metals can produce superoxide ions (02*~) which can damage D N A and cells in vivo. F o r example, in the presence o f i ron, oxygen wi l l undergo reduction and produce superoxide ion ( 0 2 " ) , hydrogen peroxide ( H 2 0 2 ) , and hydroxyl radical (*OH) as described by the equations below: 2.0 Literature Review 43 2 0 2 + 2 F f o F e 2 + + 0 2 <^ F e 2 + + 0 2 " o F e 2 + + F £ 2 0 2 o F e 2 + + * 0 H o F e 3 + + OFT + ' O H F e 3 + + O H " H 2 0 2 + 0 2 F e 3 + + 0 2 -F e 3 + + H 2 0 2 (Eq 2.2) (Eq2.3) (Eq 2.4) (Eq2.5) (Eq2.6) The net result o f the equations ((Eq 2.2) and (Eq 2.4) wi l l therefore, be : F e 3 + + 0 2 " o F e 2 + + H 2 0 2 o F e 2 + 0 2 F e 3 + + OFT + * 0 H (Eq2.3) (Eq2.5) 02" + H 2 0 2 ^ 0 2 + O H " + * 0 H (Eq2.7) In this set o f reactions, reaction (Eq 2.5) is known as the Fenton reaction and reaction (Eq 2.7) is called as Haber-Weiss reaction or superoxide driven Fenton reaction. The presence o f a reducing agent, such as ascorbate, recycles the reactants on the right side o f the equation back to the left side thus, continuously driving the Fenton reaction. Since M R P s are known to possess metal chelating as wel l as reducing properties, it wou ld be o f interest to explore the possible role o f M R P s in participating in the metal driven Fenton reactions both in vitro as wel l as in vivo. St ruc tu re - An t i ox i da t i ve Ac t i v i t y Re la t ionsh ip Pure reductones prepared from hexose and secondary amines have been demonstrated to possess antioxidative properties (Evans et al., 1958). These observations have led to the conclusion that the antioxidative activity o f M R P s is associated with the presence o f reductones in melanoidins (Lingert and Er icksson, 1981). Further work by Eichner (1981) also indicated that 1,2-enaminols, formed f rom Amador i rearrangement products which possess reductone-like structures, may be responsible for the antioxidative activity o f M R P s . A m o n g the different types o f reductones, amino reductones were reported to possess more antioxidative activity than do enamino-like reductones (Evans et al., 1958; Itoh et al., 1975). However , the presence o f reductones, under certain conditions has also been shown to enhance lipid oxidation (Yamaguchi, 1969). This contradiction was shown to be especially 2.0 Literature Review 44 dependent on the moisture content o f the system. F o r example, ascorbic acid, a typical reductone and a hydrogen donor, could act as an antioxidant in a non-aqueous or l ow moisture system while it could also behave as an oxidant in an oil-water system (Namik i , 1988). Contrary to these findings, Yamaguch i (1986) observed that although the ozonolysis o f melanoidin caused a decrease in reducing activity, the process did not, however, affect the antioxidative activity. Fractionation o f the ozonolysed product produced a l o w molecular weight colourless product wi th very high antioxidant activity which had synergistic antioxidant activity with tocopherol. A s discussed in the previous section, melanoidins are known to possess high metal binding activity. This affinity for metal ions has been suggested as another reason for the observed antioxidant activity o f melanoidins since copper, iron, and other heavy metals play an important role in enhancing lipid oxidation. A s such, chelation o f such metals wi l l retard the metal catalyzed l ipid oxidation, except under certain circumstances. F o r example, oxidation promoted in a copper ascorbate system is due, in part, to the production o f some active oxygen species (Martel l , 1982). However , Yamaguchi and Fuj imaki (1974b) observed that the antioxidative activity o f tocopherol was decreased in the presence o f copper ion while that o f melanoidin was not significantly affected. Since the structures o f melanoidin compounds have never been completely determined, and the donor groups that participate in the antioxidative mechanism have never been identified, the relationship between structure and activity is as yet unknown. 2.7.3 Antimutagenicity Friedman (1996) classified antimutagens into two groups; i) desmutagens and ii) bioantimutagens. Desmutagens inactivate mutagens by chemical or enzymatic modif ication whi le bioantimutagens suppress the mutagenesis after the D N A has already been modif ied by a mutagen. Chan et al. (1982) reported that the mutagenic activity o f N-methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) and anatoxin 2.0 Literature Review 45 B l was suppressed by fructose-lysine browning reaction products. Y e n and Chau (1993) and Y e n and Hs ieh (1994; 1995) further explored the effects o f M R P s on the mutagenicity o f 2-amino-3,8-dimethyl imidazo [4,5-f] quinoxaline ( M e l Q x ) and 2-amino-3-methylimidazo-4,5-f quinoline (IQ), respectively. M R P s were found to suppress the mutagenicity o f M e l Q x and IQ effectively. That research wo rk also revealed that the inhibitory effect toward the above compounds occurred by formation o f inactive adducts through M R P s reacting with IQ, rather than direct inhibition o f hepatic microsomal activation. Therefore, the M R P s o f this example fall into the category o f desmutagens ( Y e n and Hsie ih, 1995). M R P s may also exhibit antimutagenic effects by scavenging free radicals and also by inhibiting enzyme activity o f S9 microsomal enzyme mixtures. F o r example, Ki t ts et al. (1993b) reported that glucose-lysine M R P s exhibited a chemotherapeutic protective activity against a chemical carcinogen, benzo(a)pyrene [B(a)P] by modifying xenobiotic intestinal enzyme activity in the gastrointestinal tract. Y e n et al. (1992) studied the antimutagenic activity o f 12 M R P s formed by 4 amino acids (glycine, L-lysine monohydrochloride, tryptophan, and arginine monohydrochloride) and three sugars (glucose, xylose, and fructose). The results o f that study indicated that xylose-lysine M R P s had a strong antimutagenic effect against the mutagenic compounds IQ , 2-amino-6-methyldipyrido[l,2-a:3',2'-d]imidazole (G lu-P-1) , and 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in Salmonella typhimurium T A 9 8 strain. It has also been shown that antimutagenic effect o f these M R P s correlated wel l wi th antioxidative activity and reducing power. 2.8 N u t r i t i o n a l Consequences of M R The M R that occur during food processing or home cooking can influence the nutritional value o f food products. Since the reaction by itself involves amino groups o f proteins, peptides, and amino acids, even in situations where browning is less evident, protein quality o f foods can be decreased. One o f the dominant consequences o f this reaction is the destruction o f lysine and other essential amino 2.0 Literature Review 46 acids which occur through a reaction with pre-melanoidins during advanced M R (Hurrel l and Carpenter, 1981). Moreover , premelanoidins can also react with and destroy certain vitamins (Ford et al., 1983) and also chelate certain trace elements (Rendleman, 1987), thus contributing to potential nutritional consequences. 2.8.1 Des t ruc t i on a n d Loss o f Essent ia l A m i n o A c i d s Lys ine destruction is the most significant consequence o f the M R in most foods. F o r example, Hurre l l (1990) demonstrated the loss o f lysine in whole milk powders during storage at 60° and 70°C. Over a period o f 9 weeks o f storage at 60°C, almost 4 0 % o f lysine was progressively transformed to lactulosyl-lysine without any noticeable brown colour formation. In addition, the storage o f product at 70°C caused 5 0 % o f the lysine to convert to lactulosyl-lysine after only two weeks, and the product turned brown three weeks onward. The metabolic fate o f fructosyl-lysine was studied by Finot and Magnenat (1981), who observed that the compound was wel l absorbed from the gut, but rapidly excreted in the urine (64%) and faeces (14%) while the remaining 2 2 % were assumed to be metabolised by intestinal flora. Sterilization (121°C) o f an experimental product containing 3 % protein, 2 . 5 % reducing sugar, and 2 .2% sucrose, decreased the Protein Eff ic iency Rat io ( P E R ) by 4 0 % whi le lysine loss was about 1 5 % (Hurrell, 1990). M R could also occur during processing o f weaning foods and in specialized infant formulae. Therefore, processing conditions used for those foods need to be controlled carefully. F o r example, lactose hydrolysed milks in formulae containing glucose, for lactose intolerant children, represent a potential problem in quality assurance (e.g., sensory character), nutritional value (e.g., available amino acids) and safety (e.g., allergenicity) o f the products. Thus, the food industry has to take special care when processing these formulae. In addition, the ability o f pre-melanoidins to react wi th certain other 2.0 Literature Review 47 amino acids such as arginine, histidine, tryptophan, and cysteine (Hurrel l , 1990), may cause severe amino acid losses in foods, rich in those amino acids, during thermal processing. 2.8.2 Protein Digestibility Whi le destruction o f essential amino acids is one o f the primary consequences o f M R , the development o f protein cross linkages due to reaction with reducing sugars may further decrease protein digestibility (Kato et al., 1986; 1987). Such indirect effects o f M R on protein util ization have been suggested by Adr ian (1974) who claimed that water soluble pre-melanoidins decreased protein digestibility and also affected util ization o f absorbed amino acids. Val le-Riesta and Barnes (1970) attributed the increased uptake o f a severely heated glucose-egg albumin mixture to a decreased pancreatic enzyme secretion. However , no direct evidence for the responsible mechanism was presented in that study. 2.9 Physiological Consequences of MR 2.9.1 Influence of MR on Vitamin Content V e r y few studies have been conducted to investigate vitamin loss due to M R . L o s s o f ascorbic acid due to M R was studied by Archer and Tannenbaum (1979). It is very unlikely that these reactions could attain nutritional significance since most processing and storage conditions destroy ascorbic acid wel l before the commencement o f M R . T w o other vitamins that could theoretically participate in the M R are thiamine, which possesses an amino group and pyridoxine (vitamin B 6 ) wh ich possess an aldehyde group. Fo rd et al. (1983) demonstrated that thiamine, pyridoxine, pantothenic acid, and vitamin B i 2 reacted with pre-melanoidins while nicotinic acid and biotin did not create M R . Vi tamin B 6 , wh ich is a critical nutrient for humans, is therefore potentially at risk due to M R . 2.0 Literature Review 48 2.9.2 Effect of MR on Digestive Enzymes M R P s are also known to inhibit digestive enzyme activity. Oste (1989) reported the inhibitory activity o f M R P s on trypsin activity at a concentration o f 1 mg /mL and carboxypeptidase A and B activity at a concentration o f 0.5 mg/mL. Oste (1989) further showed that a glucose-lysine reaction compound, 2-formyl-5-(hydroxymethyl) pyrrole-1-norleucine, was a strong competit ive inhibitor o f aminopeptidase in vitro. Intestinal disaccharidase activity also appears to be affected by M R P s . Lee et al. (1977) reported that the specific activities o f lactase, sucrase, and maltase were decreased in rats fed a diet containing 7 1 % browned apricots for 2 months. In contrast to the above, G o m y o and M i u r a (1986) reported that oral administration o f glucose-glycine melanoidins increased the specific activity o f small intestine disaccharidases in rats. 2.10 Toxicological Aspects of MR 2.10.1 Mutagenicity Considerable research has been directed toward the mutagenic properties o f M R P s in yeast (Saccharomyces cerevisiae) (Rosin et al, 1982), bacterial assays (Bjeldanes and Chew, 1979; Shinohara et al, 1980; Powr ie et al, 1981; W e i et al, 1981; Cuzzon i et al, 1988; 1989; Ki t ts et al, 1993a) and mammalian cells in vitro (Stich et al, 1980; Powr ie et al, 1981; L y n c h et al., 1983; Vagnarel l i et al, 1991; Ki t ts et al, 1993a). M o d e l systems containing creatine and creatinine amino acids and sugars have been used to demonstrate the origin o f some genotoxic heterocyclic amines (Jagerstad et al, 1984; Gr ivas et al, 1985; 1986) with carcinogenic potential (Sugimura and Sato, 1983). In some model systems, a correlation between browning and mutagenic activity was also demonstrated (Cuzzon i et al, 1988). In contrast to the above findings, desmutagenic activity o f l ow molecular weight melanoidins against mutagenic heterocyclic amines, such as anatoxin B i , B(a)P, 2-aminofluorene, 4-aminobiphenyl, and 2-aminonaphthalene, using S. typhimurium T A 9 8 strain in the 2.0 Literature Review 49 presence of S9 mix has also been reported (Lee et al., 1994). MacGregor et al. (1989) observed significant mutagenic activity in fructose-lysine browning products in TA100, TA2637, TA98, and TA102 Salmonella strains. The same mixtures, however, did not show a significant effect in micronucleated erythrocyte cells when administered to rats by the oral route indicating that the genotoxic compounds present in the reaction mixtures either did not reach the bone marrow cells in sufficient quantities to induce micronuclei or they were inactivated during the passage through the gut. Kitts et al. (1993a) studied the mutagenic and clastogenic activity of high, intermediate, and low molecular weight MRPs derived from glucose-lysine MR. All three MRP fractions exhibited mutagenic activity in both TA-98 and TA-100 strains without S9 pre-treatment, but such mutagenicity was totally eliminated in the TA-98 strain with added S-9 mix. A similar result was also noticed with hamster ovarian cells. Both intermediate molecular weight and high molecular weight melanoidins exhibited clastogenic activity without S-9 but the activity decreased in all three fractions with added S-9 mix. Based on the above, one basic question that arose, is: what relevance does the data from the reverse mutation Ames assay have in assessing carcinogenicity ? Compounds shown to be mutagenic in the Ames test should be extensively studied in animal and human systems prior to drawing any definitive conclusions about their relative toxicities. 2.10.2 In vivo Gly cation of Proteins Similar to the derivation of MRPs in food systems, M R also takes place inside the human body. Under physiological conditions, body proteins and enzymes can be modified through formation of ARPs and other MRPs by reacting with glucose or with other reducing sugars. This process is known as protein glycation and its derivatized products are referred to as advanced glycated end products (AGEs) which subsequently lead to impairment of physiological function of proteins and early ageing. As a result, patients having diabetes with elevated blood glucose levels are prone to have more AGEs 2.0 Literature Review 50 in tissues or body fluids when compared to healthy persons. Due to the limited information available, the impact o f the formation o f M R P s on the changes observed during ageing and diabetes is yet to be quantified, thus warranting further investigation (Sengel et al, 1989). 2.10.3 Toxic Compounds Formed through the MR Toxic i ty o f M R P s has been the subject o f extensive research work for many years. The toxicity o f several M R P compounds was tabulated by Mauron (1981). A review o f the reported toxicities o f M R P s indicate that the L D 5 0 values o f heated glucose-amino acid and heated glucose-nucleic acid mixtures were 5 0 % lower than the toxicity o f free amino acids and nucleic acids used to prepare them. Some o f the potential difficulties that have impeded the examination o f nutritional and toxicological aspects o f M R P s , as summarized by O 'B r ien and Morr issey (1989) include : 1) the difficulty o f isolating and purifying many individual M R P compounds, and 2) the difficulty o f purifying a sufficient quantity o f pure M R P s to conduct meaningful studies. Ideally, a proper examination o f M R P s wou ld require identification and isolation o f all products from the crude M R P mixtures and detailed study o f such isolated pure fractions. 2.11 Thesis Development 2.11.1 Hypothesis The extent o f browning in Mai l lard reactions is known to greatly depend on the reaction conditions and the type o f reactants. Different Mai l lard reaction products ( M R P s ) derived under such conditions could potentially be considered to exhibit a variety o f physico-chemical as wel l as genotoxic and cytotoxic properties. Moreover , the affinity o f M R P s for metal ions together with their reducing activity may also al low these compounds to modulate the Harber-Weiss cycle in the presence o f polyvalent metal ions. 2.0 Literature Review 51 2.11.2 Aims • to study the influence o f reaction conditions on the metal chelating, antioxidant, and D N A nicking effects o f model M R P mixtures synthesized using two reactant sugars (£)-glucose and D-fructose) wi th Z-lysine. • to study the behaviour o f model and food derived M R P mixtures in modulating l ipid oxidation, D N A breakage, and cytotoxicity in the presence and absence o f metal ions. 2.11.3 Objectives • to synthesize glucose-lysine and fructose-lysine M R P mixtures using variable reaction conditions and to determine their elementary composit ion, metal chelating activity, antioxidant activity, and D N A nicking activity. • to study the ability o f M R P mixtures having the highest metal chelating and antioxidant activities in retarding l ipid oxidation reactions in a linoleic acid emulsion and a prototype food system with or without the presence o f copper ions. • to study the antioxidative effectiveness o f the same M R P mixtures in a D N A model system as wel l as in a cell culture system, again, wi th or without the presence o f metal ions. • to compare the outcome o f the above experiments with those obtained wi th a food derived M R P components. 52 3.0 Study I Metal Chelating and Anti / Pro-oxidant Activity of Glucose-Lysine and Fructose-Lysine Model Maillard Reaction Product Mixtures 3.1 INTRODUCTION Non-enzymat ic interactions between reducing sugars wi th amino acids, peptides, or proteins in foods typify carbonylamine browning reactions, collectively referred to as the Mai l la rd browning reaction ( M R ) . Mai l lard React ion Product mixtures ( M R P mixtures) result from a complex network o f chemical reactions which include: (a) Amador i rearrangement, (b) Strecker degradation, (c) deoxyosone degradation, (d) cyclization, and (e) polymerization reactions. These reactions ultimately lead to the production o f high molecular weight polymeric brown coloured compounds known as melanoidins (Whistler and Daniel , 1984). Format ion o f melanoidins and their intermediate compounds is largely influenced by both the source o f reactants and reactant conditions (Cammerer and K r o h , 1995), and, even fixed reactants and reaction conditions are also known to produce a variety o f M R P mixtures (Namik i , 1988). The chemical reactions involved in the derivation o f different M R P mixtures have been the subject o f considerable investigation (Feather, 1981; Led l , 1990). However , the most advanced separation and analytical techniques currently available do not completely enable the characterization o f all melanoidin structures or associated intermediate products. Rather, a more general formula (e.g. sugar + amino acid - 2_3 H 2 0 ) has been used to describe melanoidins (Kato and Tsuchida, 1981) and an empirical formula (e.g. CgHnNOe) has also been proposed (Mota i and Inoue, 1974). Furthermore, a fundamental structure for melanoidin has been defined by K a t o and Tsuchida 3.0 Study 1 (1981) and by Cammerer and K r o h (1995). Since M R affect the colour, flavour, functional properties, and nutritional value o f foods, as wel l as a potential for a multitude o f bioactive properties (Rendleman, 1987; Terasawa, et al, 1991; K i t ts et al, 1993a; b) this reaction has been the subject o f considerable research for many years. In many respects, antioxidant, metal chelating, and reducing activities o f M R P mixtures have been understood to be the underlying cause or effect responsible for some biological properties. Direct incorporat ion o f M R P mixtures into food systems (Lingert and Er iksson, 1981; Bedinghaus and Ockerman, 1995), or indirect yielding o f M R P mixtures as a result o f food processing practices (Ch iou, 1992; Smith and Al fawaz, 1995), have indicated improved oxidative stability o f some food systems (Bressa et al, 1996). In addition, metal binding studies o f M R P mixtures have suggested that melanoidins have a very high affinity to chelate metal ions. Rendleman (1987) and Rendleman and Inglett (1990) proposed that two FT ions are released for each C u 2 + or C a 2 + bound to derived melanoidin, and that the amount o f metal bound to melanoidin, on the basis o f nitrogen content o f melanoidin, was C a / 4 N and C u / 4 N . This value signifies the number o f C a 2 + or C u 2 + ions bound per four nitrogen atoms in melanoidin molecule. However , a relatively small number o f studies have investigated the influence o f reaction condit ions on the significance o f metal chelating affinity and antioxidant activity o f model M R P mixtures. Moreover , no studies, so far, have been conducted to investigate the significance o f metal chelating affinity o f model M R P mixtures on associated antioxidant activity potential. M o s t o f the research conducted on the bioactivity o f M R P mixtures appears to have focused primarily on either the metal chelating affinity or the antioxidant activity in isolation, and not on the two aspects in combination. 3.0 Study J 34 The objective o f the present study therefore was to investigate and compare the elementary composit ion, metal chelating affinity, and antioxidant activity o f model M R P mixtures synthesized at varying reaction times, p H , water activities, and temperature combinations, using two model M R systems. One set o f model M R P mixtures was prepared by heating the basic amino acid Z,-lysine wi th the aldo sugar £)-glucose, and the other was prepared by heating Z-lysine wi th the ketose sugar D-fructose. The reaction conditions selected were chosen to simulate typical thermal processes involved in many cook ing situations. The derived M R P mixtures synthesized as above were designated as: (a) glucose-lysine Mai l la rd reaction product mixture (Glu-Lys MRP mixture,); and (b) fructose-lysine Mai l la rd reaction product mixture (Fru-Lys MRP mix ture/ The metal chelating activity o f model M R P mixtures was examined using the methods o f atomic absorption spectroscopy and the tetra-methyl murexide method, whereas antioxidant activity was assessed using the thiobarbituric acid method and an oxygen electrode employing a linoleic acid emulsion system. In addition, the genotoxicity o f the derived M R P mixtures was analysed using a D N A assay method. 3.0 Study! a 3.2 HYPOTHESIS Definit ive anti-/pro oxidant properties as wel l as specific metal chelating activities o f model M R P mixtures could be dependent on the reaction conditions used for synthesis as wel l as the source o f reactants used. 3.3 OBJECTIVES • T o derive two model Mai l la rd reaction mixtures by heating D-glucose or D-fructose with L-lysine under mi ld reaction conditions using a randomly selected experimental design that varies the experimental conditions o f the reaction simultaneously. • T o assess the elementary composit ion, metal chelating activity and anti-/pro oxidant activities o f these derived model M R P mixtures using different assay methods. • T o separate crude M R P mixtures into components based on their specific metal chelating activities and to determine their primary component composit ion. 3.0 Study! DO 3.4 MATERIALS 3.4.1 Production of Model Maillard Reaction Product Mixtures L-lysine, D-glucose, D-fructose were purchased f rom Sigma Chemical Company (St. Lou is , M O ) . Glycero l and sodium hydroxide ( N a O H ) were purchased f rom Fisher Scientif ic Company (Fair L a w n , N J ) and B D H Chemical Company (Toronto, O N ) , respectively. A l l solvents used were o f analytical grade and all the glassware used were soaked in I N HC1 (24 hours) and rinsed 3-4 times wi th deionized distilled water before use. Dialysis tubing (Molecular weight cut of f ( M W C O ) = 6 k D and 3.5 k D , diameter = 5 cm) was obtained from Spectrum Scientific Company (Houston, T X ) . Dist i l led water used to prepare model M R P mixtures was further purified by a Barnstead E-pure system, and used throughout the study. Water activity (a w ) and the p H o f the medium was measured using a R o -tronic Hygroskop D T relative humidity detector (Rotronic Instrument Corp . , Hunt ington, N Y ) and Fisher Accumet model 620 p H meter, respectively. Reactant solutions were heated in a Perma V i e w hot air oven and the reaction temperature was recorded by inserting a Type -T copper constantan thermocouple into the reaction solution. Decipher (Data Electronics, 1987) program was used for the thermocouple data acquisition. 3.4.2 Measurement of Copper Binding Activity of Model MRP Mixtures and Fractionation of Model MRP Mixtures by Chelation Chromatography Copper sulphate ( C u S 0 4 . 5 H 2 0 ) , tetra methyl murexide ( T M M ) , ethylene di-amine tetra acetic acid ( E D T A ) , standard copper solutions for atomic absorption spectroscopy, and hexamine were purchased from Fisher Scientific Company (Fair L a w n , N J ) . Hydrochlor ic acid, di-basic potassium phosphate, sodium acetate, sodium chloride, and potassium chloride were obtained from B D H Chemical Company (Toronto, O N ) . Chelating Sepharose fast flow resin was purchased f rom 3.0 Study 1 3/ Pharmacia Fine Chemicals (Uppsala, Sweden). B i o - R a d E c o n o pump controller and B i o - R a d E c o n o U V detector (B io- rad laboratories, R ichmond, C A ) were used in the chelation chromatography to adjust the p H gradient o f the mobile phase and to measure pre-melanoidins in the eluent. 3.4.3 Assessment of Antioxidant Activity of Model MRP Mixtures in a Lipid Model System Using an Oxygen Electrode and Thiobarbituric Acid (TBA) Method Lino le ic acid, butylated hydroxytoluene ( B H T ) , 1 , 1 , 3 , 3 - tetraethoxy propane, 2-thiobarbituric acid ( 2 - T B A ) were purchased from Sigma Chemical Company (St. Lou is , M O ) . C i g - S e p Pak cartridges and 1 0 cc syringes were obtained from Waters Associates (Mi l fo rd , M A ) and Bec ton-Dick inson (Rutherford, N J ) , respectively. Sulfuric acid and copper sulphate were purchased from Fisher Scientific Company (Fair L a w n , NJ ) . D i fco Laboratories (Detroit, M I ) was the source o f Tween 8 0 . Incubation o f M R P mixtures with linoleic acid emulsion was performed in a Precision Scientific, shallow form shaking bath and the oxygen consumption measurements were performed using a Y S I M o d e l 5 3 0 0 B io logical oxygen monitor (Ye l low Springs, O H ) . 3.4.4 Assessment of Toxicity of Model MRP Mixtures in a DNA System Production of PM2 Phage DNA Magnes ium sulphate ( M g S 0 4 . 7 H 2 0 ) , cesium chloride (CsCl ) , sodium chloride (NaCl ) , potassium chloride (KC1), calcium chloride (CaC l 2 ) , sodium dodecyl sulphate ( S D S ) , and Tr izma base were purchased from Sigma Chemical Company (St. Lou is , M O ) . Bac to nutrient broth and bacto agar were purchased from D i f co Laboratories (Detroit, M I ) . Guage 1 4 needles were obtained from Becton-Dick inson (Rutherford, NJ ) . Phenol and hydrochloric acid were purchased from Fisher Scientif ic Company (Fair L a w n , N J ) and B D H Chemical Company (Toronto, O N ) , respectively. Polyal lomer tubes were purchased from DuPont C o . (Wilmington, D E ) . Pseudomonas Bal 31 bacteria 3.0 Study! :>» and P M 2 phage virus were a gift from Dr . S.S. Tsang (Cancer Research Institute, B C ) . B lue M shaking water bath (Blue M , B lue Island, IL) , Beckman (Palo-Al to, C A ) Type 2 l a n d Type 50 rotors, and Son/al l R C - 5 B (DuPont, U S A ) Type SS-34 and G S A rotors were used throughout during the growth and harvesting stages o f D N A . F o r the preparation o f loading dye, bromophenol blue and xylene cyanol F F were purchased from Fisher Scientific Company (Fair L a w n , N J ) . F ico l l (Type 400) was bought from Pharmacia Fine Chemicals (Uppsala, Sweden). Gel Electrophoresis of DNA Glycine, E D T A , and ethidium bromide were obtained f rom the Sigma Chemical Company (St .Louis , M O ) . Electrophoresis grade agarose was purchased from B i o - R a d Laborator ies (Richmond, C A ) . H C 1 , potassium phosphate, and acetic acid were obtained from B D H Chemical Company (Toronto, O N ) . Incubation studies o f D N A were conducted in a B l u e - M (Blue Island, I L ) water bath and the gels were scanned using a B i o - R a d model G S 670 imaging densitometer. A Polaro id M E M camera and Type 55 (positive) and Type 57 (positive/negative) professional films were used to take the photographs. 3.5 M E T H O D S 3.5.1 P r o d u c t i o n o f M o d e l M R P M i x t u r e s M R P mixtures were prepared by heating a solution o f 0 . 8 M Z)-glucose or 0 . 8 M D-fructose wi th 0 . 8 M L-lysine (Kitts et al., 1993a; b) in a hot air oven. Since it is difficult to isolate the effect o f a single factor influencing the production o f M R P mixtures, due to the complex interactions ongoing among different reactants under a variety o f reaction conditions (Lingert, 1990), this study was performed using a random experimental design generated using Random-centro id optimization program ( D o u et al, 1993), which varied the conditions o f the thermal processes simultaneously. Fou r 3.0 Study J variable experimental factors including, time, temperature, water activity (a^,), and p H were used in the R a n d o m Centroid design with lower and upper limits ranging f rom 30-120 minutes, 80-160 0 C , 0.4-0.95, and 6-8, respectively. The initial reaction conditions (e.g. p H and a*,) as wel l as the reaction times and oven temperatures used to produce M R P mixtures, together wi th final a« and p H values o f the experiments are given in Table 3.1. The initial a« o f reactions were adjusted according to Eichner and Kare l (1972) and the initial p H values were adjusted with 1 M N a O H . A l l experiments were carried out in 500 m L round bottom flasks with aluminum foil caps, containing 100 m L o f reactants. The internal heating pattern o f the reaction solution and oven temperatures were recorded wi th sheathed Type-T copper/constantan thermocouples. A t the end o f the reaction time, the resulting brown solution was rapidly cooled on ice, dialysed against several changes o f double distilled deionized water at 4 °C for seven days and lyophil ised. The derived Glu-Lys MRP mixtures and Fru-Lys MRP mixtures were weighed and stored in a desiccator at 0 °C until further use. 3.5.2 C o r r e c t i o n fo r T h e r m a l L a g s Since temperatures given for various experiment numbers in Table 3.1 represent oven temperatures, but not reaction solution temperatures, the temperature histories were corrected for heating lag, and an equivalent reaction time (U) was calculated according to the method described by Hayakawa et al. (1977) and Ramaswamy et al. (1989) for a reaction temperature o f 120 °C (393 K ) . The equivalent process heating times (U) calculated for each individual experiment using the reference temperature o f 393 K is given in Table 3.2. In addition to the heat penetration data acquired using thermocouples, two other parameters namely, activation energy (E a ) , and z value [i.e. temperature change required to change the decimal reduction time (D) by a factor o f 10] were 3.0 Study J 60 3 c .2 '-a c o C J -a c IE C o c o u •o 3 S3 . a X d. I f 7 CU cn CD •B ~ CS OO 1—1 f- rt o rt oo vq CS r-^  ro" cs rt" CS rt rt" cs ro cs rt" ro' ro +1 -H -H -H -H +1 -H -H -H +) +1 -H in «n o m m oo o in m m vo o VO O ON o o o O ~ in o o o vd O o cs" cs o rt cs o m ro rt o 00 ro rt vd CS .—« ro cs rt" rt" c --<" iS cs +1 -H +1 +1 +1 +1 -H +1 -H +1 +1 +1 +1 f—• o in ro o 00 O O ro O o o ro o ro CS o m cs cs o t--' o CS o o o o m oo r-00 VO m VO m r-VO ON ON t» r-oo 00 VO ON r— ro t— wo 00 o" o" o O o" o o o o o* o o O o o cs VO cs m >n r- •rr »n o ro ro m vq cs cs in ro m 00 VO m ro ro ro ro' ro ro ro ro ro ro ro ro ro VO m o 00 r-m VO ro m VO VO ro ON cs ON vo r-t-00 ON in O 00 00 o o o O o O O o o" o o O o o o cs m m cs VO ro m cs CO «n o vq cs in ro in vo oo oo m ro ro ro ro ro ro ro ro ro ro ro ro 0.58 oo 00 vo r-m 00 vo ON m ON O 00 00 00 CS VO 00 oo 00 00 0.58 © o o o O O o d o o O o" O 6.37 00 f-ON m 00 ON VO 00 ON 00 r-m VO VO 6.37 vd vd vd oo' r-' vd vd r-" vd oo" r-" 00 o VO r~ cs CS t-in VO 00 ON ro •3-ON ON cs ON m ON cs o 00 VO o ON ON VO 00 r— o «n 00 m 00 in VO ro ro o ro 00 o • — ' f \ i r o T f m v o r ~ o o o \ O rt cs ro rr 3.0 Study I 61 S3 4) V O f II o S I C D r— -5 c •5 u co o o O N oo ° . TT co' °. vo —' CS CN vc O N T}- "3" o o co ~ —: — o o O S O N O <— 00 o co V O - *n C N r ^ . — 1 r —i O N ^ i n O N CN CN «—1 •—1 N . « T f O — CS CN —' V O O N O O • — i >—i V O M N ^ 00 m ^ i -i — • •—< O O CN CN —< 00 T j " O N '—' °. co' ^ co' - - - ( N MD t~~ r — V O 00 V O O O oo TJ- oo o o ^ O r - < v r i O O C N r - T J - O N vo «n >—i O vd O «/-» oo O O N C O «/"> »—• v> o O N O O N vo oo O N «n co —1 O CN U~t O O t — i r f v o o — | v o o o c N o r - - o n n "t v) *o o co o ^ CN m D O O N r - rH rH ^ r * H co O N O N CN O N O N II 1 H '3 O N 3 i — i e o <fcj 4> C •a <-> J O O N U , C U 3 13 > "8 r r oo CN II N a tn C D 3 13 > «> 2? J3 — 73 U 3 13 U H -3 3.0 Study J 62 required to calculate the U values. The E a values necessary for the calculation were chosen from the data presented by Labuza and Baisier (1992) (Table 2.2). T w o substantially different E a values (147 and 96.6 U/mo le ) were selected for the computation o f U , in order to determine the effect o f E a on U at a given reference reaction temperature. The E a value o f 96.6 U /mo le represented the activation energy o f a glucose-glycine model M R ( a ^ L O ; reaction temperature = 60-100 0 C ) whi le E a value o f 147 kJ/mole represented the activation energy for protein quality loss in a soy-glucose model M R (aw=0.3-0.8; reaction temperature = 80-130 ° C ) . Bo th these Mai l lard reactions were conducted with aw and reaction temperatures similar to the present study. The appropriate z value necessary for the calculation o f U for the corresponding E a , was assumed to be a certain numerical value (i.e. 10, 20, 30, etc.) and the temperature T 2 (temperature z degrees less than a reference temperature T ^ ) was calculated for a reference temperature o f 393 K , employing the fol lowing equation (Lund, 1975). z = ( 2 . 3 0 3 . R . T ! . T 2 / E a ) (Eq. 3.1) where: R = universal gas constant (8.314 J/mole K ) , T i = selected reference temperature (393 K ) and T 2 = a temperature z degrees less than T i . The assumed z value that was closest to the difference between T i and T2(i.e. z « T i - T 2 ) was taken as the proper z value for the computation o f U using the fo l lowing equation: t = tb TJ= A E l O ^ At (Eq. 3.2) where, T i = reference temperature (393 K ) , T = reaction temperature and tb = unmodif ied heating time (minutes). U was thus the equivalent time o f the total reaction at 393 K for a reaction wi th E a o f 147 and 96.6 kJ/mole. 3.0 Study] 63 3.5.3 Spectral Characteristics of Model MRP Mixtures Absorpt ion spectra o f the crude M R P mixtures (0.01 mg/mL) and absorbance readings o f the final M R solutions at 420 nm were measured using a Shimadzu U V - 1 6 0 U V - V i s spectrophotometer. Hunter chromaticity parameters o f L which measures lightness to darkness, a wh ich measures the red to ye l low axis, and b which measures the blue to green axis o f the reaction solutions were obtained using the Hunter L a b tri-stimulus colorimeter (Hunter Associates Laboratory, Reston, V A ) . Fibre optic light source and one centimeter diameter aperture size was used. These measurements provided an objective measure o f the extent o f browning reaction. 3.5.4 Metal Chelating Affinity of Model MRP Mixtures The metal chelating affinity o f model M R P mixtures was determined by the combined use o f atomic absorption spectroscopy and copper chelation (Terasawa et al, 1991). A tom ic absorption spectroscopy measured total copper while the tetramethyl murexide ( T M M ) method measured the amount o f free copper present. The amount o f copper bound to M R P mixtures was determined from the difference between the two methods. 3.5.4.1 Binding Activity of Copper Ions to MRP Mixtures Solutions consisting o f C u S 0 4 (0.05- 0.4 m M ) , M R P mixtures (100 ug /mL) , and T M M (1 m M ) were prepared in 10 m mol hexamine HC1 ( p H 5) buffer containing 10 m M K C 1 . The M R P samples (1 m L ) were individually mixed with 1 m L o f C u S 0 4 (0.05 m M to 0.4 m M ) for 10 minutes at room temperature. The amount o f total copper present in each solution was measured by atomic absorption spectrophotometry. Free copper in the same mixture was measured by adding 0.1 m L o f T M M reagent to the solution mixture and measuring the absorbance at 460 and 530 nm. The amount o f free copper in the solution was attained from a standard curve derived from an absorbancy ratio 3.0 Study! 64 (A460/A530), in a solution o f 1 m L Q 1 S O 4 (0.05-0.4 m M ) , 1 m L o f hexamine HC1 buffer and 0.1 m L o f T M M . The amount o f copper bound to M R P mixtures at different copper concentrations were calculated as the difference between the amount o f total and free copper present in the solution. A l l absorbance readings were corrected by subtracting the absorbance readings taken for M R P solutions wi th equivalent concentrations. The binding activity o f melanoidin to copper ( C u 2 + ) ions was developed by plotting the bound C u 2 + versus the ratio o f bound C u 2 + to free C u 2 + . The number o f association sites (n) and the dissociation constant (Kd) ( p M ) o f melanoidin was calculated by extrapolating the plotted line to the x-intercept and from the slope o f the Scatchard curve, respectively. 3.5.4.2 Fractionation of Model MRP Mixtures by Copper Chelation Chromatography Fractionation o f crude M R P mixtures into copper chelated components was conducted according to the method given by Terasawa et al. (1991). Chelating Sepharose 6 B column (3.5 x 7cm) was filled approximately with 30 m L o f 0. I M C u S 0 4 solution and washed wi th several volumes o f deionized distilled water at a flow rate o f 3.2 mL/min. The eluate was collected at different intervals and analysed by atomic absorption spectrophotometry for detectable copper ions eluted f rom the column. A 250 m L volume o f water was sufficient to wash the column without leaving residual free copper ions. The resulting C u 2 + chelating column was subsequently equilibrated wi th a 250 m L vo lume o f 0 . 0 5 M p H 7.65 phosphate buffer (Buffer A ) containing 0 . 5 M N a C l . M R P mixtures (250 mg) were dissolved in 2 m L o f Buffer A and l m L o f the sample was applied onto the column and washed wi th a 250 m L volume o f the same buffer. The remaining aliquot (1 m L ) was applied again onto the column and washed wi th the same volume o f Buffer A as detailed above. Th is procedure was effective in removing compounds that did not bind onto the copper chelating Sepharose-6B column 3.0 Study J 65 material, as wel l as, concentrating the compounds that did bind to the column material at p H 7.65. E lu t ion o f the bound M R P mixtures from the column was performed at the same flow rate, wi th a p H gradient ranging f rom p H 7.65 (Buffer A ) to p H 5.5 (Buffer B ; 0 . 0 5 M acetate buffer wi th 0 . 5 M N a C l ) . The p H gradient was adjusted using a pump controller and the eluent was detected by a U V detector set at 280nm. B r o w n coloured compounds which did not elute during the gradient p H fall, could be removed by washing the column with 0 . 0 5 M E D T A containing 0 . 5 M N a C l . Eluents from the column containing peaks detected at 280 nm were collected and dialysed ( M W C O = 3.5 k D ) against several volumes o f deionized distilled water at 4 °C, and then lyophilised. Before application o f the next M R P sample, the column was flushed with several volumes o f deionized distilled water and resaturated with C u S 0 4 , as described above. Mo lecu la r weights o f the fractionated components were later determined by Ma t r i x Assisted Laser Desorpt ion Ionization mass spectrophotometer ( M A L D I - M S ) . 3.5.5 E l e m e n t a r y C o m p o s i t i o n and M o l e c u l a r We igh ts of M o d e l M R P M i x t u r e s and M R P M i x t u r e C o m p o n e n t s F rac t i ona ted by Che la t i on C h r o m a t o g r a p h y Elementary composit ion o f the crude M R P mixtures and fractionated M R P mixtures was obtained by pyrolysing the M R P compounds at 1100 °C in helium using a E A M o d e l 1108 (Italy) elemental analyser (Dept. o f Chemistry, U B C ) . Gasses that escaped from the compound during pyrolysis were detected using a gas chromatograph. Mo lecu la r weights were determined by a Kratos Kompac t M A L D I - M S (Dept. o f Chemistry, U B C ) using sinnapinic acid (3,5-dimethoxy-4-hydroxy cinnamic acid) as the matrix. M a s s accuracy was ± 0 . 1 - 0 . 0 1 % . 3.0 Study J 66 3.5.6 Assessment of Antioxidant Activity of MRP Mixtures 3.5.6.1 Oxygen Consumption Measurements Oxygen depletion in a linoleic acid emulsion system with added C u 2 + ions and in the absence or presence o f M R P mixtures was measured according to the methods o f M c G o o k i n and August in (1991) and Lingert et al. (1979), using a biological oxygen monitor. The reaction mixture consisted o f 1.5 m L o f linoleic acid emulsion (1.5 g linoleic acid mixed wi th 0.4 g o f Tween 80), and 0.6 m L o f M R P solution (3 mg/mL) , in a 0.1 M phosphate buffer ( p H 6.8) containing 2 m M C u S 0 4 . The reaction mixture was pumped into a jacketed reaction vessel containing an oxygen electrode at room temperature. Oxygen depletion in the reaction solution was recorded immediately after the reaction mixture was introduced into the vessel. Fo r the measurement o f oxygen depletion in the absence o f antioxidant compounds, the experiment was performed in an identical manner wi th the exception that 0.6 m L o f buffer replaced the M R P solutions. B o t h antioxidant and prooxidant activity o f M R P mixtures were expressed in terms o f a protective index (PI), defined as: [Time for 50% 02 depletion with test compound] PI= (Eq3.3) [Time for 50% 02 depletion without test compound] where: P I = protective index; P I < 1 denotes prooxidant activity; P I = 1 denotes no activity; P I > 1 denotes antioxidant activity (Lingert et ai, 1979). 3.5.6.2 Measurement of Thiobarbituric Acid Reactive Substances (TBARS) M R P mixtures (2 mg) were dissolved in linoleic acid emulsion system (1.5g o f linoleic acid and 0.4 g Tween 80) in a 10 m L capped test tube, and incubated for 48 hours at 60°C in a water bath. Af ter incubation, the solution was diluted 10 times with 25 m mol Tr is buffer ( p H 7.4), containing 0 .02% sodium azide and passed through a C\g Sep-pak cartridge several times, to remove residual 3.0 Study 1 67 lipids. Clarified homogenate was assayed for the M D A content using the T B A method described by Buege and Aust (1978). One mL of TBA reagent, containing 0.02 % freshly preparer] BHA, was added to 2 mL of clarified homogenate in test tubes with marble caps, and immersed in a boiling water bath for 15 minutes. After cooling, absorbance readings of the reaction solutions were made at 532 nm, using a UV-Vis spectrophotometer. Quantification of M D A content in samples was made from a standard curve, prepared from 1,1,3,3-tetraethoxypropane in 1% H2SO4. All results were expressed as percent antioxidant activity, defined as: [(TBA value of the control - TBA value of the test sample) xJOOJ %AO= (Eq3.4) [TBA value of the control] where: % AO = percent antioxidant activity; control = emulsion without MRP. 3.5.6.3 Assessment of DNA Nicking Activity of model MRP Mixtures Effect of MRP mixtures on D N A nicking was determined in order to investigate the potential genotoxic behaviour of model MRP mixtures. 1) Production and yield of PM2 phage DNA I. Production of PM2 bacteriophage D N A Production of PM2 phage D N A was performed as described by Espejo and Canelo (1968). Media and solutions • Bal Broth: Bal broth was prepared by mixing 12 g MgS04.7H 20, 26 g NaCl, 8 g Bacto nutrient broth in IL of deionized distilled water and autoclaved. When the medium was cooled, filter sterilized 10 mL of I M CaCl 2, 3.5 mL 20% KC1, and 10 mL I M Tris HC1 (pH 7.5) were added per IL of Bal broth. 3.0 StudyJ 68 • Bal top agar: B a l top agar was prepared by supplementing I L o f Bal-broth with 5 g o f Bacto-agar. • Preparation of Bal plates: A n autoclaved mixture o f B a l broth ( I L o f Bal-broth supplemented wi th 23 g bacto-agar) was poured into petri dishes until the plate surface was covered. II. Preparation o f P M 2 phage stocks Pseudomonas Bal 31 bacteria were grown in a 250 m L Erlenmeyer flask containing 25 m L o f Bal -broth at 28°C, under vigorous shaking (40 rpm). A t a bacterial density o f 2 x 10 7 /mL, the bacteria were infected with P M 2 virus and incubated overnight at room temperature. Bacter ial density was read from a standard curve prepared by plotting the optical density (O .D) o f the medium at 600 nm against the total bacterial count. Bacterial debris and unlyzed bacteria were removed by centrifuging the medium in a Sorval l R C 5 B G S A rotor for 10 minutes at 16,300 x g (where g is the gravitational force). Supernatant was used as the phage stock. The phage yield was calculated by performing a plaque assay. U l . Plaque assay to detect P M 2 phage yield Pseudomonas Bal 31 bacteria were grown overnight at room temperature in a shaker bath. A n aliquot o f 0.2 ml bacterial culture was mixed with O . lmL o f P M 2 phage stock (diluted in Bal-broth) and 2.5 m L o f B a l top agar (heated to 50°C). The mixture was poured onto B a l plates and left overnight at room temperature. Plaques appear approximately after 8 hours. Phage yield was found to be 1 0 1 1 phage /mL . 3.0 Study] 69 IV . Harvest ing bacteria and phage D N A Pseudomonas Bal 31 bacteria were grown in 1.5 L o f B a l broth in a 3 L Erlenmeyer flask incubated at 28 °C and vigorously shaken (40 rpm). When bacterial cells reached a density o f 2 x 10 7 /mL, cultures were infected with P M 2 phage at a multiplicity o f infection o f 10"3 (1 phage virus per 10 3 bacterial cells). The infected culture was incubated overnight and the bacterial and cell debris were removed by centrifuging at 16,300 x g for 15 minutes at 4 °C using a Sorval l R C 5 B G S A rotor. Remova l o f the phage particles from the supernatant was performed by centrifuging the supernatant at 60,000 x g for 3 hours using Beckman Type 21 rotor at 4 °C. The phage pellet was resuspended in about 2 - 3 m L o f R B buffer (1 M N a C l per I L 20 m M T r i s - H C l ; p H 8) and stored overnight in a cold room (5 °C). Phage suspended R B buffer in all the tubes were collected into one tube and suspended again in 12 m L o f R B buffer and centrifuged again for 15 min in a Sorval l R C 5 B Type SS-34 rotor at 12,100 x g. The C s C l was then added to the phage solution to a density o f about 1.28 g/cc (0.3588 x weight o f phage solution) and the phage solution was distributed into two polyal lomer tubes and centrifuged for 24 hours at 196,000 x g at 20 °C using a Beckman Type 50 rotor. Fo l low ing C s C l gradient centrifugation, one major band o f phage particles was evident in the middle o f the polyallomer tube (Fig. 3.1) and this fraction was collected using a gauge 14 syringe through the side o f the tube. The purified phage was dialysed against I L o f B E buffer (0.1 M N a C l / 1 m M E D T A / I L 20 m M T r i s - H C l ; p H 7.5) for 3 or more hours. A t room temperature, phage was lyzed by adding 10% S D S dropwise until the solution was cleared and the D N A was extracted from the aqueous phase with an equal volume o f phenol saturated B E buffer. The aqueous phase referred to here as "aqueous 1" was re-extracted wi th 1/2 volume o f B E saturated phenol, referred to here as "phenol 2" . The phenol phase was re-extracted with 1/2 volume o f B E buffer referred to here as "aqueous 2" . "Aqueous 2 " was 3.0 Study 1 70 Fig. 3.1: Appearance of PM2 phage band in cesium chloride following gradient centrifugation. 3.0 Study] 71 extracted wi th "phenol 2" and combined with "aqueous 1". Us ing this extraction scheme every phase was extracted twice. The extracted D N A pellet was dissolved in 10 m mol T r i s - H C l ( p H 7.5), 0.1 m M E D T A and stored at 4 °C. The concentration o f the phage D N A was determined by measuring the absorbance at 260 nm. The molar extinction coefficient o f D N A ( "^E ic , ) at 260 nm used was 6.5 x 10 3 . The extracted D N A was stored at -30 °C until further use. 2) Agarose gel electrophoresis of DNA A l l reactions were conducted in potassium phosphate buffer ( p H 7.4, 50 m mol) under ambient oxygen pressure. T o detect the possible genotoxic effect o f M R P mixtures on D N A , at different M R P concentration levels, 2 u L o f phage D N A was mixed with 2 u L o f buffer and 2 u L each o f M R P solutions (10 " 4 , 10"3, 10"2, and 10"1 %, w/v) in a 500 u L Eppendor f tube. The final volume o f the reaction mixture was brought to 10 u L with deionized distilled water and incubation was conducted for 1 hour at 37°C. A t the end o f the incubation period, 2u l o f loading dye (0 .25% bromophenol blue, 0 .25% xylene cyanol F F , and 1 5 % Fico l l in water) was added to the incubated mixture and 10 u L o f this mixture was loaded onto a agarose gel wel l . Electrophoresis was conducted at 60 volts in Tr is acetate ethylene diamine tetra acetic acid ( T A E ) buffer [0.04 M Tris acetate ( p H 7.4), 0.001 M E D T A ] . The agarose gel was stained with ethidium bromide (0.5 \xg/mL deionized distilled water) for 20 minutes. D N A bands were visualized under illumination by U V light. 3) Quantification of DNA breakage by densitometry The percentage o f super coiled, nicked circular, and linear D N A was quantified with an imaging densitometer using B i o R a d Molecular Analyst ™/PC image analysis program (version 1.0). The area under each fractionated D N A band was quantified by drawing a line across each respective 3.0 Study 1 72 lane and the area under each band was determined using the program. Background noise was eliminated in all the readings by subtracting the area under the test line f rom the area under the background line. Rout ine runs o f original D N A were conducted daily throughout the experimental period to acquire an accurate estimate o f variability required for making comparisons across experiments. 3.5.7 Statistical Analysis One-way analysis o f variance ( A N O V A ) , fol lowed by multiple range test (Tukey) or student t-test was used in the data analysis. Systat for Windows statistical data package was used and the level o f confidence required for significance was selected at p<0.05. E a c h experiment was replicated three times, wi th internal controls. 3.6 RESULTS 3.6.1 Yield of GIucose-Lysine (Glu-Lys) and Fructose-Lysine (Fru-Lys) MRP Mixtures The initial and final p H and aw values, reaction times, and oven temperatures used in the product ion o f M R P mixtures, along with their non-dialysable melanoidin yields, are given in Table 3.1. The final p H o f all experiments performed was acidic (e.g. range 3.12 - 3.84), regardless o f the initial p H value recorded. The a*, values remained relatively unchanged f rom initial values. It was also noted that the yields o f detectable M R P mixtures varied depending on the combination o f reaction conditions and the source o f reducing sugar. The minimum and maximum yields o f M R P mixtures ranged f rom 0.1 - 7.5 g for the Glu-Lys reactions and 0.05 - 6.65 g for the Fru-Lys reactions. Non-dialysable melanoidin could not be recovered from the Glu-Lys experiment number 7 and experiment numbers 7 and JO o f the Fru-Lys reaction. The two experiments in the Glu-Lys reaction which produced the greatest M R P yields were experiment numbers 3 and 13. In both experiments although similar initial 3.0 Study! 73 oven temperatures (127° and 129 °C) and water activities (0.74 and 0.78) were used, both the initial p H (6.14 and 8.41) and reaction times (119 and 71 min.) were substantially different. In the case o f Fru-Lys reactions, the highest M R P yields obtained in experiment numbers 5 and JI were characterized by having similar heating temperature (157° and 159 °C), and initial p H (8.51 and 8.57) values, but large differences in reaction times (107 and 43 minutes). Plots o f heating time versus reaction temperature for two Glu-Lys and Fru-Lys MRP synthesis experiment numbers 5 and 11 are given in the appendix. Accord ing to those graphs, reaction temperatures seemed to be largely dependent on the oven temperatures used, for each experiment. However , for a given experiment, both Glu-Lys and Fru-Lys had similar heating patterns. The effect o f reaction parameters, equivalent reaction time, a ^ and p H on the product ion o f non-dialysable M R P mixtures are presented in Figs. 3.2a and b, respectively. In the present study, since all four experimental conditions varied together simultaneously, it was not possible to draw a direct relationship between the melanoidin yield and any o f the reaction parameters studied above. However , in general, when all fourteen synthesis experiments were taken into consideration, the experiments that consisted o f both l ow reaction times and l ow oven temperatures (e.g., experiment numbers 6 and 7) produced a minute quantity o f non-dialysable M R P mixtures. In addition, experiments either having a short reaction time or l ow oven temperatures (Experiment numbers 9, 12, and 14) as wel l as the experiments that possessed a a™ value greater than 0.8 ( experiment numbers 2, 7-9, 10, 12, and 14) in general, produced low quantities o f non-dialysable M R P mixtures. U values for both E a parameters, however, showed little variability indicating the relatively minor importance o f E a i n reaction kinetics at this reference temperature (393 K ) . In addition, U values 3.0 Study] 74 0.5 0.55 0.6 0.65 0.7 0.75 0.8 Initial Water Activity (aw) 0.85 0.9 0.95 > 0s-2 3 • • ° W o i l o5 • -11 • . 1 . 5 • 10 1 3 o . 2 ° - 2 D 1 0 7 o4 S 8 4 . -12 1 o 14 • I4 « 6-. —, 0 n 1 13 0 , 6.5 7 7.5 Initial pH 8.5 Fig. 3.2a: Yield of crude non-dialysable MRP rnixtures versus the initial water activity (a„) (A), and initial pH value (B). Number beside symbols denote experiment number for set of conditions used in MRP synthesis. ° = Fru-Lys MRP mixtures, • = Glu-Lys MRP mixtures. 3.0 Study! 75 10 20 30 40 SO Reaction time (m inu tes ) F ig . 3.2b: Y i e l d o f crude non-dialysable M R P mixtures versus the calculated equivalent reaction times (U) . Equivalent reaction times were calculated for a reference temperature o f 393 K ; E a = 147 kJ/mole; Z = 19.1 °C. Number beside symbols denote experiment number for set o f conditions used in M R P synthesis, o = Fru-Lys MRP mixtures, 1 • = Glu-Lys A ^ R P mixtures. 3.0 Study! 76 calculated for Glu-Lys MRP mixtures were similar to U values calculated for Fru-Lys MRP mixtures for a given M R P synthesis experiment 3.6.2 Spectral Characteristics of MRP Mixtures 3.6.2.1 Absorption Spectra Spectral patterns for both Glu-Lys and Fru-Lys MRP mixtures at complet ion o f reaction, are given in F ig . 3.3. The spectral pattern shown in F ig . 3.3 A had one absorption shoulder around 320 nm whi le the spectral partem shown in F ig . 3.3 B had two absorption shoulders, i.e. one at 280 nm and the other at 320 nm. The Glu-Lys experiment numbers 4, and 6 had similar spectral patterns observed in F i g 3 .3B. A l l other Glu-Lys experiments and all Fru-Lys experiments had spectral patterns similar to F ig . 3.3 A, even though the magnitude o f absorption for each M R P at a given wavelength varied. 3.6.2.2 Absorption at 420 nm The absorbance o f M R P solutions at 420 nm were measured as an indicator o f soluble melanoidins produced in the different reaction conditions, and also as a measure o f the extent o f M R . The results are given in F i g 3.4 along with their final non-dialysable M R P yields. The absorption at 420 nm increased as M R P yield increased, thus indicating a possible increase in non-dialysable soluble melanoidins in the reaction solution. This observation was common for both Glu-Lys and Fru-Lys reactions. Experiments yielding less than 1% non-dialysable melanoidin had similar absorption readings for both Glu-Lys and Fru-Lys model experiments. A s the non-dialysable yield o f M R P increased above 1%, a relative difference between the 420 nm absorbance readings between the Glu-Lys and Fru-Lys model systems was observed. 3.0 Study! F i g . 3.3: • Spectral patterns o f crude Glu-Lys and Fru-Lys MRP mixtures (A) = Spectral pattern o f all Fru-Lys MRP mixtures and Glu-Lys MRP mixtures except Glu-Lys MRP mixtures 4 and 6 (B) = Spectral patteni o f Glu-Lys MRP mixtures 4 and 6. 3.0 Study I oo I D > 2 "a. C a CO <• o co - j co cn <U § 1 cn ^ ) CO CD <u 5 o o co c • "-a co , II o £ co P"> •9 3 c i uiu QZf JK aoucqjosqy •a 00 cu 3.0 Studyl 79 3.6.2.3 Hunter Lab L, a and b Values Hunter lab L, a, and b values were also recorded as an indicator o f the extent o f browning in different M R solutions. The results o f Glu-Lys and Fru-Lys L, a, and b values plotted against the yield o f M R P in each experiment is presented in F ig . 3.5 (A) and (B), respectively. L value, wh ich is a measure o f lightness (L=100) to darkness (L=0), decreased as the non-dialysable melanoidin yield increased. The a and b values, which respectively reflect the green (-a) to red (+a) and blue (-b) to ye l low (+b) colour co-ordinates o f the non-dialysable M R P , exhibited positive values for both a and b values. 3.6.3 Metal Chelating Affinity of Model MRP Mixtures 3.6.3.1 The Copper Binding Activity of MRP Mixtures The total amount o f copper bound to different M R P samples generated f rom each synthesis experiment is given in Table 3.3. The plot o f amount o f copper bound to M R P versus the yield o f non-dialysable melanoidin is presented in F i g 3.6. M a n y o f the M R P mixtures derived f rom different experimental conditions possessed detectable copper binding activity, albeit the relative amount o f copper bound to each M R P varied according to the specific conditions used in the experiment. The lowest and the highest amount o f copper bound to M R P mixtures ranged f rom 0.03 to 1.57 u M per m g for Glu-Lys MRP mixtures and 0.02 to 2.27 u M per mg for Fru-Lys MRP mixtures. The few experiments (e.g. Glu-Lys MRP mixtures 3, 13; Fru-Lys MRP mixtures 5, 11) that resulted in high M R P yields also exhibited relatively high metal chelating affinity. Dissociat ion constants (Ka) and the number o f copper binding sites (n) calculated for one mil l igram o f crude M R P mixtures by Scatchard plot analysis in each synthesis experiment are presented in Table 3.3. M a n y o f the synthesis experiments used for Glu-Lys MRP mixtures were 3.0 Study 1 80 8 on 0) J 3 6 4 -a c 03 2 ^ — 0 -2 Percent Yield (w/v) 8 cu -a 0 Percent Yield (w/v) Fig. 3.5: Hunter Lab 'L', 'a' and 'b' values against the yield of crude Fru-Lys (A) and Glu-Lys (B) MRP mixtures. Values represent mean ± S.D. • =UL" value, A = "a" value, • ="b" value. 3.0 Study! 8 1 eu o, O o g c 5 5 -2 .a S3 ro r—< O O vo cs cs o O N ro V O t-~ cs ro O o © t 1 i O O i i V O ro w-i wo V O ro cs in m ro r- in o wo oo wo O N ro cs V O ro o 1 — 1 o o • 1 o i i © © O O O T—< o *? o O o © X X X X X X X O N O N rt O N O 0 0 O O 0 0 WO 1 ' cs i 1 i oo" i cs i i o T O © o O O o T O o f—< X X X X X X X X X ro t- o O N CO rt1 r-in cs ro oo" i 1 ro" i i rr' 0 0 CS cs" CS in O cs o vo T T V O o wo V O 0 0 ro O o o o rt" O o" rt o o o +1 +1 +1 +1 +1 -H -H +1 -H +1 +1 ro o o V O r-cs V O V O o o o o wo O O N o o cs V O cs o 0 0 o O o o cs o o o o O o O o o O ro CS rt wo rr ro o cs V O o ro O CS o ro O N O O o o O rt o o rt cs O o +1 -H -H -H +1 -H +1 -H -H -H -H ro O o O N ro V O o o o V O O ro © t- ro vo t-» WO vo o O o O o o o o O o o o CS ro wo V O •a 0 0 « O N «f O rt cs ro 3.0 Study J 82 00 v© > 2 <NI cu (d)lW 2«i /n3 j\[n) jaddco punog 3.0 Study] 83 characterized wi th a Kd value that was approximately ten times greater than the corresponding Fru-Lys MRP mixtures. The number o f binding sites also varied with specific M R P synthesis conditions. Specif ic conditions used in experiment numbers 3, and 13 wi th the Glu-Lys reaction and in experiment numbers 5 and 11 wi th Fru-Lys reactions were found to have greater than one binding site per mil l igram M R P mixtures. Since crude M R P mixtures are comprised o f a mixture o f compounds, crude M R P mixtures synthesized from the above experiments were further fractionated into components using copper chelation chromatography to further understand the definitive affinities o f specific M R P components to chelate C u 2 + . 3.6.3.2 Fractionation of Model MRP Mixtures by Chelation Chromatography Copper chelation chromatography was used as a technique to fractionate non-dialysable model M R P mixtures from each synthesis experiment into several components based on copper chelating power. On ly the crude M R P mixtures that possessed a chelating value o f greater than 0.08 p M C u / m g M R P were used for fractionation by chelation chromatography. Component number 0, eluted from the copper chelation column during the wash phase with neutral buffer A ( p H 7.65), preceded component numbers 1- 5, which were eluted during a gradient p H fall from 7.65 to 5.5. The last component number (6), was recovered only by flushing the column wi th E D T A . The percent recoveries o f the various components from the synthesis experiment numbers 3, 13 for Glu-Lys M R and 5, 11 for Fru-Lys M R are given in F ig . 3.7. The results indicate that the percentage o f melanoidin recovered from the copper chelating column increased as the p H o f the mobile phase decreased. C o m m o n to all four experiments were the component numbers 0, 2, 4-6 ( E D T A fraction), wi th component numbers 5 and 6 having the first (80%) and the second (60%) largest M R P concentrations, respectively. This trend was observed with all four crude M R P mixtures tested. 3.0 Study 1 84 60 50 --F Component Number F ig . 3.7: Percentage o f M R P mixtures recovered in components fractionated by chelation chromatography. H = Fru-Lys MRP mixture 5, m = Glu-Lys MRP mixture 3. • = Fru-Lys MRP mixture 11, U = Glu-Lys MRP mixture 13. 3.0 Study 1 85 The low recovery of certain fractionated components from crude Glu-Lys and Fru-Lys MRP mixtures and the association of EDTA in component number 6, further precluded the measurement of bound copper in those components. As such, only the component numbers 1 and 5 were used for further characterization by MALDI-MS in this study. Therefore, the Kd values and the number of binding sites (n) in eluted component numbers 1 and 5 are specifically given in Table 3.4. A comparison of the Kd and n values of crude MRP mixtures with those of their fractionated components, indicated higher Ka and n values in the fractionated components of each MRP mixture. These results demonstrate that compounds with more copper binding activities present in the crude MRP mixtures were concentrated in the fractionated components. In addition, the chelating affinity of these different components increased, with elution from the column at lower pH values (i.e. MRP fractionated component number 5 has higher number of binding sites compared to component number 1 for both Glu-Lys and Fru-Lys MRP mixtures). The MALDI-MS spectrums of fractionated component numbers 1 and 5 obtained for both Glu-Lys and Fru-Lys MRP mixtures are given in Figs. 3.8 a-b. Both component numbers 1 and 5 in Glu-Lys and Fru-Lys MRP mixtures contained two common principal peaks: a major one with a molecular weight of approximately 5,700 daltons and a minor one with a molecular weight of 12,400 daltons. Accompanying these two peaks were many other smaller peaks (Fig. 3.8 c) indicating the presence of other trace compounds produced during different stages of the polymerization reaction, again possibly indicating differences in component composition. 3.6.4 Elemental Analysis of Crude MRP Mixtures and MRP Mixture Components Results of the elemental analysis together with the estimated empirical formulae for both crude MRP mixtures and fractionated MRP component mixtures of both Glu-Lys and Fru-Lys MRP 3.0 Study 1 86 CD c 0 1 cfc § CD -a -i o c co CD 4-» "53 GO c c CD a. a. o o o 1— CD co C o o c o '5 o co co CD H CO CD H CO CD i2 c CD c o cx 6 o o CD g CD CD a. x t -ro CO t -•<d- CN CN O VO TT O CN CN m CN m oo *~' o\" *—' vd OV ~ CO* vo CN oo' T O T o O T O o o -9 O V I o T O O o o X X X X X X X X X X X X TT CN r - CN VO ON •—• 00 co vq CN vd in CN CN CN vd O CN CO — O O o o o CD 2 CO CD r~ cN <n •O o o —' o o CD •a a u CD — CN vn VO O O vd o o CD 5 u CD CN — CN o o —; o o CD -a a o CD ac ac .5 .c IT! *-< 3.0 Study J 87 206.4 l— So C3 C CU jo 5719.3 1140.0 2860.6 (A) kL i 2,000 206.9 4,000 6,000 8,000 Mass/charge 10,000 12,000 CO - 4 - « 1-1 J? -C <u (B) 5721.7 U 1161.9 3241.4 12354.1 2,000 4,000 6,000 8,000 10,000 12,000 Mass/charge F ig . 3.8a: M A L D I - mass spectra o f component number 1 o f (A) Glu-Lys MRP mbrture 3 and (B) Fru-Lys MRP mixture 5 fractionated by chelation chromatography. 3.0 Study 1 88 206.9 (A) c G 3 -4—» IS 0 3 co »«-C c ca C O M • C O C CU 5725.3 1141.0 4-3792.9 12361.1 2,000 4,000 6,000 8,000 206.7 Mass/charge 10,000 12,000 5724.1 1162.9 2902.4 4113.7 2,000 4,000 6,000 8,000 Mass/charge 10,000 12,000 F ig . 3.8b: > M A L D I - mass spectra o f component number 5 o f ( A ) Glu-Lys MRP mixture 3 and (B) Fru-Lys MRP mrxture 5 fractionated by chelation chromatography. 3.0 Study J 89 12463.9 12300 12350 12400 124SO 12S00 Mass/charge 12J79.0 1 2 K 0 12300 123SO 12400 12<S0 12S00 Mass/charge F ig . 3.8c: M A L D I - mass spectra o f fractionated component number 1 around 12,000 Da l ton molecular weight range. ( A ) Glu-Lys MRP mixture 3 mid (B) Fru-Lys MPP nuxture 5 3.0 Study J 90 mixtures are presented in Tables 3.5 and Table 3.6, respectively. Fru-Lys MRP mixtures consistently exhibited a lower number o f carbon atoms compared to their Glu-Lys counterpart. Despite this difference, the C : O ratio was approximately 2:1 for all eight experiments. In the case o f M R P fractionated components, a C:0 ratio o f approximately 2 : >1, wi th a lesser number o f carbon atoms in the empirical formulae was observed. The C : N ratio also varied depending on the type o f reactant sugar and experimental condit ion used to generate different M R P mixtures. Fru-Lys MRP mixtures exhibited higher C : N ratios compared to Glu-Lys counterpart, although this difference was not noticed in metal chelated M R P component mixtures. 3.6.5 Assessment of Antioxidant Activity of MRP Mixtures A s a result o f different copper chelating affinities observed for different M R P derivation experiments, it was assumed that they could possess variable anti-/pro-oxidant activities. In order to evaluate this character o f model M R P mixtures, three different antioxidant assays were performed, using an oxygen electrode, thio-barbituric acid assay method ( T B A ) and a model D N A nicking assay method. 3.6.5.1 Assessment of Antioxidant Activity by TBA Method The generation o f T B A R S in a model metal free linoleic acid emulsion system containing Glu-Lys and Fru-Lys MRP mixtures are presented in F ig . 3.9. A l l non-dialysable water soluble products derived from the experimental conditions used to synthesize both Glu-Lys and Fru-Lys MRP mixtures lowered lipid peroxidation, as assessed by M D A measurement in the l ipid emulsion system. It is noteworthy, that despite the finding that all experiments that yielded detectable amounts o f M R P mixtures also exhibited antioxidant activity, as assessed by T B A R endpoint measurement, many o f the products derived from different Fru-Lys experiments produced relatively l o w (e.g. below 5%) 3.0 Study I 91 "z o o cj J5 g o -a o "S 'a. £ 5 0 S -O x C N m t o •<-J- N O T r t -O O o o o o o o O o o IT) O N o o r - r - o O O N o «—< •—< o o ~ o" o <—* C N C N C N C N C N C N c s CN" r s OO O o o o vO O r o O o o o r N o o Os r o wo, v » r f r o O O z £ Z z z z z z m r o o o o o r o r f ( N CN r o —. r n o \ r -r o EC X X X X X o o VD v o •<* ON r - o i n o ON t-' o\ oo oo 0 0 O CJ o CJ CJ u u cj N O C N C O V O vo V O C O m C O V O r - V O 0 0 C O V O vd r-' V O r-' vd o o ' C O C N O N ,_ C N vo o o O N vq o C N 0 0 vq m ' c o ' c o " o o " r-' i n c o ' C O C O C O C O C O C O C O C O T T C N r - C N m O N O r - r - T T C O C N vo vd vd vd vd vd r-' r-' r-' r - C N o o ,_ 0 0 C O V O O N o o V O C N no V O O C O ,_" ,_<" c o " C O o o ' r-»' o O « n m m m m i n z I CJ" II S Z <§ c3 II 11 73 33 CJ CJ 0 1 53 CO II 3.0 Study! 92 o CJ •c '5. 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O 13 +3 O S t £ < PQ F—1 cu 3 CU cu PH I O o »-, c2 -o cu 3 3 § s ,cu co "a OH «g 8 | c2 P cu on 3 <-H .3 cu CO ±2 o •a 2 g 3 ,13 ^ --3 cu kH 3 X £ 3 l II C O <u X i—i '£ II "oo 5 £ B ~ W -3 13 co -rO >, o -Q CO -a -5 P 3 ON ob cu £ C O c-l cu g I cu S v CU co >> "3 3.0 Study] 94 antioxidant activity. In the case o f Glu-Lys reactions, M R P mixtures derived from two synthesis experiment numbers, specifically 3 and 13, had antioxidant activities as high as 26 and 22 percent, respectively. 3.6.5.2 Assessment of Antioxidant Activity by Oxygen Consumption Measurement In the present study, oxygen consumption measurements were also used as a chemical measure for evaluating free radical reactions in metal supplemented linoleic acid emulsion systems. The percentage o f oxygen remaining in the reaction vessel over a period o f time was recorded as a measure o f calculating the protective index (PI) values o f crude M R P compounds. F i g . 3.10 illustrates the percentage o f oxygen left in the reaction vessel wi th respect to time as the reaction progressed in the presence o f two different M R P mixtures. Generally, the percentage o f oxygen remaining in the vessel depleted rapidly in the control emulsion, when compared to the emulsion wi th added M R P mixtures. There were, however, specific M R P mixtures (e.g. Glu-Lys MRP mixtures derived from experiment numbers 1, 8, 9, and Fru-Lys MRP mixtures derived from experiment numbers 1, 6, 8, 12, 13, 14), that accelerated the rate o f oxygen consumption in the vessel, thereby indicating a potential prooxidant activity not detected using the T B A R measurement. The P I values calculated for M R P mixtures obtained for different experiments are given in F ig .3 .11. Experiments wi th P I values greater than 1 were considered to possess antioxidant activities whi le P I values less than 1 were considered to have prooxidant activity. Compounds without anti- or prooxidant activity had a P I value equal to 1. In summary, Glu-Lys experiments produced 6 antioxidant, 3 prooxidant and 4 inactive M R P mixtures, compared to Fru-Lys experiments yielding 5 antioxidant, 5 prooxidant, and 3 M R P mixtures with no activity. A m o n g the different model M R P 3.0 Study 1 95 o cu a cu a •a . — i_ 3 £ JoqaiBq3 aq; ni Suiuiciuay uaSXxQ inaoaaj 3.0 Study] O + Tt 4- CM 4- © oo o + u E -c s cU Q< w (» "cw <S i-H O Id 3.0 Study] 97 derivation experiments employing aldo- and keto- sugars, antioxidative activity was found to occur in more instances wi th Glu-Lys MRP mixtures compared to Fru-Lys MRP mixtures. P I values plotted against the yield o f non-dialysable M R P mixtures derived f rom individual Glu-Lys and Fru-Lys reaction conditions are presented in F ig . 3.12. N o absolute relationship was found between the yield o f non-dialysable M R P mixtures derived in different model Glu-Lys and Fru-Lys experiments and characteristic antioxidant or prooxidant activity. Rather, identical experimental conditions used to generate M R P mixtures in both Glu-Lys and Fru-Lys systems were individually unique in producing a relationship between antioxidant or prooxidant activity and overall yield o f non-dialysable material produced. F o r example, identical conditions employed in specific experiment numbers 3 and 11, although producing contrasting comparative yields for Glu-Lys and Fru-Lys MRP mixtures, respectively, manifested similar antioxidant activities. Similar yields o f M R P mixtures obtained under duplicate conditions for synthesis o f Glu-Lys and Fru-Lys reactants, in experiment numbers 13, and 14 respectively, resulted in mild antioxidant activity for the Glu-Lys reactants, but prooxidant activity for the Fru-Lys products. A plot o f P I values versus the amount o f bound copper is shown in F ig . 3.13. A l though M R P mixtures wi th higher copper binding activity had P I values greater than 1, it is difficult to draw definitive general conclusions regarding this association since only two model systems have been studied in detail. However , it is noteworthy that the four experiments which produced the highest non-dialysable melanoidin yields in both model systems (i.e., Experiment numbers 3 and 13 in Glu-Lys reaction and Experiment numbers 5, and 11 in Fru-Lys reaction), also exhibited greater copper binding affinities whi le at the same time having different antioxidant activities. 3.0 Study 1 9 8 o 2,* o o V o o " 2 S f C " . fN SO o S O n OO > 2 e ^ CM CM cs IT) »-< IT) © c o 2 *-C cu CA o CO <5 cu cu .tJ o Id 3.0 Study] 99 : v> i o m O CM © o ta o U c s o CQ Id CO OJ 3.0 Study] iuu 3.6.5.3 In vitro D N A N i c k i n g Studies Three different types o f D N A strand scissions that could be visualized in a D N A gel electrophoresis are illustrated in F ig . 3.14 for two M R P mixture types at four different M R P concentration levels. The results indicated that the original supercoiled (S) D N A used for the experiment (Lane 1) was gradually broken down to nicked circular ( N C ) and linear (L ) forms as the added M R P concentration increased. Wi th certain M R P mixtures, at the highest concentration level tested (i.e. 0 .1%, w/v) , original supercoiled D N A was completely degraded and appeared as a smear. This particular effect was predominently observed with Fru-Lys MRP mixtures compared to Glu-Lys MRP mixtures. T o explain this behaviour in more detail, percentages o f supercoiled, nicked circular, and degraded D N A remaining after incubating D N A with different M R P concentrations were quantitated and presented in Figs. 3.15 - 3.17, respectively. The percentage o f original supercoiled D N A left after incubation o f D N A with three different M R P concentrations is given in F ig . 3.15. Acco rd ing to this figure, the relative efficacy o f different model M R P mixtures to break supercoiled D N A to nicked circular, or degraded forms, was shown to be specific to the individual synthesis conditions used in experiments to produce both Glu-Lys and Fru-Lys model M R P mixtures. Moreover , D N A breakage was dependent on the concentration o f individual M R P mixture added to D N A (Figs 3.15 A - C ) . In both instances, exposure o f D N A to higher concentrations o f both Glu-Lys and Fru-Lys model M R P mixtures resulted in enhanced D N A strand breakage. A greater breakage o f supercoiled D N A generally occurred when Fru-Lys M R P mixtures were used. D N A exposed to low M R P mixture concentrations [e.g. 10"3 % (w/v) F ig . 3.15 A ] produced similar D N A strand breakage (approx. 1-10%) for both Glu-Lys and Fru-Lys across all experiments. A s the concentration o f M R P mixtures incubated wi th D N A was increased to 10" 1 %, not only did the supercoiled D N A breakage 3.0 Study J 101 2 3 4 5 6 7 8 9 10 F i g . 3.14: Concent ra t ion dependent P M 2 D N A nicking observed for t w o M R P mixtures. Lane 1 = supercoi led P M 2 D N A ; Lane 2 = supercoi led P M 2 D N A treated w i th M s p l ; Lanes 3 and 7 = D N A + 0 . 0 0 0 1 % (w/v) M R P mixture; Lanes 4 and 8 = D N A + 0 . 0 0 1 % (w/v) M R P mixture; Lanes 5 and 9 = D N A + 0 . 0 1 % (w/v) M R P mixture; Lanes 6 and 10 = D N A + 0 . 1 % (w/v) M R P mixture; Lanes 3-6 represent D N A n ick ing patterns observed w i th Fru-Lys M R P mixture IL Lanes 7-10 represent D N A nick ing patterns observed w i th Glu-Lys M R P mixture 3. S = superco i led D N A , N C = n icked c i rcular D N A , L = l inear D N A . 3.0 Study! 102 100 80 60 40 20 0 lOO < O 80 -a C U 60 U La C U C 40 3 C M o 20 0 100 80 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Synthesis Experiment Number Fig. 3.15: Percentage of supercoiled DNA remaining after incubating PM2 phage DNA for 1 hour at,37 °C with different concentrations of crude MRP mixtures derived from different synthesis experiments. Dotted line represent initial percentage of supercoiled DNA in control preparation. Values represent mean + S.D. * = significant with respect to similar Fru-Lys experiment (p<0.05). ® = No remaining supercoiled DNA ND = non-dialysable MRP not detected. (A) 0.001% (w/v) MRP, (B) 0.01% (w/v) MRP, (C) 0.1% (w/v) MRP. • -Glu-Lys MRP mixture, • = Fru-Lys MRP mixture 3.0 Study 1 < Q 60 D 40 + T3 C U "2 C M o 20 0 1 7 8 9 10 11 12 13 14 Synthesis Experiment Number Fig. 3.16: . Percentage of nicked circular DNA remaining after incubating PM2 phage DNA for 1 hour at 37 °C with different concentrations of crude MRP mixtures derived from different synthesis experiments. Percentage of supercoiled DNA at the beginning of the reaction = 82.4%. Values represent mean ± S.D. * = significant with respect to similar Fru-Lys experiment (p<0.05). ND = non-dialysable MRP not detected. (A) 0.001% (w/v) MRP, (B) 0.01% (w/v) MRP, (Q 0.1% (w/v) MRP. • = Glu-Lys MRP mixture, • = Fru-Lys Admixture. 3.0 Studyl 104 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Synthesis Experiment Number Fig. 3.17: Percentage of degraded DNA remaining after incubating PM2 phage DNA for 1 hour at 37 °C with different concentration levels of crude MRP mixtures derived from different synthesis experiments. Percentage of supercoiled DNA at the beginning of reaction = 82.4%. Values represent mean ± S.D. <8> = No remaining circular DNA. * = significant with respect to similar Fru-Lys experiment (p<0.05). ND = non-dialysable MRP not detected. (A) 0.001% (w/v) MRP, ( B ) 0 . 0 1 % (w/v) MRP, (Q 0 .1% (w/v) MRP. '•= Glu-Lys MRP mixture, • =Fru-Lys MRP rruxtare. 3.0 Study J 105 increase (Fig. 3.15 B) for both model M R P mixtures, but the spread between common experiments within different Glu-Lys and Fru-Lys reactants also increased. Exposure o f D N A to the highest M R P concentration used in this study (10" l o/o, F ig . 3.15 C ) resulted in the greatest magnitude o f supercoiled D N A breakage and the highest degree o f variation in activity among all experiments. F o r example, at the 1 0 _ 1 % level, M R P mixtures f rom Fru-Lys experiment numbers 3, 4, 5, 12, and 14 caused maximal supercoiled D N A breakage at the end o f the incubation period. It is noteworthy, that whi le breakage o f supercoiled D N A by Glu-Lys MRP mixtures remained relatively constant for all synthesis experiments, considerable variation in supercoiled D N A breakage was observed wi th Fru-Lys MRP mixtures derived from different conditions used in experimental synthesis. In addition, complete breakage o f supercoiled D N A observed with Fru-Lys MRP mixtures, generated in experiment numbers 3, 4, 5, 12 and 14 at the highest concentration which reflected prooxidant activity, was not seen with corresponding Glu-Lys MRP mixtures. Similarly, the percentages o f nicked circular and degraded forms o f D N A formed after exposure o f D N A to different crude M R P mixture concentrations for each synthesis experiment number are respectively given in F i g 3.16 - 3.17. The results indicate that the amount o f nicked circular as wel l as degraded forms o f D N A increased as the level o f added M R P concentration increased. A t a M R P concentration o f 0 .1% (w/v), certain D N A samples (e.g. M R P mixture o f Fru-Lys experiment numbers 4, and 14) only carried nicked circular and degraded forms o f D N A material. 3.0 Study] lUb 3.7 DISCUSSION 3.7.1 Effect of Reaction Conditions on the Intensity of Browning between Glu-Lys and Fru-Lys Model Reactions A. Yield of non-dialysable MRP mixtures In this study, the effect o f reaction conditions used to synthesize M R P mixtures were assessed on non-dialysable melanoidin formation in Glu-Lys and Fru-Lys browning mixtures by collectively monitoring yield o f non-dialysable melanoidin, chromatic character at 420 nm, and co lour quality using Hunter L a b tristimulus colorimetry. The results confirmed former studies by Eichner and Ka re l (1972), Resnik et al. (1979), and Cerrutti et al. (1985), that collectively indicated the yield o f non-dialysable M R P mixtures to be greatly influenced by both the initial reaction conditions and the type o f reactant sugar. In all experiments, although similar reaction conditions were used for both model M R P experiment numbers, the resulting yields were substantially different. Similar observations were reported by Baxter (1995). In his study, a mixture o f glucose wi th amino acid produced more absorbing material at 420 nm compared to a mixture o f fructose wi th the same amino acid when the mixtures were stored at 50 °C at p H 7.5. The generally higher yields o f M R P from glucose points to the greater reactivity o f this reducing sugar compared to fructose, which is a less reactive reducing ketose sugar. In general, aldose sugars are more reactive than ketose sugars, as a consequence o f more sterically hindered carbonyl group o f ketose (O 'Br ien and Morr issey, 1989). K e t o sugars are known to proceed through imine intermediates which lead to the formation o f Heyns products while aldose sugars proceed through Amador i products (Hodge, 1953). The extent o f Amador i or Heyns product formation is primarily determined by the p H , a^,, and temperature o f the reaction medium which subsequently determines the product distribution originating from either pathway. In addition, 3.0 Study! 107 the reported fact that browning rates o f Heyns products were slower than those o f Amador i products (P i lkova et al., 1990), further explain the reason for the observed lower product ion o f l o w M R P yields in Fru-Lys reactions in many instances. Moreover , the results o f this study f irmly indicate that both the rate o f M R and the type o f intermediates formed are largely dependent on both the source o f reactant sugar as wel l as the initial reaction conditions. R Absorption spectra and intensity of colour The typical b rown colour obtained in a model system, or in a food subjected to heat treatment, usually indicates some degree o f occurrence o f the M R (Spingarn and Garv ie, 1979). Therefore, measurement o f co lour quality in a reaction solution was found to be valuable in objectively predicting the extent o f browning reactions. Accord ing to O'Br ien and Mor issey (1989) M R P mixtures are a composite o f several discrete chromophores that exhibit decreased absorbance with increasing wave lengths. The first stage o f the M R P can be characterized by an absorption maximum at 280 nm, which has been attributed to the presence o f heterocyclic derivatives (Cuzzoni et al, 1988). A s the reaction advances, the absorbance values at 280 nm progressively decline due to the appearance o f more complex compounds which are known as soluble pre-melanoidins having an absorbance maximum at 320 nm. A s such, the shoulder detected around 320 nm in the U V - V i s spectra for both Glu-Lys and Fru-Lys MRP mixtures in this study was an indication o f the presence o f more complex soluble M R P compounds in the reaction solution. These precursors o f complex melanoidins are expected to accumulate to a measurable extent long before any measurable amount o f high molecular weight melanoidin appears (Rendleman and Inglett, 1990). In addition, the absence o f a definitive absorption maximum in all the M R P mixtures tested herein could be attributed to the simultaneous formation and condensation o f chromophores on 3.0 Study] lus a single polymeric molecule, previously referred to as the mixed chromophore hypothesis (Clark and Tanenbaum, 1974). In this study, although all M R P mixtures although had similar spectral patterns, they all exhibited different absorption readings at a given wavelength. This observation therefore indicates that not only did the extent o f M R differ between the two model systems, but also the concentration o f individual intermediate compounds formed could be different. R e w i c k i et al (1994) reported, that under similar experimental conditions, model fructose-amino acid M R gave rise to a greater pyrazine content compared to similar glucose-amino acid M R . Measurement o f final M R solutions at 420 nm in this study was used as an end point measurement for quantifying the amount o f melanoidins present in different final M R P solutions. Higher absorbance readings were consistently noticed for Glu-Lys model M R P mixtures compared to Fru-Lys model M R P mixtures, indicating a higher concentration o f melanoidins formed in the former reaction. Pigment formation, or the darkening o f the medium during heating, is the result o f polymerization o f the many highly reactive compounds that are formed during the advanced stages o f the reaction. The unsaturated carbonyl compounds and furfural (Reynolds, 1965) wh ich give rise to heterocyclic amines (Mauron, 1981) with molecular weights above 1,000 daltons are some good examples o f this. Therefore, lower absorbance readings at 420 nm and lower non-dialysable yields observed for many Fru-Lys experiments is a further indication that the rate o f polymerization in Fru-Lys M R occurred at a slower rate, or was less complete compared to the reaction occurr ing wi th Glu-Lys M R . Aga in this result may be explained on the basis o f steric hindrance o f some o f the compounds formed during the Fru-Lys model reaction (O 'B r ien and Morr issey, 1989) wh ich wou ld decrease overall yield o f the product. 3.0 Study! 109 The Hunter lab L, a, and b values were also used as an objective measurement for describing the extent o f browning reaction. The yield versus the L, a, and b values for different M R P mixtures, clearly showed that experiments producing higher non-dialysable melanoidin yields possessed lower L values (darker) and positive a and b values. These results further confirmed the finding o f 420 nm absorbance readings, indicating that the concentration o f non-dialysable melanoidins formed in a specific M R P synthesis experiment can be related to its L value as wel l as to its absorbance at 420 nm. 3.7.2 Characterization of Metal Chelating Affinity of Model MRP Mixtures 3.7.2.1 Metal Chelating Affinity of Crude MRP Mixtures The experiments in this study were conducted to examine the metal chelating affinity o f both crude M R P mixtures and fractionated components. The results obtained wi th crude M R P mixtures indicated that most crude M R P mixture types had appreciable but variable affinity to chelate C u 2 + ions, indicating the importance o f the reaction conditions in determining the chelating affinity o f the derived M R P mixtures. 3.7.2.2 Separation of Model MRP Mixtures by Chelating Chromatography A s the results indicate, chelation chromatography was useful for separating crude M R P mixtures into components. Crude M R P mixtures tested herein were fractionated into 5-7 distinct components confirming the results o f a similar study conducted by Terasawa et al. (1991) using glucose-glycine derived melanoidins. A s suggested by Hashiba (1985), chelation properties o f M R P mixtures could be attributed to the presence o f hydroxypyranone and hydroxypyridone residues present in melanoidin polymers. Thus, the degree o f chelation can be assumed to vary depending on the number o f such groups present in different crude M R P mixtures. 3.0 Study 1 110 The observed fact that M R P components fractionated by chelation chromatography possessed higher copper binding activities and more association sites when compared to crude M R P mixtures, indicates that chelation chromatography can be a valuable method for separating and concentrating specific M R P components, wi th specific chelating affinity, f rom the crude mixtures. In addition, the present study further showed that components eluted from the chelation column were not totally saturated wi th copper, but rather had the affinity to chelate more copper ions. 3.7.3 Mass Spectroscopy M A L D I - M S was used to determine the molecular weights o f M R P components eluted f rom the copper chelating column. E v e n though this technique has been used to quite an extent for analyzing proteins, its use in carbohydrate chemistry has been developed only recently (Stahl et al, 1994). So far, no work related to M R has been reported in the literature using this spectroscopic method. M A L D I mass spectra o f fractionated M R P component numbers 1 and 5 o f Glu-Lys and Fru-Lys experiment numbers 3 and 5, respectively, showed that both Glu-Lys and Fru-Lys MRP mixtures contained fragmented constituents to a similar degree. The study revealed that both component numbers 1 and 5, which were eluted from the chelation column during the gradient p H change, had two primary peaks in the molecular weight range o f 5,700 and 12,400 daltons. A higher intensity was always observed wi th the 5,700 molecular weight peak compared to the 12,400 molecular weight peak indicating the availability o f a complex mixture o f compound(s) wi th chelating affinity in the 5,700 molecular weight range. In addition, a large number o f intermediate compounds at a lower intensity level were also detected indicating the presence o f a number o f polymerization end products in the crude M R P mixtures. These intermediary compounds varied from sample to sample and between individual components derived from different sugar amino acid model reactions. These results further 3.0 Study] 111 demonstrate that the M R P mixtures synthesized herein are composed o f complex mixtures o f compounds having principal constituents, with molecular weights o f approximately 5,700 and 12,400 daltons which possess copper chelating potential. Yamaguchi et al. (1981) l ikewise reported the presence o f a M R P component having a molecular weight o f 4,500 daltons in a glucose-glycine model system that possessed strong antioxidant activity, although no studies regarding the metal chelating affinity o f this compound was reported. In the present study, the two component numbers, 1 and 5, o f the same M R P mixture, although possessing different C u 2 + chelating affinities and similar principal constituent composit ion, both eluted from the chelating column at two different p H values. This indicates that the metal chelating affinity o f these Mai l lard components is p H dependent. In addition, binding o f C u 2 + by M R P mixtures reported herein could be similar to the binding o f F e 3 + by maltol and 4-hydroxy-2,5-dimethyl-3(2H)-furanone ( H D M F ) as reported by Hashiba (1985). That scientist suggested the ketone and the hydroxyl group in the ortho-position o f these compounds enable the formation o f F e 3 + chelates and product ion o f chromophores. 3.7.4 Elemental Analysis of Crude MRP Mixtures and the Fractionated MRP Components M R P mixtures synthesized using different reaction conditions in this study possessed a variety o f empirical formulae. In addition, empirical formulae o f the Fru-Lys crude M R P mixtures consistently had one less carbon atom than the Glu-Lys MRP mixtures, and possessed a lower C : N ratio compared to their Glu-Lys counterparts. Mo ta i and Inoue (1974) reported an empirical formula o f C g H n N O e for xylose-glycine melanoidins and indicated that this crude mixture was composed o f eight different melanoidin components possessing different antioxidant activities, although they all had the same empirical formula. The results o f the present study, therefore, support the above results by suggesting 3.0 Study] 112 that the number o f active compounds within one type o f M R P can not be explained by its empirical formula or by its molecular weight. Further evidence for these findings comes from results o f Cammerer and K r o h (1995). Those scientists reported that the fundamental composit ion o f derived M R P polymers was largely dependent on the reaction conditions used for synthesis whi le molar ratio o f the reactants had a negligible influence. In addition, they also reported a difference between the ratio o f nitrogen to carbon in the melanoidins o f the two hexose sugars investigated, glucose and fructose. Fru-Lys MRP mixtures possessed a higher percentage o f nitrogen and lower percentage o f carbon whi le an opposite trend was observed with Glu-Lys MRP mixtures. These observed composit ional differences, in the above as wel l as in the present study, could be attributed to the different thermal fragmentation abilities and different reactivities o f the two sugars toward one single amino acid. A s such, the fundamental structure o f melanoidin, the backbone and side chains, could vary depending on the reaction conditions used for synthesis. Moreover , it is also feasible that two similar molecular weight compounds possess several different structures exhibiting various potential bio-active properties. 3.7.5 Antioxidant / Prooxidant Activity of Model MRP Mixtures 3.7.5.1 Measurement of Antioxidant Activity by Oxygen Consumption Measurements M a n y studies in the literature have been conducted to determine the preventative action o f M R P mixtures on lipid peroxidation. However , very little attention has been given in determining the effect o f reaction conditions on the antioxidant activity potential o f the derived M R P mixtures. Un l i ke many studies reported in the literature, the present study demonstrated both prooxidant as wel l as antioxidant character o f model M R P mixtures using oxygen consumption measurements. These differences in anti-/pro-oxidant activities o f M R P produced from the two sugars suggest that the 3.0 Study J 113 two sugars not only reacted to different extents in browning, but also generated different intermediates with characteristically different antioxidant activities. Due to the complexity of the reactions involved in MR, and the uncertainty of common melanoidin end point formation, the exact structures of intermediate and final products responsible for the antioxidative effect observed herein were not fully determined. In addition, the observation of strong antioxidant activities in both low yielding (e.g. MRP mixture of Glu-Lys experiment number 5 and MRP mixture of Fru-Lys experiment number 3) as well as high yielding (e.g. Glu-Lys MRP mixtures 3, and 13; Fru-Lys MRP mixtures 5, and 11) MRP synthesis experiments, make it particularly difficult to draw definitive conclusions regarding a possible relationship between antioxidant activity and non-dialysable melanoidin yield. Melanoidins are also known to possess reductone and amino reductone structures and these structures have been confirmed to possess reducing and metal chelating activities. Therefore, the antioxidant activity of MRP mixtures tested herein could also be attributed to the presence of reductone structures in the MRP mixtures. However, the reason for the prooxidant activity detected with some MRP mixtures is not yet known. Neinaber and Eichner (1995) also reported prooxidant activity of fructose -arginine, -alanine, -histidine Amadori rearrangement products. In addition, in the present study, the Cu 2 + used in the oxygen consumption study, as a promoter of lipid oxidation, is also a consideration when explaining the anti-/pro-oxidant activity of the MRPs. 3.7.5.2 Antioxidant Activity of MRP Mixtures as Measured by the TBA Method In the present study, an additional method for evaluating antioxidant activity of different MRP mixtures in a lipid model system involved measuring the TBAR substances. The results indicated that all the MRP mixtures possessed variable antioxidant activity despite the absence of copper. MRP mixtures from specific Glu-Lys experiment numbers 1,3,4,5,13, and 14, and MRP mixtures derived 3.0 Study] 114 f rom specific Fru-Lys experiment numbers 4, 5, and 77, respectively exhibited the greatest antioxidant activities (e.g. antioxidant activities greater than 10%). Since these studies were conducted without C u 2 + supplemented model systems, the effectiveness o f different M R P mixtures to display antioxidant activity in a l ipid emulsion system could signify some affinity to scavenge free radicals. This activity in turn wou ld decrease the propagation step o f l ipid autooxidation reactions. Support for this conclusion is given by the study conducted by Hayase et al. (1990), who used electron spin resonance to demonstrate the effectiveness o f glucose-glycine conjugate derived melanoidins to scavenge superoxides generated by nicotinamide-adenine-dinucleotide phosphate-reduced form ( N A D P H ) -phenazine methosulphate ( P M S ) - nitroblue tetrazonium ( N B T ) reaction system, under aerobic conditions. Un l i ke the oxygen depletion measurements which, identified both antioxidant or prooxidant activity for individual Glu-Lys and Fru-Lys model experiments, a potential prooxidant effect o f M R P mixtures could not be identified using the T B A R measurement. The determination o f malonaldehyde ( M D A ) by reaction with 2-thiobarbituric acid ( 2 - T B A ) is commonly used in evaluating lipid peroxidation reactions. However , while the consumption o f oxygen is a true indicator o f total activity o f reactive oxygen species, T B A on the other hand is limited in evaluating free radical reactions (Draper and Handley, 1990). Reactive oxygen species attack unsaturated fatty acids producing lipid peroxides which subsequently decompose into reactive aldehydes, such as M D A . Autooxidat ion studies conducted with soybean oil revealed a linear relationship between oxygen consumption and peroxidation o f unsaturated fatty acids (Kishida et al., 1993). Therefore, the protective Index (PI) values calculated from oxygen-electrode studies was a definitive measure for distinguishing between antioxidant and prooxidant activities o f crude M R P mixtures. 3.0 Study! 115 Factors including pFL a^,, reaction time, temperature, and concentration o f reactants have been regarded as important parameters influencing the composit ion o f derived M R P mixtures (Pomeranz et al, 1962; Rendleman and Inglett, 1990). The study presented herein, further demonstrated that varying the initial reaction conditions used to produce M R P mixtures are also important in yielding functional M R P mixtures with characteristic antioxidant or prooxidant activity. A possible explanation for the finding o f anti-/pro-oxidant activity o f M R P mixtures in this study could be the characteristic abilities o f different M R P mixtures to sequester transition metals that are required in the Fenton reaction (Paller and Hedland, 1994), as wel l as the possible retardation o f autooxidation propagation reactions by scavenging o f free radicals. 3.7.5.3 Genotoxicity of Model MRP Mixtures Results o f the M R P dose-response studies identified the ability o f both model Glu-Lys and Fru-Lys MRP mixtures to nick D N A . This result supports previous findings that have shown mutagenecity o f model browning mixtures (Shinohara et al, 1980; Ki t ts et al, 1993a). Compar ison o f these results wi th the apparent yield o f M R P mixtures generated in each experiment revealed little indication o f a relationship between D N A breakage and the yield o f material generated f rom individual M R P synthesis experiments. A l though the precise chemical nature o f the complex mixture o f compounds produced in these specific model M R P mixtures remain unknown, the present findings do show that varying the conditions to derive different M R P mixtures can result not only in generation o f specific antioxidant properties, but also potential prooxidant and genotoxic activities as wel l . Reduc ing monosaccharides have been shown to be involved in enolization and autooxidation reactions, wi th the concomitant production o f oxygen-derived free radicals (Thornalley, 1984), inducing D N A strand breaks and virus inactivation during several hours o f incubation. Further to these 3.0 Study J 116 findings, the results o f the present study showed that M R P mixtures which are the net result o f reducing sugars reacting with amino groups, at higher concentrations, can also nick D N A . This effect was more pronounced with specific Fru-Lys MRP mixtures than with Glu-Lys MRP mixtures. Such an effect could be the result o f strong reactivity o f Heyns products resulting from ketoses compared to Amador i products resulting from aldoses, facilitating a faster conversion o f these groups to f luorophores as suggested by Suarez et al. (1988). In this respect, ketose sugars appear to be more important reactants in non-enzymatic glycation reactions than aldoses. Moreover , H i ramoto et al. (1993) reported that M R P mixtures from glucose - amino acid mixtures heated at 200 °C induced single strand breaks in D N A when D N A was incubated with M R P mixtures at 37 °C at p H 7.4. The authors suggested that the chemiluminescent ( C L ) M R P compounds exert the D N A breaking effect by generating singlet oxygen, which in turn attacks D N A . Kurosak i et al. (1989) reported that the C L activity and browning reactivity o f sugars fo l low the same order, namely, aldopentoses (ribose, arabinose, xylose) > aldohexoses (glucose, galactose) > ketohexose (fructose). One exception was D- glucose, although possessing high browning reactivity compared to fructose, which also possessed low C L activity. The greater D N A breaking effect noticed in the present study with Flu-Lys MRP mixtures compared to Glu-Lys MRP mixtures thus may be explained by the above finding. In this study, the M R P mixtures that had high metal binding affinity whi le exhibiting strong antioxidant properties in l ipid emulsion systems also manifested marked prooxidant activities when applied to a D N A model system. The above differences, therefore, make it difficult to establish a definitive relationship between the extent o f browning and specific antioxidative activity potential. The results o f this study further demonstrate that evaluating the prooxidant activity o f M R P mixtures is 3.0 Studyl 117 l ikely complicated by the nature o f their metal sequestering activity. F o r example, although many o f the M R P mixtures synthesized in this study had C u 2 + chelating affinity, some o f them showed pro-oxidant activity in oxygen electrode measurements conducted with supplemented copper ions. 3.8 C O N C L U S I O N S T h e results o f this study demonstrated that the elementary compos i t ion , metal chelat ing affinity, and antioxidant activity o f model M R P mixtures are largely inf luenced by the experimental condi t ions and the type o f reactant sugar used in the react ion, though the molecular weights o f the pr incipal constituents present in the fractionated metal chelated components were similar. These f indings indicate that similar molecular weight M R P mixtures cou ld stil l possess different structured compounds. The compounds that are present in minute quantit ies may also have played key roles in governing the bioact ive properties o f M R P mixtures synthesized under different water activi ty, p H , and temperature condit ions. 118 4.0 Study II Assessment of Anti- / Pro-oxidant Activity of Glu-Lys and Fru-Lys Model Maillard Reaction Products in a Linoleic Acid Emulsion System and a Model Cookie Dough Food System. 4.1 INTRODUCTION L ip id oxidation is a common deteriorative reaction in foods which can occur irrespective o f fat content. Transit ion metal ions, such as iron and copper are present in most food systems in trace amounts and are wel l known to act as catalysts o f lipid oxidation (Castel and Spears, 1968; Ci l lard et ai, 1980a; b; G r a f et ai, 1984; Mahoney and Graf, 1986). Antioxidants, whether naturally present, or when added to the food system, retard but do not completely inhibit l ipid oxidation reactions. Antioxidants exhibiting radical scavenging activity [e.g. butylated hydroxy toluene ( B H T ) , butylated hydroxy anisole ( B H A ) , tert-butylated hydroquinone ( T B H Q ) , propyl gallate ( P G ) , ascorbic acid and ce-tocopherol (a-Toc) ] (Mahoney and Graf, 1986) directly react wi th free radicals formed during oxidation and convert them to less reactive compounds rather than blocking the initial free radical generation reaction. A second category o f antioxidants consists o f additives such as citrate, diethylenetriamine pentaacetic acid ( D T P A ) , ethylene diamine tetra acetic acid ( E D T A ) (Mahoney and Graf, 1986), malic acid, and tartaric acid (Lemon et ai, 1950; Mo r r i s et ai, 1950) which express antioxidant activity by forming chelates with metal ions. Meta l chelating antioxidants either chelate metal ions or suppress ion reactivity by occupying all co-ordination sites on the metal ion (Mahoney and Graf, 1986) and are, thus, effective antioxidants only in retarding metal catalyzed l ipid oxidation (Lemon et ai, 1950). Under specific conditions, however, metal chelating compounds exhibit pro-4.0 StudyII l i y oxidant activity (Mahoney and Graf, 1986) as evidenced by the model ascorbate-copper systems that have yielded hydroxy radicals and malonaldehyde (Maior ino et al, 1993). Increased attention has recently been given towards using natural antioxidants in food systems (Namik i , 1988). The non-enzymatic Mai l lard browning reaction products ( M R P s ) that are derived, during processing, packaging, and storage, via the reactions between amino and carbonyl compounds in foods are regarded as components common to many food systems. Products o f M R wi th strong reducing potential (Yamaguchi et al, 1964) have antioxidant potential as a result o f reductone content produced at an early stage o f the reaction (Yamaguchi et al, 1964) and melanoidin content derived during a latter stage o f the reaction (Kir igaya et al, 1968). Several studies have demonstrated the ability o f M R P mixtures to act as radical scavengers (Hayase et al, 1989; 1990) and as metal chelators (Gomyo and Hi rokosh i , 1976; Johnson et al, 1983; Rendleman, 1987; Asakura et al, 1990; Rendleman and Inglett, 1990; Terasawa et al, 1991) thus potentially providing dual activity as free radical scavenger and metal chelator in inhibiting lipid oxidation. The presence o f antioxidant character associated with M R P s has been considered as a value added component since M R P mixtures are naturally synthesized during many food processing practices (Lingert and Er iksson, 1981). Al though many studies to date have focused on extending the shelf life o f heat processed foods by incorporating reactants that are essential for initiating the M R in the food systems, very little attention has been given towards studying the potential o f adding pre-formed M R P mixtures to food systems for the primary purpose o f providing antioxidant activity. Significant drawbacks o f employing the former strategy include loss o f nutritional value (e.g. available lysine loss) and the relative non-specific nature in which M R P mixtures are produced in all food systems using different processing conditions. Therefore, adding defined M R P products to foods for value-added purpose precludes a situation whereby M R P mixtures formed within a food system during processing 4.0 StudyII 120 are not known or characterized, and the reaction conditions may not be optimal to produce those MRP mixtures with the greatest antioxidant activity. In Study I, two model MRP mixture types, synthesized by heating Z-lysine with D-glucose or Z)-fructose under several different experimental conditions, were examined for their potential metal chelating and antioxidant activity. The results demonstrated the efficacy of those MRP mixtures to chelate metal ions, as well as to act as possible antioxidants or prooxidants, depending on the type of reactant sugar and reaction conditions used for the synthesis. This chapter presents a further evaluation of the antioxidant effectiveness of some of those specific MRP mixtures in decreasing metal catalyzed lipid oxidation reactions in a model linoleic acid emulsion system and a model, uncooked, prototype food system (cookie dough). Based on a review of the previous results, the MRP mixtures derived from Glu-Lys experiment numbers 3 and 13 and Fru-Lys experiment numbers 5 and 11 were selected for this study due to relatively greater antioxidant and copper chelating activities. The experiments reported herein were conducted in order to measure the effectiveness of these four MRP mixtures at minimizing lipid oxidation in model systems containing different MRP mixture concentrations. 4.0 StudyII 121 4.2 HYPOTHESIS Antioxidant compounds that possess metal chelating affinity can exhibit prooxidant activities under specific conditions. As such, MRP mixtures that exhibit antioxidant activity may possess potential prooxidant character depending on the composition of MRP used. 4.3 OBJECTIVES • to assess the antioxidative effectiveness of model MRP mixtures in the presence and absence of copper ions in a linoleic acid emulsion and a cookie dough model system; • to assess the antioxidative effectiveness of oc-tocopherol in the above mentioned two model systems with and without copper supplementation; • to study the potential synergistic and antagonistic effects of model MRP mixtures together with oc-tocopherol in two copper supplemented and unsupplemented model systems. 4.0 Study II 122 4.4 MATERIALS A linoleic acid emulsion was used as a model lipid system and employed vitamin E stripped soy oi l purchased f rom V a n D e n Bergh F o o d Ingredient group ( U S A ) . Cook ie dough for the prototype food system contained Fraser Val ley butter (Dairyworld Foods, B . C ) , Rob in H o o d all purpose flour (Mul t i Foods Inc., O N ) , B lue R ibbon baking powder (Thomas J . L ip ton , Canada), and Windsor salt (The Canadian Salt C o . , Canada). Z ip lok plastic bags ( D o w Brands, Canada) were bought f rom a local retail store ( B . C , Canada), a- tocopherol (a -Toc ) with a purity o f 9 9 % was purchased f rom I C N Biochemicals (Cleveland, O H ) . Potassium ferricyanide, di-sodium hydrogen phosphate, and d i -hydrogen sodium phosphate were purchased from B D H Chemical Company (Toronto, O N ) . Ferr ic chloride, and trichloroacetic acid were obtained from Fisher Scientific Company (Fair L a w n , N J ) . 4.5 METHODS 4.5.1 Preparation of Model MRP Mixtures M o d e l non-dialysable M R P mixtures synthesized from Experiment numbers 3 and 13 o f the Glu-Lys reactions and Experiment numbers 5 and 11 o f the Fru-Lys reactions were used to perform the model cookie dough experiments. These synthesized M R P mixtures were termed as Glu-Lys MRP mixture 3, Glu-Lys MRP mixture 13, Fru-Lys MRP mixture 5 and Fru-Lys MRP mixture 11, respectively. 4.5.2 Assessment of Lipid Oxidation in a Linoleic Acid Emulsion System in the Absence and Presence of Copper Lino le ic acid emulsion was prepared as described previously (3.5.5.2). One set o f experiments were conducted with added C u S 0 4 ( 3 m M , 1 mL/100 m L o f emulsion) and wi th model M R P mixtures (3 x lvTVo , 3 x 10"3, 3 x 10"2 and 3x 10""%, w/v) or oc-tocopherol (0.03%, 0 .3%, 2% and 5%, w/v). The next set o f experiments were conducted without added copper ions but only wi th added M R P 4.0 Study II 123 mixtures or oc-tocopherol. The combined effect o f M R P mixtures with cc-tocopherol was determined by adding 0 .003% (w/v) o f M R P mixtures and 0 .03% (w/v) o f oc-tocopherol to the emulsion system. A l l reactant combinations were incubated for 48 hours in a water bath set at 60 °C using 10 m L capped test tubes. A t the end o f incubation period, aliquots (2 m L ) o f emulsion were removed and the M D A content was quantitated by the method described previously (3.5.6.2). 4.5.3 P r e p a r a t i o n of C o o k i e Doughs The preparation o f basic cookie doughs contained the fol lowing ingredients: wheat f lour (138.2 g), butter (14.0 g), v i tamin-E stripped soy oil (12.0 g), water (46.0 g), salt (0.5 g) and baking powder (0.5 g). Sugar was not incorporated into the dough since it interferes wi th the T B A assay. Doughs were hand mixed in a plastic bowl . The different treatments used in this study are reported in Table 4.1. The addition o f copper ions to the dough was performed by incorporating 0.1 ml o f C u S 0 4 (3 m M ) solution to each 100 g o f dough. Thus, the final concentration o f copper in the dough was 0.3 p M per 100 g o f the dough. This concentration o f copper was selected in order to represent the concentrations typically found in foods (Underwood, 1971), and also to maintain it within the smallest recommended daily dosage (1.0 mg) for an individual (Food and Drugs, 1989). The addition o f a - T o c and M R P mixtures to the cookie doughs were based on a dry weight basis. Prepared doughs were kept inside Z ip lok plastic bags and stored at 4° C for 10 days or more. 4.5.4 Assessment o f L i p i d O x i d a t i o n in C o o k i e Doughs The generation o f l ipid oxidation products, using malonaldehyde ( M D A ) as an end point measure was determined by the T B A method described in section 3.5.6.2 wi th some minor modifications. A 2.0 g portion o f cookie dough was homogenized in 8.0 ml o f ice cold 25 m M Tris buffer at p H 7.4, containing 0.02% (w/v) sodium azide using a polytron (type P T 10/35 Kinemat ica G m b H , Switzerland) set at 12,500 r.p.m. for 10 minutes thrice. The homogenate was diluted 10 times 4.0 Study II 124 Tab le 4 .1 : Exper imenta l des ign used i n the preparat ion o f cook ie doughs. A ) C o o k i e aough (Cont ro l - I ) a B ) C o o k i e dough + 0 . 3 m M C u S 0 4 (Cont ro l - I I ) b C ) C o o k i e dough + 0.0001 % Glu-Lys MRP mix tures 3C, 13d or Fru-Lys MRP mixtures 5e, I / + 0 . 0 0 1 % Glu-Lys MRP mixtures 3, 13 or Fru-Lys MRP mixtures 5, 11 + 0 . 0 1 % Glu-Lys MRP mix tures 3, 13 or Fru-Lys MRP mixtures 5, 11 + 0 . 1 % Glu-Lys MRP mix tures 3, 13 or Fru-Lys MRP mixtures 5, 11 D ) C o o k i e dough + 0 . 0 1 % oc-Toc g + 0 . 1 % a - T o c + 2 % a - T o c + 5 % a - T o c E ) C ) + 0 . 3 m M C u S 0 4 F ) D ) + 0 . 3 m M C u S 0 4 G ) C o o k i e dough + 0 . 0 0 1 % Glu-Lys MRP mix tures 3, 13 or Fru-Lys MRP mixtures 5, 11 + 0 . 1 % a - T o c H ) C o o k i e dough + 0 . 0 0 1 % Glu-Lys MRP mixtures 3, 13 or Fru-Lys MRP mixtures 5,11 + 0 . 3 m M CuS04 + 0 . 1 % a - T o c Dough without supplemented copper ions Dough with supplemented copper ions Crude M R P mixtures derived from Glu-Lys experiment number 3 Crude M R P mixtures derived from Glu-Lys experiment number 13 Crude M R P mixtures derived from Fru-Lys experiment number 5 Crude M R P mixtures derived from Fru-Lys experiment number / / 8 a - T o c o p h e r o l 4.0 StudyII 125 with the same buffer, centrifuged for 10 minutes at 3,000 rpm, and the supernatant (6 m L ) was passed through a Cig Sep-pak cartridge (previously conditioned with methanol) to remove residual lipids. Clari f ied homogenate was assayed for the M D A content as described previously (3.5.6.2). 4.5.5 Colour Measurement of Cookie Doughs Three grams o f prepared cookie dough was flattened on a 2.5 cm petri dish (Fisher Scientific C o . , Fa i r L a w n , NJ) to a thickness o f 1 cm, and L, a, and b Hunter colour co-ordinates were measured using a 0745° Hunter L a b Labscan II tri-stimulus colorimeter (Reston, V A ) attached to a D-65 illuminant. Aperture size used was 6.35 mm. A l l readings were taken in triplicate using randomly drawn samples. 4.5.6 Assessment of Reducing Activity of Model MRP Mixtures Reduc ing activity o f model M R P mixtures was assessed by the method o f Y e n and Chen (1995). One m L o f model M R P mixtures or ascorbic acid (10 - 1000 pg /mL) standards were mixed with 2.5 m L o f phosphate buffer ( 0 .2M, p H 6.6), 2.5 m L o f potassium ferricyanide (1%, w/v) , in 10 m L test tube and incubated for 20 minutes in a water bath set at 50 °C. A t the end o f incubation, 0.5 m L o f ferric chloride (0.1%, w/v) and 2.5 m L o f deionized distilled water were added to 2.5 m L o f incubated reaction mixture and the absorbance was read at 700 nm. One way analysis o f variance fo l lowed by Tukey test was used in data analysis. 4.6 RESULTS 4.6.1 Model Linoleic Acid Emulsion System 4.6.1.1 Assessment of Lipid Oxidation in the Absence of Copper Ions The concentration o f malonaldehyde formed in the absence o f copper ions expressed as an index o f percent antioxidant activity (% A O , section 3.5.6.2) is given in Table 4.2 for the emulsion model system. A l l compounds exhibited antioxidant character at all four M R P concentrations tested. 4.0 Study II 126 Table 4 .2 : Assessment o f l ip id oxidat ion in a l inoleic acid emuls ion wi th added M R P mixtures in the absence and presence o f copper. M R P T y p e o f M R P Concentrat ion (% w/v) % A O a % A Ob Control 0.0 0.00 0.0 Glu-Lys MRP 0.0003 18.0 ± 0.1 20.5+0.3 mixture 3 0.003 19.1 ± 0.5 27.2 ± 1.0 0.03 22.0 ± 0 . 9 34.1 ± 0 . 3 * 0.3 23.2 ± 1 . 1 35.2 ± 0 . 3 * Glu-Lys MRP 0.0003 17.4 ± 2 . 1 20.2 ± 1.2 mixture 13 0.003 17.4 ± 0.4 19.6 ± 0 . 2 0.03 19.5 ± 0.6 33.1 ± 0 . 5 * 0.3 -15.2 ± 0 . 1 * 32.8 ±0.5* Fru-Lys MRP 0.0003 13.4±0.2 16.0 ± 0 . 2 mixture 5 0.003 -9.1 ± 0 . 7 * 18.2 ± 1.0 0.03 -14.1 ± 1.2* 19.5 ± 0 . 3 0.3 -15.2 ± 0 . 0 * 19.5 ±0 .3 Fru-Lys MRP 0.0003 14.7 ± 1.0 2 1 . 9 ± 0 . 7 mixture 11 0.003 -15.1+1.0* 22.6 ± 0 . 8 0.03 -17.4±0.5* 29.8 ± 0 . 5 * 0.3 -18 .7±0 .1* 30.1 ± 0 . 3 * a -Toc 0.03 23.6 ± 1.4 27.0 ±0 .5 0.3 28.3 ±0.5 33.0 ± 0 . 4 * 2.0 -29.1 ± 0 . 9 * 35.3 ± 0 . 1 * 5.0 - 3 1 . 9 ± 0 . 1 * 4 2 . 9 1 0 . 1 * Glu-Lys MRP mixture 3 ( 0.003%) + oc-Toc (0.03%) 21.4 ± 0 . 1 34.1 ± 0 . 2 Glu-Lys MRP m i x t u r e ^ (0.003%) + ocToc (0.03%) 19.2 + 0.3 34.0 ± 1 . 3 Fru-Lys MRP mixture 5 (0.003%) + ocToc (0.03%) 16.3 ± 1.0 28.0 ± 0 . 1 Fru-Lys MRP mix ture/ / (0.003%) + ocToc (0.03%) 16.5 ± 0.3 31.0 ±0 .5 Values represent mean ± S.D for n = 6 ^ with supplemented copper without supplemented copper * = Significantly different relative to lowest concentration of M R P used (p<0.05). % A O = ( T B A value o f the control without test compund - T B A value with the test compound) x 100 T B A value o f the control 4.0 StudyII 127 Addi t ion o f a - T o c and model M R P mixtures individually or together significantly (p<0.05) enhanced the antioxidative effect o f the emulsion compared to the control. Despi te the observed antioxidant enhancement effect, no significant (p>0.05) differences in antioxidant activity were observed between the different M R P mixtures derived f rom different synthesis experiments. 4.6.1.2 Assessment of Lipid Oxidation in the Presence of Copper Ions The relative antioxidant efficacy o f different M R P mixtures under copper supplementation is also given in Table 4.2. A negative value for antioxidant activity represented a prooxidant activity o f the test compounds. A l l compounds with the exception o f Glu-Lys MRP mixture 3 exhibited prooxidant activity at higher concentration levels. F o r example, both Fru-Lys MRP mixtures exhibited prooxidant activity above 0.003% (w/v) and oc-tocopherol above 2 % (w/v) whi le Glu-Lys MRP mixture 13 only exhibited such activity above 0 .3% (w/v) level. 4.6.2 Model Cookie Dough Experiment 4.6.2.1 Intensity of Dough Colour Co lou r measurements obtained for fresh and low temperature stored doughs using Hunter Lab colorimeter are given in Table 4.3. A n index (Colour Index; C I ) for comparing the co lour o f doughs was derived by multiplying a and b values and expressing the product as a fraction o f the L value parameter. Since L value decreases as the colour gets darker, a raised C I value corresponds to cookie doughs containing an increased amount o f model M R P mixtures. Between two M R P mixture types, characteristically high C I values were obtained for the doughs containing Glu-Lys MRP mixtures at a maximum concentration level (0 .1% w/w) while no difference in colour was observed in doughs containing a - T o c at all concentration levels tested compared to the control. 4.0 Study II 128 Tab le 4 .3 : C o l o u r intensity o f cook ie doughs measured using Hunter lab tr i -st imulus color imeter . M R P Treatment Concentrat ion L a (%L Con t ro l 0.0 94.1 ± 0.2 1.1 ± 0 . 4 10.6 ± 0 . 2 12.4 ± 0 . 3 Glu-Lys MRP mixture 32 0.0001 0.001 0.01 0.1 90.0 ± 0 . 3 84.3 ± 0 .0* 6 3 . 2 ± 0 . 1 * 48.5 ± 0 . 4 * 1.9 ± 0 . 4 1.8 ± 0 . 2 2.4 ± 0 . 3 * 3 . 4 ± 0 . 1 * 11.1 ± 0 . 3 10.4 ± 0 . 1 9.00 ± 0.4 8.80 ± 0 . 8 23.4 ± 0 . 3 * 22.5 ± 0 . 1 * 3 4 . 2 ± 0 . 1 * 61.7 ± 0 . 1 * Glu-Lys MRP mixture 13 0.0001 0.001 0.01 0.1 89.9 ± 0 . 3 * 85.3 ± 0 . 3 * 62.1 ± 1.0* 43.9 ± 0 . 7 * 1.0 ± 0.1 1.9 ± 0 . 6 2.8 ± 0 . 6 * 3.9 ± 0 . 5 * 10.80 ± 1.1 9.60 ± 0 . 7 8.80 ± 0 . 2 * 8.70 ± 0 . 3 * 12.0 ± 0 . 2 21.4 ± 0 . 0 * 39.7 ± 0 . 2 * 77.3 ± 0 . 1 * Fru-Lys MRP mixture 54 0.0001 0.001 0.01 0.1 92.3 ± 1.3 91.2 ± 0 . 9 85.8 ± 0 . 1 58.2 ± 0 . 2 * 1.3 ± 0 . 5 1.6 ± 0 . 2 1.9 ± 0 . 6 3.0 ± 0 . 3 * 13.0 ± 0 . 7 * 12.4 ± 0 . 2 11.1 ± 0.1 8.6 ± 0 . 1 * 18.3 ± 0 . 3 2 1 . 7 ± 0 . 7 * 24.6 ± 0 .4* 44.3 ± 0 . 3 * Fru-Lys MRP mixture 11s 0.0001 0.001 0.01 0.1 91.1 ± 1.0 86.9 ± 0 . 3 * 60.3 ± 0 .7* 48.4 ± 2 . 0 * 1.5 ± 1.2 1.8 ± 0 . 4 2.7 ± 0 . 5 * 3.6 ± 0 . 9 * 12.8 ± 0 . 1 11.9 ± 0.5 8.9 ± 0 . 8 * 8.7 ± 0 . 4 * 21.1 ± 0 . 3 * 24.6 ± 0 .0* 39.8 ± 0 . 3 * 64.7 ± 0 . 2 * a - T o c 6 0.01 0.1 2.0 5.0 94.1 ± 0 . 4 93.9 ± 0 . 6 92.0 ± 0.6 9 1 . 8 ± 0 . 1 1.0 ± 1.2 1.0 ± 0 . 5 1.3 ± 0 . 7 1.1 ± 0 . 8 10.6 ± 0 . 1 10.3 ± 0.7 10.1 ± 0 . 4 10.0 ± 0 . 1 11.3 ± 0 . 7 11.0 ± 0 . 3 14.0 ± 0 . 3 12.0 ± 0 . 2 1 Colour Index (CI) = (a x b) x 100/Z 2 Crude M R P mixtures derived from Glu-Lys experiment number 3 3 Crude M R P mixtures derived from Glu-Lys experiment number 13 4 Crude M R P mixtures derived from Fru-Lys experiment number 5 5 Crude M R P mixtures derived from Fru-Lys experiment number 11 6 a - T o c o p h e r o l Va lues represent mean ± S.D fo r n = 9 * = Signi f icant ly different (p<0.05) w i th respect to contro l . 4.0 StudyII 129 4.6.2.2 Changes in Lipid Oxidation in Control Cookie Doughs Format ion o f malonaldehyde in two control doughs both in the presence and absence o f added copper is presented in F ig . 4.1. Storage o f doughs for more than five days resulted in a significant (p< 0.05) increase in the production o f malonaldehyde ( M D A ) between the two doughs. A faster rate o f l ipid oxidation was always observed in doughs containing added copper. F o r example, a M D A concentration o f 100 nMoles /g dough was reached by the copper supplemented dough within 12.5 days, compared to 16.5 days taken by the copper unsupplemented dough. 4.6.2.3 Effect of Added MRP Mixtures and a-tocopherol on the Rate of Lipid Oxidation Occurring in Cookie Doughs without Added Copper a) Lipid oxidation in cookie doughs with added MRP mixtures M D A formation in doughs without copper but with added Glu-Lys and Fru-Lys MRP mixtures is shown in Figs. 4.2 (a and b) and 4.3 (a and b), respectively. B o t h model Glu-Lys and Fru-Lys MRP mixtures effectively decreased the malonaldehyde formation in doughs at 7, 10, and 14 days o f storage compared to the control dough. In addition, the effectiveness in controll ing M D A formation was dependent on the level o f M R P added. Wi th both Glu-Lys MRP mixture types, the effectiveness at decreasing M D A formation significantly (p<0.05) increased as the added M R P mixture concentrations increased, although there were no significant difference (p>0.05) in the malonaldehyde formation at a given storage time among the four concentration levels tested. The antioxidant activity o f Glu-Lys MRP mixture 3, however, was greater compared to Glu-Lys MRP mixture 13 (Fig. 4.2a and 4.2b) M D A formation in cookie doughs with added Fru-Lys MRP mixtures in the absence o f added copper ions is presented in Figs. 4.3 a and b. Fru-Lys MRP mixture 11 produced a trend similar to Glu-Lys MRP mixtures in effectively decreasing the level o f l ipid oxidation as the concentration o f added M R P mixtures increased. However , an opposite effect was observed wi th Fru-Lys MRP 4.0 Study II 130 o C N 00 CU £ CU OX) o 15 o o CN o so o CN o 00 o (qSnop S/saiopvu) Vdl\[ -a >. c - 3 CO c _ CO « ep J5 oo O CO "° >, cu co 3 -° - o cu o E O C3 , , CO O cu is J= o c O o cu •4-* — co ^ E c2 CU L- VO cu 2 - +1 CO co ~ " C CO Q oo cu -5 CO co C CU O 3 15 "cO S E > E CU a. CL, o 0 1 JS U) 3 O CM O . c co CO CU co CU l_ CO p, *-CL, o, co — O £> CO o 3 u, TS o-o ^ CL, <L> i> C 3 cu cu H E cu Ii 3 TD co / r^ n' * i W-> * § p 00 5 VI •rr > Cc PH > *—' CU o. o. o o + JS 00 3 o p II 4.0 StudyII o • I. • ir. irt C/3 >, a Q cu E cu ex « o C/3 _ cO •S > . CJ V i •O ta - r-O O o 00 o VO o o (qSnop S/S3[op\[a) VGM 4.0 Study II 132 o oo c/3 Q B -2 CD ox CQ o co ^ rr* CU vP i/-> O 3* o I 0 s -o O O 00 o o o (qSnop §/S3|0j\iu) VQIA[ 4.0 Study II 133 o 00 a u © C/3 15 o o o 00 o SO o o (qSnop S/S3{op\[n) VQPM CO 4) 4.0 Study II o CM 00 s ••a cu OX) _ U 2 ° CO r*- ^ rrt m O O _1 _ *\ CU cx o O O 00 o NO o o (qSnop S/saiop\[u) VQJ\[ 4.0 StudyII 135 mixture 5. F o r example, Fru-Lys MRP mixture 5 at concentrations o f lO 'Vo and 10" 1 % produced a greater rate o f M D A formation than 10" 3 % and l C V o Fru-Lys MRP mixture concentrations. A n interesting observation made with this particular M R P mixture was that although it produced an increased amount o f M D A at higher concentrations, it was able to maintain the M D A formation at a significantly l o w (p<0.05) level compared to the control dough, at all four concentrations tested during storage. Conversely, although it did not exhibit any prooxidant activity Fru-Lys MRP mixture 11 was unable to significantly decrease M D A formation when compared to the control dough. In general, a comparison between Fru-Lys and Glu-Lys MRP mixtures indicated much stronger antioxidant activity for the Glu-Lys MRP mixtures at all four concentrations used in the preparation o f doughs. b) Lipid oxidation in doughs with added a-tocopherol(oc-Toc) The relative efficacy o f a - T o c to stabilize cookie doughs against l ipid oxidation is presented in F i g . 4.4. There was a greater antioxidant activity in doughs containing higher levels o f added a - T o c . In addition, all four concentrations o f a - T o c produced the greatest antioxidant activity when compared to both Glu-Lys and Fru-Lys MRP mixtures. There were no significant differences in temporal M D A formation in a - T o c supplemented doughs or the Glu-Lys MRP mixture added doughs when kept up to 10 days. There was, however, a significant (p<0.05) decrease in M D A formation in oc-Toc containing doughs, when compared with Fru-Lys MRP supplemented doughs throughout the test period. These findings therefore suggest that the antioxidative effectiveness o f Glu-Lys MRP mixtures was comparable wi th a - T o c in a cookie dough model system up to 10 days o f refrigerated storage. 4.0 Study II 136 o CM o o o 00 o vo o o CM c/i >> 03 Q cu S cu OX C3 J -o -«-> C/5 a co •a -o E T3 ccj <S co j - a> .*ti -*-» £ c co ° J S 2 op • — -•»-» cu c o ,<u o ta .a j= §'5 \3 co cd CU E 3 !- cd c2 > cu • •a 2 8 J £ E E Ct-l cu ° u c e-VH 3 CU co O O H I a 1> Q +1 c cd cu E 3 cu co CU I — Cc cu o o o A * 2 > ^ co o CU Z: 3 O II o cu O 6 <~ fg cd O ?H (qSnop 2/S9(op\[a) VQPM cd O 3- u , O cd 3 ii cu O co O. J3 £ rt S cu H rg .. cu oo § PM H cu Cu Cc O O v> 3 O o O S VI ~ cc £ o o •ti 00 v o cd O Q cN S || || 4.0 StudyII 137 4.6.2.4 Effect of MRP Mixtures and a-Toc in Retarding Lipid Oxidation Occurring in Cookie Doughs with Added Cupric Ions a) Lipid oxidation in doughs with added MRP mixtures The relative effectiveness o f model M R P mixtures and oc-Toc in decreasing the accelerated generation o f M D A in doughs supplemented with copper is shown in Figs. 4.5 (a and b), 4.6 (a and b), and 4.7, respectively. B o t h types o f model Glu-Lys MRP mixtures exhibited a similar behavioural pattern in decreasing metal catalyzed lipid oxidation with the exception that relatively greater antioxidant activity was observed for Glu-Lys MRP mixture 3 compared to Glu-Lys MRP mixture 13 at both 0 . 1 % and 0 .01% (w/v) concentrations. Similar to the behaviour observed earlier wi th cookie doughs devoid o f added copper ions, added Glu-Lys MRP mixtures effectively lowered metal catalyzed l ipid oxidation in doughs when compared to the control dough in a dose dependent manner. A prooxidant activity, however, was observed with both model Fru-Lys MRP mixtures 5 and 11 when added to copper supplemented doughs. In both cases, Fru-Lys MRP mixtures at concentrations o f 10" 2 % and 10"*% (w/v) produced M D A at a greater rate than rates observed wi th Fru-Lys MRP at 10" 3 % and 10""*%. This behaviour in particular was more notable for doughs containing Fru-Lys MRP mixture 11 than for doughs containing Fru-Lys MRP mixture 5. F o r example, the two concentration levels, 10" 3 % and l O ' V o o f Fru-Lys MRP mixture 11 produced malonaldehyde levels that were greater than that observed in the control dough. b) Lipid oxidation in doughs with added a-Tocopherol The rates o f l ipid oxidation in doughs with added a - T o c at four different concentrations are given in F i g 4.7. A t all four concentrations tested, a - T o c effectively decreased the metal catalyzed lipid oxidation compared to the control dough. However , two highest concentrations ( 2 % and 5%, w/v) o f a - T o c resulted in greater l ipid oxidation rates than lower concentrations (0 .01% and 0.1%, 4.0 Study II o <N 00 >> ° cu <J3 WD o 03 T3 1~ >^  O - C c/3 2 - 1 , 0) sP o o o vo o o 00 © (qSnop S/s3|0ivu) VGM 4.0 Study II o CM I CL) (u§nop S/s3]op\[ii) VQIM 4.0 StudyII o CM oo C/5 i—l 03 o CU ©X 03 O £ © o o o o 00 o (qSnop S/S3ioj\[u) VQPV <o +s & • • 4.0 Study II 4.0 Study II 143 w/v) . A similar behaviour was observed with Fru-Lys MRP mixture 5 in the presence o f copper ions. The rate o f M D A formation in Fru-Lys MRP added doughs was significantly (p<0.05) higher than that o f the a - T o c added doughs at equivalent concentrations. Such a difference was not observed wi th Glu-Lys MRP mixture added doughs. 4.6.2.5 Evaluation of the Possible Synergistic / Antagonistic Effect of a -Toe and MRP Mixtures in Retarding Lipid Oxidation The combined effect o f a - T o c and M R P mixtures in retarding l ipid oxidation in the presence and absence o f copper ions is presented in Figs. 4.8 and 4.9, respectively. The concentrations used in the combined study were selected to avoid the specific concentrations that could exhibit potential prooxidant activity. Thus, a concentration o f 0 .1% (w/v) for a - T o c and a concentration o f 0 .001% (w/v) for the M R P mixtures were used in this study. The presence o f M R P mixtures along wi th a - T o c were found to have no synergistic or antagonistic effect at minimizing M D A formation. 4.6.3 Reducing Activity of Model MRP Mixtures Reduc ing activity o f model M R P mixtures and ascorbic acid at different concentrations are given in F i g . 4.10. Since higher absorbance readings at 700 nm are an indication o f greater reducing power, the results show that both Fru-Lys MRP mixtures had stronger reducing powers than both Glu-Lys MRP mixtures. The respective reducing activities o f both M R P mixtures were lower than that observed for the ascorbic acid standard. Moreover , a significant difference (p<0.05) in the reducing activity between two M R P mixture types occurred only at the highest concentration (0.1%, w/v) tested. 4.0 Study II © O o fN O vo o fN o 00 o r f (qSnop 2/s3[op\[ii) VOW 00 CD CD C rt co CO „3 -C a) 15 *-O d VI Q. C cd o CO no 3 O -a CD 'Si C IG "E too o CL . s i 03 Q CU S CU OX 03 i -o c o "G CO E •I CD CD T3 </3 13 •?! c o CD t-Cd >, a ~o CD E CO CD c o co CD rt a o <? • + o o o ° ' 0 H 1 « + CD 3 •*-» X E «o CD u-3 X £ i- c-< \ ° v=> £ < ^ ^ C*-l O cct ••-» CO ^ E1— CD CD 4-> O 00 CD o c CD •-5 Q GO £ +i "5 c ^ cd co 2 CD g 3 z cd c CO . CD CD Q. O . fD O fD CD CD E c CD CD on O .5 o 6 CD 3 X o 0 H 1 8 + > Q *± il * ~ • 3 . 1 co 8 ° w H CD 3 + « J> E £ % g o 2. °-4.0 Study II 145 o CM 00 o o CM o VO o CM © 00 o (qSnop ?/S9[op\[u) VCIPV CW Q cu 6 cu DX) C/3 I C ^ •S § -g cu cd u. m O O VI Cu • M 4) 3 cd O c S i a + GO ~ J CO o « • O 3 CU o o 8 I s -3 3 55 .2 ^ c S ^ cu ° T3 to JS != « 3 cd O 1 ft! C M ^ td O Cu JS "cd O ^ Cu u-E CU JS (D 4-> P M » O 3 OO >» cd -a cd C O c o o o o 5 CL) L -3 •*-» X £ o o H « + — s > 2 > 3 CL) l_ ^5 P 5 -H ^ cd co 2 cu £ 2 Z cd C * ^ C O . CD CL) CU CU J> Cu O CU •a 13 CU to O ^ o VJ - i II a 3 o 0 H 1 H + > 0 S i—I O N CU - 3 0b g 3 cu £ 3 " •s 2 • Cu ,cu cu tt3 co T3 M <u Cu ^ Cu *Y 8 5 + e) J S II oo § < P p o cu I -« cN ^ © 3 s 4.0 Study II c 4.0 StudyII Ul 4.7 DISCUSSION 4.7.1 Assessment of Lipid Oxidation in Model Systems Containing MRP Mixtures and a-Toc without Added Copper A temporal pattern o f malonaldehyde production was measured in cookie doughs containing, or in the absence of, added copper ions to assess the rate o f l ipid oxidation in the present study. The relative antioxidant activities observed for both M R P mixtures reported herein support former studies conducted using simple model l ipid systems with glucose-lysine, fructose-lysine (Lingert and Er icksson, 1981) and xylose-lysine model M R P mixtures (Yamaguchi et al, 1981). Similar findings have been reported for l ow molecular weight carbonyl compounds, including methyl glyoxal and dihydroxyacetone reactants with different amino acids (Kawashima et al, 1977). The greater antioxidant strength o f a - T o c , compared to both M R P mixtures can be explained by the hydrophobic nature o f a - T o c which enables it to be a strong antioxidant in l ipid systems. a - T o c functions as a radical scavenger and as the results indicated, terminates the propagation o f radical chain reactions to a greater extent than M R P mixtures. The potential for prooxidant activity o f a - T o c at higher concentrations is also wel l documented (Loury et al, 1966; Ci l lard et al, 1980a; b; Baz in , et al, 1984; K o s k a s et al, 1984). Reducing agents such as a - T o c and ascorbate contribute to the product ion o f highly reactive hydroxy radicals (*OH) and correspondingly the production o f malonaldehyde in l ipid model systems (Mahoney and Graf, 1986). In the present study, this particular characteristic was not observed for a - T o c when copper ion was absent in the cookie dough formulations. O n the other hand, Fru-Lys MRP mixture 5 showed possible prooxidant activity at higher concentrations both in the cookie dough and linoleic acid emulsion model systems. It is known that some phenols used as antioxidants can promote prooxidant reactions especially at high concentrations. A concentration limit o f 0 . 1% (w/v) was reported for a - T o c in a soybean oil model 4.0 StudyII 148 system by L o u r y et al. (1966). Whitt ing et al. (1979) showed that the addition o f a - T o c as an antioxidant is ineffective i f the ratio o f a - T o c :linoleate was less than 10"3 during the autooxidation o f methyl or ethyl linoleate. Under such conditions these compounds seem to act as free radical chain breakers rather than acting as free radical scavengers (Labuza et al., 1970) as explained by the fo l lowing scheme: Ant ioxidant R O O * + A O H • R O O H + A O * Prooxidant R O O H + A * • R O O * + A H or R H + A * • R* + A H In the present study, even though this type o f behaviour did not occur wi th a - T o c in the absence o f copper ions, the above hypothesis holds true for the Fru-Lys MRP mixture 5 wh ich showed a greater amount o f M D A formation at higher concentration levels. The results presented herein do not totally agree with former studies (Kawashima et al, 1977; Yamaguch i et al., 1981) reported comparability o f antioxidant activities o f different M R P mixtures wi th that o f B H T and B H A since the present study demonstrated weak antioxidant activity o f model M R P mixtures relative to a - T o c . However , the decrease in M D A formation under specific conditions by addition o f specific M R P mixtures supports earlier findings indicating increased shelf life stability o f baked cookies made with arginine-xylose, histidine-glucose, arginine-glucose, and arginine-xylose model M R P mixtures (Lingert and Ha l l , 1986). These results may in part be attributed to the free radical scavenging activity o f M R P mixtures as evidenced by other studies conducted using electron spin resonance, where 4 7 % o f hydroxyl radicals were trapped by a 0 .03% glucose-glycine M R P mixture and 8 6 % were trapped by 0 .3% M R P mixtures (Hayase et al, 1989). These scientists further reported that M R P mixtures effectively scavenge superoxides, and at .higher concentrations also effectively scavenged hydrogen peroxides. Another explanation for the observed antioxidant activity o f model M R P mixtures tested herein, could be the neutralization o f l ipid free radicals by M R P free 4.0 StudyII 149 radicals during l ipid oxidation reactions (Lingert and E r i c k s s o n , 1981). Taken together, these findings explain the increased stability o f cookie doughs to oxidative rancidity by M R P mixtures is partially attributed to free radical scavenging, albeit the relative efficacy was not comparable wi th a - T o c . Moreover , the type o f reactant sugar, the reaction conditions used in generation o f M R P mixtures, and the concentration o f reactants used in the formulation o f M R P , all play important roles in determining anti-/prooxidant activity o f model M R P mixtures. 4.7.2 A n t i - / P r o o x i d a n t A c t i v i t y of M o d e l M R P M i x t u r e s a n d a - T o c in C o p p e r Supp lemen ted C o o k i e D o u g h s Copper is an active catalyst o f l ipid oxidation as a component o f the Haber-Weiss and Fenton reactions. Since copper concentrations in most foods range between 0.2 to 1 ppm (Underwood, 1971), copper supplemented cookie doughs in the present study were formulated to contain copper within this concentration range for examining subsequent lipid oxidation reactions. The results o f the present study revealed prooxidant activities for both Fru-Lys MRP mixtures and a - T o c at higher concentrations when in the presence o f copper ions. Me ta l chelating antioxidants function by effectively decreasing the activity o f metal ions either by precipitation o f the metal ion or by occupying all co-ordination sites on the metal ion (Mahoney and Graf, 1986). Glu-Lys MRP mixtures, which did not exhibit prooxidant activities in the presence o f copper, seemed to be better at minimizing l ipid oxidation reactions that occur in the presence o f trace amounts o f polyvalent metal ions. This further supports earlier observations made by Yamaguchi and Fuj imaki (1974a; b), who reported that antioxidant activity o f a - T o c was significantly decreased in the presence o f l o w concentrations o f cupric ions while that o f melanoidin had no significant affect. The application o f metal chelating agents such as, E D T A and citrate are also reported to be potentially ineffective antioxidants under certain 4.0 StudyII 150 conditions (Gra f et al, 1984) since they can not completely prevent the metal ions f rom participating in the Fenton reaction (Graf et al, 1984). The potential prooxidant activity characteristic o f only the Fru-Lys MRP mixtures suggests that these compounds either form complexes with copper ions which result in increased solubility at specific concentrations, or are unable to occupy all co-ordination sites on the copper ions ultimately resulting in prooxidant activity at specific concentrations. This important observation therefore indicates the importance o f careful selection o f model M R P mixtures as metal chelators for proposed antioxidant activity in food processing practices. O n the other hand, Hal l iwel l (1990) reported that the reactions o f C u 2 + ions either wi th a - T o c or l ipid hydroperoxides produce C u + which also promotes lipid peroxidation reactions by reducing hydroperoxides. Similar results have been reported by Maior ino et al. (1993) in a model micellar solution o f a - T o c in the presence o f C u 2 + ions. Thus, the transition metal catalyzed prooxidation reactions obtained in Fru-Lys and a - T o c added model dough systems could result f rom the above mentioned oxidation reduction reactions. Further support for this finding comes from the reducing activity measurements taken herein for these M R P mixtures. F o r example, both Fru-Lys MRP mixtures used in the l ipid model experiments possessed greater reducing power compared to Glu-Lys MRP mixtures. It stands to reason, therefore, that these compounds had a greater potential o f reducing C u 2 + to C u + and to enhance lipid oxidation reactions. Further, the observation that Fru-Lys MRP mixtures had a significantly (p<0.05) greater reducing activity at 0 . 1 % (w/v) concentration, compared to Glu-Lys MRP mixtures, further indicates that the underlying mechanism for its remarkably high prooxidant activity at that specific concentration may be a result o f its high reducing potential. 4.0 Study II 151 4.8 C O N C L U S I O N S Preformed M R P mixtures were effective at decreasing l ipid oxidation in resulted cookie doughs. O f the two M R P mixture types studied, Glu-Lys MRP mixtures showed greater antioxidant activity. O f particular interest was the finding that a prooxidant activity in Fru-Lys MRP mixtures occurred even in the absence o f copper ions at higher concentrations. These findings therefore suggest that the antioxidant potential o f preformed M R P mixtures are related to both the type o f reactant sugars and conditions used for the synthesis and on the M R P concentration used. Therefore, although the antioxidant activity o f M R P mixtures were less than that o f a - T o c , they may still be useful in retarding l ipid oxidation reactions under certain conditions. However , caution is required to ensure that there is no prooxidant activity. 152 5.0 Study III Effect of Glu-Lys and Fru-Lys Model Maillard Reaction Product Mixtures on Metal Induced DNA Nicking 5.1 INTRODUCTION The metal chelating and reducing activit ies o f model M R P mixtures have been character ized in Study I, and Study II, respectively. These specif ic propert ies may enable M R P mixtures to act as antioxidants or as prooxidants in l ip id model systems under speci f ic condi t ions. F o r example, prooxidant act ivi ty o f Fru-Lys M R P mixtures were noted in the presence o f metal ions and was dependent on the concentrat ion used. A similar prooxidant act iv i ty was reported for certain plant der ived phenol ic antioxidants, v iz . quercetin, gossypo l , and myr icet in (Laugh ton et al, 1989), especial ly in the presence o f metal ions. It has been suggested that such prooxidant act iv i ty cou ld occur as a result o f accelerating the metal catalyzed Fen ton react ion, wh i ch yields oxygen der ived species, such as H2O2. F o r example, M R P mixtures as we l l as many other antioxidant compounds are capable o f reducing F e 3 + to F e 2 + and part icipate in the Fen ton react ion by prov id ing a cont inuous source o f F e 2 + ions. F e 2 + ions play an important st imulatory ro le in free radical reactions by decomposing l ip id peroxides to chain propagat ing a lkoxy radicals and by react ing w i th Ff 2 0 2 to produce hydroxy! radicals ('OH) and other highly react ive species. These react ive species cause damage to D N A , pigments, and proteins. Such ant ioxidant compounds w h i c h can accelerate ox idat ive damage to var ious b io-molecules (Laughton et al, 1989; A r u o m a , 1993a; b) are referred to as a "carc inogenic ant ioxidants" (Wiseman and Ha l l iwe l l , 1993). 5.0 Study III 153 Consequent ly , it cannot be assumed that naturally occurr ing compounds w i th antioxidant activity are always 'safe ' for human use. O n the other hand, recent f indings indicate that in diabetes and ageing, a substantial modi f ica t ion o f funct ional and structural characteristics o f t issue proteins and D N A can be attributed to the covalent attachment o f glucose and other reducing sugars through the A m a d o r i react ion w i th the lysyl and terminal amino groups o f proteins, or alternatively free amino groups present on nucleot ide bases (Brownlee et al., 1988). Transi t ion metal cata lyzed ox idat ion o f monosacchar ides is also regarded as a reason for the modi f icat ion o f funct ional propert ies o f proteins ( W o l f f et al., 1989). Trace amounts o f transit ion metal ions can autoox id ize g lucose through the Haber -We iss type reaction and these ox id ized end-products o f g lucose can react w i th proteins causing tissue damage. Thus, the M R process may play an important ro le in in vivo as wel l as in in vitro systems. A l though , considerable attention has been g iven towards studying a var iety o f bioact ive propert ies o f M R , comparat ively few studies have examined the effects o f polyvalent metal ions together w i th M R P mixtures on cellular D N A . Prev ious studies showed that var ious metal ions may accelerate or decelerate the M R . F o r example, whi le copper and i ron salts accelerated the M R , t in and manganese salts inhibited the M R (Bohart and Carson , 1955; Pa t ton , 1955; K a t o et al., 1981). T o some degree this effect may be considered to be due to the changes in p H , as evidenced by the w o r k o f P o w e l l and Spark (1971). E v e n though the presence o f metal ions has long been k n o w n to influence the M R , the nature o f the interaction between specif ic metal ions and M R P mixtures is still not we l l understood. Mo reove r , very f ew studies have been conducted to investigate the combined effect o f polyvalent metal ions and M R P mixtures on such b iopo lymers as D N A . The pertinence o f this area o f research is obv ious since oxidat ive damage 5.0 Study III 154 to D N A can lead to mutat ion and cytotox ic events (A ruoma , 1993 c). Damage to D N A result ing f rom covalent modi f icat ion by a carcinogenic chemical or its metabolites is general ly regarded as the init ial b iochemica l alteration leading to neoplastic transformation in the case o f the majori ty o f chemical carcinogens (Fahl et al., 1984). Since M R P mixtures act as metal chelating agents, understanding the effectiveness o f these compounds on metal catalyzed oxidat ive damage to D N A is o f importance. K a w a k i s h i et al. (1990) reported that incubation o f bovine serum albumin ( B S A ) w i th pure A m a d o r i rearrangement products ( A R P s ) in the presence o f cupr ic ions caused marked fragmentat ion o f B S A a long w i th degradation o f histidine and tryptophan amino acid residues. It has been noted that A R P s can act in a manner similar to other reducing monosacchar ides to produce hydroxy l free radicals through catalysis by trace amounts o f polyvalent metal ions. T h e objective o f the present study was to examine the degree o f D N A n ick ing when present in an i ron dependent radical-generating system in the absence or presence o f Glu-Lys and Fru-Lys mode l M R P mixtures. In this effort, super coi led P M 2 bacter iophage D N A was used as a sensit ive measure o f evaluating oxidat ive D N A scissions. The experiments were conducted in order to determine the extent o f D N A n ick ing in the presence o f different polyvalent metal ions (e.g. F e 2 + , F e 3 + , and C u 2 + ) together w i th Glu-Lys MRP mixtures 3 and 13 and Fru-Lys MRP mixtures 5 and 11 under different experimental condit ions. 5.0 Study III 155 5.2 H Y P O T H E S I S M R P compounds that act as antioxidants in l ip id systems cou ld exhibit p roox idant act ivi t ies in non- l ip id D N A systems. 5.3 O B J E C T I V E S • T o assess the dose dependent response o f model M R P mixtures and polyvalent metal ions (e.g. F e 2 + , F e 3 + , and C u 2 + ) on P M 2 bacteriophage D N A at four p H (e.g. 7.4, 4.0, 3.2, 2.6) v a l u e s . • T o study the effect o f model M R P mixtures on D N A n ick ing in the presence o f metal ions, three experiments w i l l introduce M R P mixtures and metal ions to D N A as fo l l ows : (a) direct in t roduct ion; (b) introduct ion after preincubation o f M R P mixtures w i th metal ions at r oom temperature for 30 minutes under atmospheric oxygen; (c) in t roduct ion after preincubat ion o f M R P mixtures w i th metal ions at r oom temperature for 30 minutes in an atmosphere o f argon. • T o compare and contrast D N A n ick ing patterns observed w i th M R P mixtures w i th k n o w n antioxidants, e.g. phyt ic ac id, ascorbic acid and E D T A , in the presence o f metal ions. 5.0 Study III 156 5.4 MATERIALS E D T A , ascorbic acid, phyt ic acid, copper sulphate, ferrous sulphate, and ferr ic chlor ide ( impuri t ies < 0.01%) were purchased f rom Sigma Chemica l C o . (St. L o u i s , M O ) . Sephacry l S-200, B l u e Dex t ran 2000, Dex t ran T -10 , Dext ran T -70 , Sephadex G - 1 0 , G - 1 5 , G - 2 5 were obtained f rom Pharmac ia F ine Chemicals (Uppsala, Sweden). A l l prepared buffers were treated w i th Che lex 100 and then fi l tered through polypropylene E c o n o co lumns ( B i o - R a d Laborator ies , R i c h m o n d , C A ) . 5.5 METHODS 5.5.1 Preparation of Model MRP Mixtures M o d e l M R P mixtures der ived f rom four model M R P der ivat ion experiments (i.e. Glu-Lys Exper iment numbers 3, and 13, and Fru-Lys Exper iment numbers 5, and 11) were selected on the basis of: the availabil i ty o f information on primary component compos i t ion and molecular weights f r om metal chelat ion chromatography and M A L D I mass spectroscopy techniques (see sections 3.5.4 and 3.5.5, respect ively); the M R P mixtures derived f rom these four experiments possessed h igh metal chelat ing and antioxidant activit ies. The four Ma i l l a rd react ion mixtures were dialysed ( M W C O = 3.5 k D ) , lyophi l ised and the principal constituents w i th 5,000 D a l t o n molecu lar weight were col lected using gel f i l tration chromatography. The similarity o f the molecu lar weight fract ions col lected by the above techniques, to original compounds fract ionated by chelating chromatography (Study I) was determined by performing an elemental analysis o n each extract f o l l ow ing gel filtration. 5.5.2 Gel Filtration Chromatography T e n mi l l igrams o f crude M R P mixtures were appl ied onto a Sephacry l S -200 gel filtration co lumn (100 c m x 1 cm) and eluted wi th 0.01 M acetate buffer ( p H 5.0) at a f l o w rate o f 0.5 5.0 Study III 157 m L / m i n . E luate was col lected in 3 m L fractions and the M R P mixtures were detected by measur ing the absorbance at 420 nm. The vo id vo lume o f the co lumn was determined by B lue Dex t ran 2000 and the molecular weights o f the M R P mixtures eluted f rom the co lumn were estimated f rom a standard curve prepared by detecting the elut ion t imes o f standard compounds (i.e. Dex t ran T - 8 , T -10 , T -70 , Sephadex G - 1 0 , G - 1 5 , and G-25 resins) w i th k n o w n molecular weights. E lu t i on profi les o f the standard carbohydrate samples were determined by the phenol sulfur ic ac id method (Saha and Brewer , 1994) whi le the elut ion t imes o f M R P mixtures were determined by measur ing the absorbance at 420 nm. A t the end o f gel f i l t rat ion, the eluted components were dialysed against several volumes p f deionized dist i l led water and lyophi l l ized. 5.5.3 Preparation of Bacteriophage PM2 DNA This sect ion has been described in section 3.5.6. 5.5.4 Assessment of DNA Nicking Caused by Model MRP Mixtures and Polyvalent Metal Ions at Different pH Conditions T w o microl i t res o f P M 2 D N A (0.5 m g / p L ) and 2 p L each o f g l y c i n e - H C l buffer (50 m M , p H 2.6, 3.2, 4.0) o r 2 p L phosphate buffer (50 m M , p H 7.5) were mixed w i th 2 p L o f M R P mixtures (10" 3 , 10" 2, 10" 1, 10°%, w/v ) , and the total vo lume was made up to 10 p L by adding deion ized dist i l led water. The reaction was conducted in 250 p L E p p e n d o r f tubes and the incubat ion o f test compounds w i th D N A was conducted in a water bath set at 37 °C for 1 hour. A t the end o f incubat ion, l O p L o f the reaction mixture was loaded onto a agarose gel ( 0 .7% w/v) as descr ibed previously (3.5.6.2 - 3), and electrophoresis was performed for 90 minutes at 50 V . T o detect the D N A nick ing caused by polyvalent metal ions alone, the same procedure was per formed w i th the except ion that 2 p L each o f F e 2 + , F e 3 + , o r C u 2 + (1 - 100 p M / 2 p L ) ions replaced the 2 p L o f M R P mixtures. 5.0 Study III 158 5.5.5 Studies (a - c) on the Effect of Model MRP Mixtures on Metal Dependent DNA Nicking T o study the effect o f model M R P mixtures on F e 2 + dependent D N A n ick ing , both the max imum concentrat ion o f M R P mixtures not result ing in D N A n ick ing (e.g. 10" 3 %, w /v ) and the min imum concentrat ion o f F e 2 + result ing in D N A nick ing (10 u M at p H 7.5 and 5 p M at p H 2.6, 3.2, 4.0) were selected and three separate incubation studies were per formed as descr ibed be low. a) Study A - conducted without pre-incubation A react ion mixture consist ing o f 2 u L each o f M R P mixtures, polyvalent metal ions, buffer, and P M 2 D N A were used in these experiments. The total vo lume o f the react ion mixture was adjusted to 10 u L wi th deionized disti l led water throughout the experiments. A f te r being m ixed by gentle expuls ion f rom the micropipette t ip, the test solut ions were incubated in a water bath for 1 hour at 37 °C pr ior to undergoing agarose gel electrophoresis. b) Study B - conducted with pre-incubation at room temperature In this set o f experiments, 2 u L each o f metal ions and M R P mixtures were pre- incubated at r o o m temperature in buffer (2 u L ) and water before adding D N A . Immediately after adding D N A , the react ion mixture was incubated in a water bath for 1 hour at 37 °C. c) Study C - conducted with pre-incubation under argon T h e same experimental procedure described above was per formed w i th the except ion that the pre- incubat ion o f M R P mixtures wi th metal ions was performed in an atmosphere o f argon before adding D N A . A r g o n (impurit ies < 10 ppm) was appl ied by f lushing the gas into test tubes that were carry ing 50 u L each o f M R P and metal ions dur ing preincubat ion. In the case o f experiments using F e 3 + and C u 2 + ions, a series o f metal ion concentrat ions (i.e. 5, 10, 50 u M ) were used to study the metal catalyzed oxidat ive D N A scissions. 5.0 Study III 159 5.5.6 Effect of Ascorbic Acid, Phytic Acid, and EDTA on Metal Catalyzed DNA Nicking These experiments were conducted as described above w i th the except ion that phyt ic acid, ascorb ic ac id , or E D T A were used to replace M R P mixtures. 5.6 RESULTS 5.6.1 DNA Nicking Caused by Polyvalent Metal Ions and MRP Mixtures at Different pH Values /. Metal induced DNA nicking Induct ion o f D N A scissions by two transit ion metal ions, i ron and copper , at p H 7.5 is g iven in F i g . 5.1. The max imum percentage o f supercoi led D N A n icked in the presence o f F e 3 + and C u 2 + at the highest concentrat ion tested (i.e. 90 p M ) was less than 3 0 % . A similar degree o f supercoi led D N A n ick ing was observed wi th F e 2 + at a concentrat ion o f 10 p M . T h e effect o f p H on the F e 2 + induced D N A n ick ing is presented in F i g . 5.2. Since p H had no effect on D N A n ick ing caused by C u 2 + and F e 3 + , only the results obtained w i th F e 2 + ions are g iven. A s i l lustrated in F i g . 5.2, lower p H values significantly (p<0.05) increased the degree o f D N A n ick ing caused by F e 2 + ions. F o r example, F e 2 + at a concentrat ion o f 10 p M , induced 7 7 % o f supercoi led D N A n ick ing at p H 2.6, compared to 3 0 % o f n ick ing observed at p H 7.5, at an equivalent concentrat ion. E v e n a F e 2 + concentrat ion o f 1 p M , induced a signif icant (p<0.05) difference in D N A n ick ing among the four p H values tested, even though the degree o f breakage at this concentrat ion level cou ld only be visual ized by a densitometer scan. Therefore, the min imum F e 2 + i on concentrat ion to be appl ied to bacteriophage D N A to obtain a not iceable level o f D N A n ick ing was chosen to be 10 p M at p H 7.5 and 5 p M at the other three p H values. 5.0 Study III 5.0 Study III 5.0 Study III 162 //. MRP induced DNA nicking Percent supercoi led D N A remaining after incubating D N A w i th different M R P concentrat ions at four p H values are shown in F igs . 5 J a and b. The results indicated that M R P mixtures alone caused D N A n ick ing in a dose dependent manner and that the degree o f n ick ing was not signif icantly (p>0.05) affected by the p H o f the medium. The degree o f D N A n ick ing caused by M R P , at all p H values was found to be more intense when the M R P concentrat ion reached 0 . 1 % (w/v) . A t this concentrat ion o f M R P , both Fru-Lys MRP mixtures 5 (F ig . 5.3b A ) and 11 (F ig . 5.3b B ) converted all the supercoi led D N A to degraded form. One except ion to this effect occur red in the case wi th Fru-Lys MRP mixture 77 at p H 7.5. H o w e v e r , both Glu-Lys MRP mixtures 3 and 13, at an equivalent concentrat ion only p roduced a supercoi led D N A breakage o f 50 -60%. In general, w i th except ion to the D N A n ick ing not iced w i th Fru-Lys MRP mixture 77 at 0 . 1 % (w/v) at p H 7.5, both MRP mixtures der ived f rom Fru-Lys experiments and MRP mixtures derived f rom similar Glu-Lys experiments produced equivalent D N A n ick ing patterns. 5.6.2 D N A N i c k i n g i n the P resence o f M R P M i x t u r e s T o g e t h e r w i t h F e 3 + o r C u 2 + I o n s /. The presence of Cu2+ ions and MRP mixtures The effect o f M R P mixtures (0 .001% w/v) on D N A n ick ing in the presence o f C u 2 + ions at three C u 2 + c o n c e n t r a t i o n s (i.e. 0.1, 10, 50 p M ) is shown in Table 5.1. In general , both Glu-Lys MRP mixtures and corresponding Fru-Lys MRP mixtures exhibited similar D N A n ick ing patterns. A t two lower Cu 2 + concen t ra t i ons (i.e. 5 and 10 p M ) tested, both the direct addi t ion as we l l as the preincubat ion o f Glu-Lys MRP mixtures wi th C u 2 + decreased the amount o f supercoi led D N A n ick ing compared to n ick ing caused by C u 2 + alone, albeit the values were not signif icantly different. H o w e v e r , the degree o f n ick ing observed by apply ing C u 2 + at 5 p M and 10 p M 5.0 Study III 163 100 n 40 4-20 0 J 1 1 — 0 0.001 0.01 0.1 Concentration of MRP mixture (%, w/v) F i g . 5.3a: D o s e dependent D N A n ick ing in the presence o f Glu-Lys MRP mixture 3 ( A ) and Glu-Lys MRP mixture 13 (B) at four different p H values. Va lues represent mean ± S D for (n=3Y S D < 1% is not shown. • = p H 2 . 6 , • = p H 3 . 2 , A = p H 4 . 0 , • = p F £ 7 . 5 . Fig. 5.3b: Dose dependent DNA nicking in the presence of Fru-Lys MRP mixture 5 (A) and Fru-Lys MRP mixture 11 (B) at four different pH values. Values represent mean ± S.D for (n=3). S.D < 1% is not shown. • =pH2.6, • = pH3.2, A =pF£4.0, • =pH7.5. 5.0 Study III 165 CD i X i B r o Jo CD ^? 3 55 3 ^ x c CD 6 cu CN O CN «n O oo o d d d d +1 +1 +1 +1 oo o CN ro *—< d o\ vd 00 vd OO 00 CN O CN m O 00 o d d d d +1 +1 +1 +1 o CN CN d ON VO 00 vd 00 00 CN O CN o 00 o d d d d +1 +1 +1 +1 CN o *-—« d ON vd 00 vd 00 00 CN O CN <n O 00 o d d d d +1 +1 +1 +1 ro CN d ON vd 00 vd 00 00 < z Q "c3 .S M • c o A 3 3 3 U U O ZH ZH 3- 3-«n + rt Q o «n + Q o +1 ON o oo' 0O o +1 ro ro vd oo O +1 o CN oo o +1 CN ro oo' oo £ s o o d + < Z Q CN vo O O d d +1 +1 CN O O 00 oo CN vo O O d d +1 +1 o t -— m »n ro' 00 00 CN >° o o d <=> +1 +1 rt ro ro vq vd 0 0 oo oo CN >0 d o +1 +1 rt o W0 O vo oo oo PL, J= O a 3 3 3. 3. «n in + + 3 & > > o o d + o o d + o o o d d +1 +1 rt VO rt -rf ro oo oo O O d d +1 +1 00 TJ" r- CN oo oo o o d d +1 +1 T f r 00 00 00 o o d d +1 +1 o *-, rt ro >n vd oo oo PL, fN fN 3 3 u u 3. 3. O O + + £ £ S 2 > > is £ o O d + < Z Q o o d + Q * * oo r» rt O d d +1 +1 O rt f- ro ON vd oo r~-rt O d d +1 +1 CN r-vo rt-d t-' oo r-~ oo rt; 0 <=> d +1 +1 £ ro ON s; 00 oo o d o +1 o T f -CN ro <N oo oo rt O PH 3 3 o u 3. 3. O O >n >n + + £ £ > > vP x° O V flV O O d + < z Q o o d + < a c _o cd X) C 3 O CD ^3 t; ^ rt O £ O Wi ii r> PH II O PH 5.0 Study III 166 concentrat ion levels w i th M R P mixtures, after a pr ior incubat ion step at r o o m temperature in atmospher ic oxygen, was lower than that was observed by direct appl icat ion. In contrast, addi t ion o f 5 0 p M C u 2 + together w i th Glu-Lys MRP mixtures caused addit ive D N A n ick ing effect wh i ch occur red independent o f the pr ior incubation. In the case w i th Fru-Lys MRP mixtures, regardless o f a pr ior incubat ion step, all concentrat ions o f C u 2 + studied enhanced the degree o f D N A n ick ing in compar ison to n ick ing caused by C u 2 + alone. The degree o f D N A nick ing however, was not signif icant (p>0.05) at 5 p M and 10 p M concentrat ions o f C u 2 + . In contrast, 50 p M C u 2 + exhibi ted a signif icantly (p<0.05) greater D N A n ick ing when applied after preincubating w i th Fru-Lys MRP mixtures at r o o m temperature. //. The presence ofFe3+ and MRP mixtures Resul ts o f a D N A study conducted w i th F e 3 + , at three different concentrat ions (i.e. 5, 10, 50 p M ) o f F e 3 + are g iven in Table 5.2. In contrast, to the results obtained w i th C u 2 + , the addit ion o f both Glu-Lys and Fru-Lys MRP mixtures when preincubated w i th F e 3 + enhanced the degree o f D N A n ick ing more so than F e 3 + alone. The results were statistically signif icant (p<0.05) for Fru-Lys MRP mixtures w i th all concentrat ions o f F e 3 + , and for Glu-Lys MRP mixtures w i th only 50 p M F e 3 + w i th and wi thout preincubation treatment. In general, Fru-Lys MRP mixtures induced more n ick ing compared to Glu-Lys MRP mixtures and the intensity o f D N A n ick ing was dependent on the concentrat ion o f F e 3 + added. In addit ion, the degree o f D N A n ick ing observed w i th r o o m temperature preincubated samples o f 50 p M F e 3 + w i th all four M R P mixtures was greater than that was observed w i th C u 2 + at a same concentrat ion after a preincubat ion step. 5.0 Study III 167 r-I -3.a i E 1 cn to CD a .a "5 <n CD 5o u, -5 £ c CD e •s CD in O o" +1 o o o cn m o o +1 C N c n in o o +1 O N O m O N m O o +1 r -o cn O N I Q .9 00 • c O o c n O o © O o o +1 +1 +1 +1 C N •xT C N NO o m r - ' o o m ' o o NO 0 0 NO" o o o c n O o o o o o +1 +1 +1 +1 c n C N o C N c n 0 0 m o o NO* 0 0 NO 0 0 -XT o c n o o NO o o o o +1 +1 +1 +1 c n C N •xT o 0 0 m 0 0 NO 0 0 t— o o o c n O o o o o o o +1 +1 -H +1 ~ C N C N *—i o c n r - ' 0 0 m ' 0 0 NO 0 0 N O 0 0 A CD CD U lb h 5 s s S =L 3. - 1 - O • n + t Q o in + < '6 > •5 o o o + $ Q * * C N NO C N O o o +1 +1 C N o cn C N O OO o o * o * o o o +1 +1 - ~ 0 0 c n -xf C N ON oo r~ o CN c n o o " +1 +1 c -o c n O VO t - -o o o o vo ° o +1 cn in NO o o o o O +1 c n O OH JD o sr CD <L> PH PH s s m in + + CD l> e s > > o o o + o o o + < z D * * * C N 0 0 C N cn o • — 1 o d o O o +1 +1 +1 +1 o o NO C N o C N >—1 cn o ON m o o * * * * m C N O cn •—1 o o o o +1 +1 +1 +1 C N 0 0 C N ^H >—' ' — ' cn »—H cK in t -* , , NO 0 0 o — c o o C N o o o o +1 +1 +1 +1 , , C N o o m NO cn o o ^ r ' C N O 0 0 OO 0 0 o o * t - t—H C N o o C N o o o O +1 +1 . +1 +1 ,_ C N o o o cn o rr m" in cn «-H o o 0 0 0 0 0 0 ^ ^ OP C u ^ — OH cEe> + + + + "CD PH UH PH UH s s s 3. O o o o «"H «n in + + + + CD u CD CD IH u. 3 IH 1 s 6 6 OH C , > > is ? o o o + < Q o o o + D > > o o o + -< Z D o o o + < Z Q c o 3 O 3 t5 "CD - D U ; 3 O H O , s : 3 "S O . - 3 O H II !> P H O f 5.0 Study III 168 5.6.3 DNA Nicking in the Presence of MRP Mixtures Together with Fe 2 + Ions According to the results shown in Fig 5.2, the minimum concentration of iron that was required to visualize DNA scissions (i.e., « 20 - 30% of supercoiled DNA nicking) was 10 uM at pH 7.5, and 5 uM at the other three pH values tested. On the other hand, the maximum MRP dose that could be applied to the assay without causing a significant amount of DNA nicking (non-toxic dose) was found to be 0.0001% (w/v) for all four MRP mixtures. Therefore, the highest MRP concentration (0.0001%, w/v) that did not result in DNA nicking together, with the MRP concentration (0.001%, w/v) that resulted in minimum amount of DNA nicking were used in this study to determine the role of MRP mixture in mitigating or enhancing Fe2+ catalyzed oxidative DNA scissions. Since all the experiments conducted with a preincubation step at room temperature exhibited a significantly (p<0.05) smaller supercoiled DNA nicking, an additional preincubation study was conducted in an atmosphere of argon for the purpose of eliminating oxygen. This experiment was conducted to further clarify the reason for the noticed protection. Results of those DNA nicking assays (5.5.5 a, b, c) that were designed to determine the behaviour of MRP mixtures and Fe2+on DNA nicking activity are discussed below. 5.6.3.1 Study Conducted with a MRP Concentration of 0 .0001% (w/v) The percentages of supercoiled DNA remaining after exposing original DNA to different incubation treatments with 0.0001% (w/v) of MRP mixtures at four pH values are presented in Fig. 5.4a and b, respectively. The results indicate, that direct addition of Glu-Lys MRP mixtures together with Fe2+ did not enhance the degree of DNA nicking more so than that was observed with Fe2+ alone. Direct addition of Fru-Lys MRP mixture 5 with Fe2+ however, significantly (p<0.05) increased the degree of DNA nicking at pH 4.0 to a greater extent than when Fe2+ alone was added. However, in all treatments, the percentage of supercoiled DNA breakage was 5.0 Study III 169 100 OX) B .5 40 + 03 S CU < Q 'o u. CU a. s t/i -*-> a CU CJ CU OH GL3 GL13 FL5 F L U Type of MRP mixture F i g . 5.4a: Ef fec t o f 0 . 0001% (w/v) Glu-Lys and Fru-Lys MRP mixtures on F e 2 + catalyzed D N A n i ck ing at p H 7.5 ( A ) and p H 4.0 (B). Va lues represent mean ± S . D for (n=3). F e 2 + concentrat ions used were 10 u M and 5 u M at p H 7.5 and p H 4.0 , respectively. R T = R o o m temperature. * = Signif icant ly (p<0.05) different relative to F e 2 + . GL3 = Glu-Lys MRP mixture 3, GL13 = Glu-Lys MRP mixture 13, FL5 = Fru-Lys MRP mixture 5, F L U = Fru-Lys MRP mixture 11. = C o n t r o l , fJ = 0 . 0 0 0 1 % (w/v) MRP mixture, • = F e 2+ = F e 2 + +MRP mixture w i thout preincubat ion, = F e 2+ n = F e + + MRP mixture preincubated in argon at R T +MRP mixture preincubated in atmospher ic 0 2 a t R T 5.0 Study III 170 G L 3 G L 1 3 F L 5 F L U T y p e o f M R P m i x t u r e F i g . 5.4b: Ef fect o f 0 .0001% (w/v) Glu-Lys and Fru-Lys MRP mixtures on F e 2 + catalyzed D N A n ick ing at p H 3.2 ( A ) and p H 2.6 (B ) . Va lues represent mean ± S . D f o r ( n = 3 ) . F e 2 + concentrat ion used was 5 u M at both p H values. R T = R o o m temperature. * = Signi f icant ly different (p<0.05) relative to F e 2 + . G L 3 = Glu-Lys MRP mixture 3, G L 1 3 = Glu-Lys MRP mixture 13, F L 5 = Fru-Lys MRP mixture 5, F L U = Fru-Lys MRP mixture 11. • = C o n t r o l , i l = 0 .0001% (w/v) MRP mixture, • = F e 2 + , @ = F e 2 + +MRP mixture w i thout preincubat ion, E = F e 2 + + MRP mixture preincubated in atmospher ic 0 2 at R T FU = F e 2 + + MRP mixture preincubated in argon at R T . 5.0 Study III 171 remarkably lowered after a preincubation step performed at r o o m temperature under atmospheric oxygen. In addi t ion, at certain p H values, the preincubation o f Glu-Lys MRP mixtures w i th F e 2 + in argon also decreased the D N A breakage (e.g. Glu-Lys MRP mixture 3 at p H 7.5, 4 .0 , and 2.6; Glu-Lys MRP mixture 13 at p H 4.0 and 3.2) compared to the breakage caused by F e 2 + alone. Th is breakage was similar to or slightly lower than that was observed when M R P mixtures and F e 2 + ions were added to D N A directly. Howeve r , these observat ions were not signif icant when compared w i th n ick ing caused by F e 2 + alone. In particular, the presence o f Glu-Lys MRP mixture 3 showed a greater protect ion against transit ion metal induced D N A n ick ing than that observed for Glu-Lys MRP mixture 13. The relative degree o f D N A protect ion by Glu-Lys MRP mixtures in general was highest at p H 3.2 and fo l lowed the order 3.2>2.6>4.0>7.5. A l t hough , this k ind o f protect ion observed w i th Glu-Lys MRP mixtures was not signif icant (p>0.05) relat ive to the n ick ing caused by F e 2 + alone, such an effect was not observed when Fru-Lys MRP mixtures were tested. Rather , both Fru-Lys MRP mixtures exhibited similar to or higher degree o f D N A n ick ing when samples were preincubated in argon at room temperature compared to degree o f D N A n ick ing observed when samples were preincubated in atmospheric oxygen. F o r an unknown reason Fru-Lys MRP mixture 5 produced a significantly high amount o f D N A n ick ing after preincubat ing in argon at p H 4.0 and p H 2.6. Ano the r important observation worthwhi le report ing here was the relat ive increase in the degree o f D N A n ick ing in the presence o f M R P mixtures together w i th F e 2 + , when compared to that occur r ing in the presence o f M R P mixtures alone. Tox ic i t y o f all four M R P mixtures was signif icantly enhanced by the presence o f small amounts o f F e 2 + . 5.0 Study III 172 5.6.3.2 Study Conducted with a MRP Concentration of 0.001% (w/v) Repet i t ion o f the above experiments using a higher concentrat ion o f M R P (0 .001%, w/v ) p roduced markedly different results (F ig . 5.5a - 5.5b) compared to the study conducted w i th lower concentrat ions o f M R P mixtures. A t all p H levels studied, all four M R P mixtures gave an addit ive enhancement on D N A n ick ing when in the presence o f F e 2 + and wi thout a preincubat ion step, when compared to F e 2 + alone. Th is D N A n ick ing effect was signif icant (p<0.05) for both Fru-Lys MRP mixture types w i th respect to n ick ing caused by F e 2 + alone. H o w e v e r , addit ion o f both Glu-Lys and Fru-Lys MRP mixtures together w i th F e 2 + , and a pr ior incubat ion step at r oom temperature, greatly decreased the amount o f D N A n ick ing at all four p H values tested (except for Fru-Lys MRP mixture 5). In part icular, Fru-Lys MRP mixture 5 exhibi ted an addit ive D N A n ick ing effect even after preincubat ing at r o o m temperature. M o r e o v e r , the results o f the argon study showed that the percentage o f supercoi led D N A remaining by employ ing M R P mixtures preincubated wi th F e 2 + in argon was equal to the absolute level o f n ick ing observed when MRP mixtures were preincubated w i th F e 2 + ions at room temperature and under atmospheric oxygen. It is also important to note that the remarkable decrease in D N A breakage by Glu-Lys MRP mixtures, after preincubating wi th F e 2 + at room temperature under atmospher ic oxygen, was similar to, or less than the breakage caused by 0 . 0 0 1 % (w/v) M R P alone. Some o f these specif ic observat ions made wi th specif ic M R P mixtures, as v isual ized in an agarose gel bed at p H 7.5, are i l lustrated in F i g . 5.6 A and B. 5.0 Study III 173 GL3 GL13 FL5 FLU Type of MRP mixture Fig. 5.5a: Effect of 0.001% (w/v) Glu-Lys and Fru-Lys MRP mixtures on Fe 2 + catalyzed DNA nicking at pH 7.5 (A) and pH 4.0 (B). Values represent mean ± S.D for (n=3). Fe 2 + concentrations used were 10 uM and 5 uM at pH 7.5 and pH 4.0, respectively. RT = Room temperature. * = Significantly different (p<0.05) relative to Fe2 +. GL3 = Glu-Lys MRP mixture 3, GL13 = Glu-Lys MRP mixture 13, FL5 = Fru-Lys MRP mixture 5, FL 11 = Fru-Lys MRP mixture 11. • = Control, • =0.001%(w/v)A^rnixture, • =Fe 2 +, rp =Fe2 + +MRP mixture without preincubation, II = Fe2++ MRP mixture preincubated in atmospheric O2 at RT E3 = Fe 2 + + MRP mixture preincubated in argon at RT. 5.0 Study III 174 GL3 GL13 FL5 FLU Type of MRP mixture F i g . 5.5b. Ef fec t o f 0 . 0 0 1 % (w/v) Glu-Lys and Fru-Lys MRP mixtures o n F e 2 + catalyzed D N A n ick ing at p H 3.2 ( A ) and p H 2.6 (B). Va lues represent mean ± S . D for (n=3). F e 2 + concentrat ion used was 5 p M at both p H values. R T = R o o m temperature. * = Signi f icant ly different (p<0.05) relat ive to F e 2 + . GL3 = Glu-Lys MRP mixture 3, GL13 = Glu-Lys MRP mixture 13, FL5 = Fru-Lys MRP mixture 5, F L U = Fru-Lys MRP mixture 11. • = Con t ro l , M = 0 . 0 0 1 % ( w / v ) A d m i x t u r e , O = F e 2 + , S = F e 2 + +MRP mixture wi thout preincubat ion, E = F e 2 + + MRP mixture preincubated in atmospher ic O2 at R T • = F e 2 + +MRP mixture preincubated in argon at R T . 5.0 Study III 175 (A) F i g . 5.6: D N A n ick ing patterns observed after incubating P M 2 phage D N A wi th two concentrat ions o f Glu-Lys M R P mixture 3 and Fru-Lys M R P mixture 5 at p H 7.5 w i th F e 2 + ions. ( A ) Incubat ion treatments conducted w i th 0 .0001% (w/v) M R P mixtures. (B) Incubation treatments conducted w i th 0 . 0 0 1 % (w/v) M R P mixtures. Lane 1 = supercoi led D N A treated w i th M s p l ; Lanes 2 and 8 = Supercoi led D N A ; Lane 3 - 7 incubat ion treatments conducted wi th Glu-Lys M R P mixture 3; Lanes 9 - 1 3 incubat ion treatments conducted w i th Fru-Lys M R P mixture 5. Lanes 3 and 9 = D N A + M R P mixture ; Lanes 4 and 10 = F e 2 + (10 u M ) ; Lanes 5 and 11 = D N A + M R P mixture + F e 2 + (without preincubation); L a n e 6 and 12 = D N A + M R P mixture + F e 2 + (preincubated in argon); Lanes 7 and 13 = D N A + M R P mixture + F e 2 + (preincubated in atmospher ic oxygen). S = supercoi led D N A , N C = nicked circular D N A L = linear D N A . 5.0 Study III 176 5.6.4 Assessment of Metal Catalyzed DNA Nicking in the Presence of Phytic acid, Ascorbic acid, and EDTA The indiv idual response behaviour o f three common metal chelat ing agents on D N A is presented in F i g . 5.7. M e t a l chelating agents alone were found to induce very l itt le damage to D N A when compared to different MRP mixtures. T h e results produced in a preincubation study conducted w i th ascorbic acid in the presence o f C u 2 + , F e 3 + , and F e 2 + are shown in F igs . 5.8 ( A ) , (B), and (C), respect ively. The presence o f 10 u M F e 2 + or 10 u M F e 3 + significantly (p<0.05) enhanced (percentage o f n icked c i rcular D N A >90%) the D N A nick ing caused by ascorbic acid. The presence o f C u 2 + p roduced a smaller amount o f D N A nick ing when compared to F e 2 + or F e 3 + . In addi t ion, preincubat ion o f metal ions together w i th ascorbic acid at r oom temperature did not decrease the degree o f D N A n ick ing as noted w i th some model MRP mixtures. The study conducted w i th E D T A in the presence o f three metal ions p roduced results are shown in F igures 5.9 ( A ) , (B), and (C), respectively. E D T A completely mit igated metal induced D N A n ick ing at p H 7.5, but accelerated n ick ing at the other three p H values. The combined effect o f E D T A wi th metal ions exhibited a behaviour similar to mode l M R P mixtures since E D T A , in some instances, decreased the degree o f D N A n ick ing after a preincubat ion step. U n d e r very acid ic p H values, E D T A was found to induce an addit ive D N A n ick ing effect when in the presence o f F e 2 + ions. The least amount o f D N A n ick ing was observed when E D T A was appl ied to D N A in the presence o f C u 2 + ions. D N A n ick ing in the presence o f phytic acid w i th three metal ions is i l lustrated in F igs . 5.10 ( A ) , (B), and (C), respectively. Compared to ascorbic acid and E D T A , the presence o f phyt ic 5.0 Study III 177 5.0 Study III 178 F i g . 5.8: Percentage o f supercoi led D N A remaining after incubation o f D N A together w i th ( A ) C u 2 + (10 p M ) and ascorbic acid (10 p M ) , (B) F e 3 + (10 p M ) and ascorbic ac id (10 p M ) , and (C) F e 2 + (10 p M ) and ascorbic acid (10 p M ) . Va lues represent mean ± S . D for (n=3). * = Signi f icant ly different (p<0.05) w i th respect to specif ic metal ion. • = Or ig ina l D N A , • = 10 u M ascorbic acid, • = F e 3 + (10 p M ) or F e 2 + (10 p M ) o r C u 2 + (10 p M ) , = m e t a l i ° n + ascorbic acid wi thout preincubation, B = metal ion + ascorbic acid w i th preincubation. F i g . 5.9: Percentage o f supercoi led D N A remaining after incubat ion o f D N A together w i th ( A ) C u 2 + (10 u M ) and E D T A (10 u M ) , (B) F e 3 + (10 u M ) and E D T A (10 u M ) , and ( C ) F e 2 + (10 u M ) and E D T A (10 u M ) . Va lues represent mean ± S . D for (n=3). • = Or ig ina l D N A , ® = 10 u M E D T A , • = F e 3 + (10 u M ) o r F e 2 + (10 u M ) o r C u 2 + (10 u M ) , H = metal i on + E D T A without preincubation, EH = metal i on + E D T A w i th preincubat ion. F i g . 5.10: Percentage o f supercoi led D N A remaining after incubat ion o f D N A together w i th (A) C u 2 + (10 p M ) and phyt ic ac id (10 p M ) , (B) F e 3 + (10 p M ) and phyt ic ac id (10 p M ) , (C) F e 2 (10 u M ) and phyt ic ac id (10 p M ) . Va lues represent mean ± S . D for (n=3). * = Signi f icant ly different (p<0.05) w i th respect to contro l F e 2 + . • = Or ig ina l D N A , M = 10 u M phytic ac id, • ••= F e 3 + (10 u M ) o r F e 2 + (10 p M ) or C u 2 + ( 1 0 p M ) , ED = metal i on + phyt ic ac id wi thout preincubation, 11 = metal ion + phyt ic ac id w i th preincubat ion. 5.0 Study III 181 acid complete ly sustained D N A n ick ing caused by F e 2 + , F e 3 + and C u 2 + ions. M o r e o v e r , pre incubat ion o f metal ions w i th phyt ic acid did not result in further D N A n ick ing. Some o f these effects, as observed in an agarose gel electrophoresis bed are presented in F i g . 5 .11. 5.7 DISCUSSION 5.7.1 Metal Catalyzed Hydroxyl Radical Formation The results o f the present study demonstrate that transit ion metals p lay a cr i t ical role in D N A n ick ing act ivi ty and that the source and valence o f the specif ic metal i on , the amount o f metal ion present, and redox characteristics are salient factors that cont ro l the extent o f D N A n ick ing. A s indicated by the results, F e 3 + ions alone do not seem to facil i tate the generat ion o f hydroxy radicals in the presence o f d ioxygen (0 2). H o w e v e r , the F e 2 + ion in so lut ion even at very l o w concentrat ions resulted in the product ion o f Oxy-radicals. Th is difference in the two redox stages o f i ron cou ld be attributed to the format ion o f non-speci f ical ly react ive " O H by the latter compared to the former. H y d r o x y radical in general is considered to be a part icular ly damaging free radical since it may abstract hydrogen f rom most b iomolecules causing cel l injury or death (Repine et al., 1979; R o s e n and Klebanof f , 1981). A s such, it is also bel ieved that " O H is an et io logical agent for several diseases and it may also be invo lved in natural ageing (Yay layan and Huyghues-Despo in tes , 1994). Therefore, any compound that initiates this free radical may cause damage to D N A and cells. F o r example, inclusion o f reducing agents such as O2, catechol , dopamine, reduced glutathione, a - T o c , ascorbic acid as we l l as M R P mixtures together w i th F e 3 + theoret ical ly should prov ide a cont inuous source o f F e 2 + . In the presence o f d i -oxygen these 5.0 Studv III 182 1 2 3 4 5 6 7 8 9 10 11 12 131415 1 2 3 4 5 B 6 7 8 9 10 11 12 131415 1 2 3 4 5 6 7 8 9 1 0 11 12 131415 F i g . 5 .11: D N A n ick ing patterns observed after incubating P M 2 phage D N A w i th ascorbic acid, E D T A and phyt ic ac id w i th F e 2 + , F e 3 + , and C u 2 + ions at p H 7.5. ( A ) Incubat ion treatments conducted w i th ascorbic ac id. (B) Incubation treatments conducted w i th E D T A . ( C ) Incubation treatments conducted w i th phytic acid. Lanes 1, 6, 11 = supercoi led P M 2 D N A ; Lanes 2, 7, 12 = D N A + antioxidant compound ; L a n e 3 = D N A + C u 2 + (10 p M ) ; L a n e 8 = D N A + F e 3 + (10 p M ) ; L a n e 13 = D N A + F e 2 + (10 p M ) ; Lanes 4 , 9, 14 = D N A + ant ioxidant + F e 2 + (10 u M ) (wi thout preincubat ion); Lanes 5, 10 15 = D N A + antioxidant + F e2 + (preincubated in atmospher ic oxygen). S = supercoi led D N A , N C = n icked circular D N A , L = linear D N A . 5.0 Study III 183 agents w o u l d lead to oxidat ive damage o f D N A . Simi lar to the observat ions made w i th F e 3 + , C u 2 + also caused D N A n ick ing but to a lesser extent compared to F e 3 + . 5.7.2 Prooxidant Activity of MRP Mixtures in a Model DNA System The results o f the current study agree wi th previous f indings that have suggested possible proox idant act iv i ty o f antioxidants at high concentrations (Laughton et al, 1989). The not iced prooxidant D N A n ick ing activity o f M R P mixtures at higher concentrat ions cou ld be a result o f M R P der ived oxygen radicals as suggested by Aesbacher (1990) or the chemiluminescent act ivi ty o f M R P mixtures as suggested by H i ramoto et al. (1993). The observat ion made by P o w r i e et al. (1981) , that the mutagenic activity o f melanoidins get metabol ical ly deact ivated by l iver enzymes such as catalase further supports our results by suggesting the product ion o f oxygen radicals by M R P mixtures. Since catalase acts as a decomposer o f oxygen radicals, observed decrease in mutagenic act iv i ty o f M R P mixtures in the presence o f this enzyme w o u l d be attr ibuted to the diminished level o f M R P derived oxy-radicals by this enzyme. Therefore, M R P mixtures when present at high concentrat ions can generate D N A n ick ing oxy-radicals and free radicals possessing chemiluminescent activity. In addit ion, the greater degree o f D N A n ick ing not iced w i th specif ic Fru-Lys MRP mixtures could be associated to their stronger g lycat ing abil i ty ( M o r i t a and Kash imura , 1991) and greater chemiluminescent activity (Kurosak i et al, 1989) relat ive to similar Glu-Lys MRP mixtures. 5.7.3 Metal Catalyzed DNA Nicking in the Presence of MRP Mixtures I. DNA nicking observed in the presence of MRP mixtures together with Fe3+ and Cu2+ ions A s discussed above, oxidat ion o f D N A requires the presence o f oxy-radicals . Because o f their inherent reducing activity as we l l as chelating power , M R P mixtures used in this study cou ld 5.0 Study III 184 either enhance D N A nicking by driving the Fenton reaction or, alternatively, retard D N A nicking by chelation of metal ions. Specific Fru-Lys MRP mixtures could therefore impart greater D N A nicking activity after preincubating with Fe 3 +, in comparison to Glu-Lys MRP mixtures as a result of their greater reducing power. A similar result could be expected with C u 2 + ions with MRP mixtures since previous studies with Amadori rearrangement products have reported to reduce C u 2 + to more toxic Cu + (Kawakishi and Uchida, 1990) as given below: A H 2 + C u 2 + * A H ' + C ^ + F f Cu + + 0 2 •' C u 2 + + 0 2 * AH" + C u 2 + • A + Cu ' + fT — A + H 2 0 • amino acid + D-arabino-hexosulose Since Cu + ions are more toxic to D N A than Cu 2 + , preincubation of M R P mixtures with C u 2 + should enhance nicking. As such, the results obtained with Fru-Lys MRP mixtures could be explained by the above given scheme. Not withstanding, the results observed with Glu-Lys MRP mixtures are not explained by this mechanism since preincubation of these particular M R P mixtures with Cu 2 + at both 0.1 and 10 p M levels decreased D N A nicking. As such, it stands to reason that metal complexation, or chelation, may have provided the protection for Glu-Lys MRP mixtures against D N A nicking. These results, therefore, demonstrate that both the strength of chelating activity, as well as the reducing power of the test compound, play an important role in determining the metal catalyzed D N A nicking caused by a certain compound. //. DNA nicking in the presence of MRP mixtures together with Fe2+ ions Compared to Fe 3 + , Fe 2 + ions rapidly oxidized D N A and the intensity of nicking increased with a decrease in pH. In addition, at defined pH values the presence Of either a higher or a lower concentration of M R P mixtures, in the presence of Fe 2 + resulted in enhanced damage to the DNA. 5.0 Study III 185 These observat ions suggest that the sites o f metal b inding and subsequent Fen ton reactions that affect D N A susceptibi l i ty to redox damage are p H dependent. A s reported by M a r t i n and M a r i a m , (1979) and Gel ler t and B a u , (1979), i ron can be bound to the D N A surface by phosphate residues at nucleot ide bases in a groove where it is most exposed, or at the - O H groups on the deoxyr ibose molecule ( A r u o m a et al., 1989). Furthermore, i ron that is bound to phosphate groups in the F e 3 + state and as F e 2 + complex w i th nucleotide bases (Tado l in i , 1987; Tado l in i and Sech i , 1987). Therefore, when ferrous compounds were added to D N A , changes in local izat ion o f i ron in the react ion system cou ld have occurred wh ich ult imately change the pattern o f D N A n ick ing ( Y a n g and Schaich, 1996). F o r example, certain compounds cou ld shift the i ron binding site f rom bases to phosphate groups or vise versa. I ron bound to D N A phosphates promote dephosphory la t ion and i ron bound to bases could lead to base damage as we l l . Therefore, different intensities o f D N A n ick ing not iced herein under different p H values may be attributed to different loca l izat ion patterns o f i ron in the D N A molecule. It is also k n o w n that some chelating agents greatly increase the solubi l i ty o f i ron. In addi t ion, the solubi l i ty o f i ron also varies wi th the p H o f the medium. A s such, these f indings are further evidence for the different degree o f D N A n ick ing observed at different p H values wi th specif ic M R P mixtures. F o r example, higher amount o f D N A n ick ing not iced at l o w p H in the presence o f M R P mixtures and F e 2 + cou ld be a result o f increased solubi l i ty o f M R P - F e 2 + chelate at ac id ic p H . O n the other hand, the smaller degree o f D N A n ick ing noted w i th specif ic Glu-Lys MRP mixtures, compared to specif ic Fru-Lys MRP mixtures cou ld be a result o f lower d issociat ion constant values o f Glu-Lys MRP m ix tu re -Fe 2 + complexes compared to Fru-Lys-MRP mix tu re -Fe 2 + comp lexes . 5.0 Study III 186 In another study, Y a n g and Schaich (1996) reported an increase in the intensity o f D N A n ick ing by the metal chelat ing agent d e s f e r o x a m i n e ( D F O ) at high concentrat ions in the presence o f F e 2 + . A s suggested by these scientists, D F O has a strong preference fo r F e 3 + , and as such, this substrate catalyzes the site specif ic oxidat ion o f F e 2 + generating oxygen radicals at the surface o f D N A as it part ial ly binds and then removes i ron. Simi lar ly, M R P - m e t a l complexes when present at h igh concentrat ions are also l ikely to act similar to D F O in promot ing si te-specif ic ox idat ion o f F e 2 + causing damage to D N A . Throughout the study per iod, all four M R P mixtures exhibi ted a signif icant (p<0.05) decrease in D N A n ick ing after preincubating wi th metal ions. T w o plausible explanations in regard to this f inding are. 1) chelat ion o f F e 2 + i o n s by M R P mixtures; o r 2 ) ox idat ion o f F e 2 + t o less tox ic F e 3 + ions. A c c o r d i n g to the results, the degree o f D N A n ick ing observed after pre incubat ion o f M R P wi th F e 2 + in atmospheric oxygen in this study was lower than the degree o f n ick ing observed after preincubating the samples in argon, or similar to the amount o f n ick ing not iced by direct appl icat ion o f the test compounds to D N A in many instances. O n l y the specif ic Glu-Lys MRP mixtures w i th F e 2 + in argon decreased the degree o f D N A n ick ing more so than when the samples were preincubated in atmospheric oxygen. Therefore, some o f the protect ion not iced after preincubat ing the test compounds in atmospheric oxygen cou ld have resulted f rom ox idat ion o f F e 2 + t o F e 3 + ions, although the control experiments conducted w i th F e 2 + alone (under different preincubat ion treatments) d id not show any significant differences. M R P mixtures are reduc ing agents. Thus, convers ion o f F e 2 + to F e 3 + cannot be caused direct ly by M R P mixtures. H o w e v e r , a M R P - F e 2 + complex cou ld have a different solubi l i ty and ox idat ion power . In addit ion, the ox idat ion o f F e 2 + i n phosphate buffers due to the presence o f trace amounts o f metal ions have been demonstrated (Tadol in i and Sechi , 1987). Untreated phosphate buffer solut ions were 5.0 Study III 187 reported to contain approximately 0.13 p M copper and 0.7 p M iron. E v e n w i th the use o f deion ized buffer treated w i th Che lex 100 in this study, it was impossible to complete ly remove all traces o f metals f rom aqueous solutions. Therefore, one last possibi l i ty not tested in the present study cou ld be the ox idat ion o f F e 2 + i o n s by metal contaminants in buffers made herein. 5.7.4 Anti / Prooxidant Activity of Ascorbic Acid, EDTA and Phytic Acid in the Presence of Metal Ions The current data indicated that whi le the metal chelat ing agent phyt ic ac id , completely inhibited the metal catalyzed D N A nick ing, ascorbate on the other hand induced greater amount o f D N A n ick ing in the presence o f both F e 2 + a n d F e 3 + ions. Ascorbate alone d id not seem to break D N A , but, in the presence o f metal ions, ascorbate signif icantly enhanced the D N A nick ing. This f inding supports prev ious results that have suggested the genotoxic i ty o f ascorbate is contingent upon traces o f adventi t ious metals (Buettner, 1986; 1988; M i l l e r and Aus t , 1989). H o w e v e r , in this instance, ascorbate behaved differently f rom Glu-Lys MRP mixtures. Glu-Lys MRP mixtures on ly enhanced the Fen ton react ion in the presence o f F e 2 + ions, but d id not signif icant ly enhance the react ion in the presence o f l o w concentrations o f F e 3 + or C u 2 + ions. A reasonable explanat ion for the above f inding is the relatively lower reducing power and stronger chelat ing affinity o f the M R P mixtures compared to ascorbic acid. A s reported by G r a f et al, (1984) the co-ordinat ion chemistry o f the chelates is crucial in determining the relative abil i ty o f a chelating compound to produce hydroxy-radicals . I ron catalyzed " O H format ion f rom C V - and H2O2 consists o f two hal f reactions (e.g. the reduct ion o f F e 3 + by Oj~ and the oxidat ion o f F e 2 + b y H2O2). Thus, the availabi l i ty o f a co-ord inat ion site that is free or occup ied by an easily displaceable l igand such as water facil i tates the first ha l f reaction. Therefore, occupat ion o f all i ron co-ordinat ion sites by a chelator w i th displacement o f water in 5.0 Study III 188 the first co-ordination sphere, will preclude the binding of H 2 0 2 to Fe2+ chelates and decrease the subsequent Fenton reaction. As such, the total elimination of metal catalyzed DNA nicking observed with phytic acid could be attributed to the chelation of all iron co-ordination sites by phytates. Another important factor that clarifies the antioxidant to prooxidant actions of chelating agents is the chelator to iron ratio in the solutions (Gutteridge et al., 1979). At a certain critical level, metal chelating agents such as EDTA and ascorbic acid change from prooxidant action to antioxidant action. However, MRP mixtures studied herein never imposed an antioxidant effect at higher concentration levels, but rather promoted DNA scissions. In addition, MRP mixtures demonstrated significantly less DNA breakage only after a preincubation step with metal ions under atmospheric oxygen. Therefore, in general, the antioxidant activity of MRP mixtures in a DNA system seems to be a very weak interaction. 5.8 CONCLUSION Genotoxicity of MRP mixtures in the presence of polyvalent metal ions was studied using a DNA nicking assay method. PM2 bacteriophage DNA was exposed to four specific MRP mixtures (i.e. Glu-Lys MRP mixture 3, and mixture 13; Fru-Lys MRP mixture 5 and mixture 11) and metal ions (i.e. Fe2+, Fe3+, and Cu2+) in three different ways. For example, DNA was either exposed directly to metal ions and MRP mixtures, or exposed to metal ion and MRP mixture combinations preincubated at room temperature in an atmosphere of oxygen, or in an atmosphere of argon. Overall, the results indicated that Glu-Lys MRP mixtures are capable of mitigating metal catalyzed DNA nicking at all four pH values tested, and this effect could be further enhanced by preincubating MRP mixtures with Fe2+ at room temperature. However, the Fru-Lys MRP 5.0 Study III 189 mixtures, in the presence o f metal ions, had an addit ive effect in inducing D N A n ick ing, thus demonstrat ing prooxidant activity. The employment o f M R P mixtures preincubated w i th F e 2 + in argon indicated that a lesser degree o f n ick ing by Glu-Lys MRP mixtures preincubated w i th F e 2 + under atmospher ic oxygen was primari ly due to a chelation effect. T h e Fru-Lys MRP mixtures in contrast had a pr imary effect on the oxidat ion o f F e 2 + to F e 3 + . H o w e v e r , the int roduct ion o f 0 . 0 0 1 % (w/v) M R P mixture concentrat ions together wi th F e 2 + a lways p roduced an addit ive enhancement on the D N A nick ing, regardless o f the type o f M R P mixture tested. On ly the preincubated M R P mixtures o f Glu-Lys MRP mixtures 3, and 13, and Fru-Lys MRP mixture 11 wi th F e 2 + in atmospheric oxygen lowered the degree o f D N A nick ing. T h e study conducted in argon, w i th a higher concentrat ion o f M R P mixtures showed that chelat ion d id not occur w i th that M R P mixture concentrat ion dur ing preincubation. The studies conducted w i th F e 3 + ions indicated that the M R P mixtures d id not induce a signif icant difference in D N A nick ing at l o w concentrat ion levels o f F e 3 + . H o w e v e r , when 50 p M o f F e 3 + were used, M R P mixtures enhanced D N A n ick ing regardless o f the type o f M R P mixture used. In addi t ion, preincubat ion o f M R P mixtures together w i th F e 3 + a lways enhanced D N A n ick ing even though the degree o f difference was not always significant. One except ion to this t rend was observed w i th Fru-Lys MRP mixtures together w i th 50 p M o f F e 3 + . C u 2 + ions exhibited a different behaviour f rom F e 3 + , in the presence o f M R P mixtures. Glu-Lys MRP mixtures in the presence o f C u 2 + decreased D N A breakage dur ing direct appl icat ion o f these two compounds to D N A , and this effect was enhanced by a preincubat ion step. In contrast, Fru-Lys MRP mixtures promoted D N A breakage dur ing direct appl icat ion and was further increased by preincubat ion at r oom temperature. 5.0 Study III 1 9 0 These results therefore indicate that, in general Glu-Lys MRP mixtures cou ld decrease the metal induced D N A n ick ing relative to the n ick ing cause by metal ions alone or metal associated w i th Fru-Lys MRP mixtures. Howeve r , the presence o f metal ions a lways increased the degree o f D N A n ick ing relative to the n ick ing cause by M R P mixtures alone. 1 9 1 6.0 Study IV Modulation of Polyvalent Metal Ion Induced Cytotoxicity by Model MRP Mixtures 6.1 INTRODUCTION Factors such as amino acid, sugar composi t ion, temperature, react ion t ime, a w , and p H are k n o w n to inf luence the mutagenicity o f different M R P mixtures (Powr ie et al., 1981). C o m p o u n d s such as dicarbonyls, pyrazines, and furane derivatives that are formed dur ing the M R have been identif ied as causative agents in bacterial, mammal ian, and/or in vitro mammal ian cel l mutagenic i ty (Shibamoto, 1983; Omura , et al, 1982; K i t t s et al, 1993a). In addi t ion, M R P mixtures have also been reported to exhibit anticarcinogenic and/or ant imutagenic effects against heterocyc l ic amines (Ka to et al., 1987; Y e n and Chau , 1993). In previous experiments o f this thesis M R P mixtures have been shown to exhibit the metal chelating and antioxidant properties. T h e fo l l ow ing study was undertaken to study the effect o f M R P mixtures in modulat ing the metal induced cytotox ic i ty in a C 3 H / 1 0 T 1 / 2 mouse embryo fibroblast cel l cul ture system in the presence o f polyvalent metal ions. Simi lar to the experiments conducted w i th P M 2 bacter iophage D N A in S tudy III, the experiments herein wi th intact cel ls were conducted by adding M R P mixtures and metal ions to the cel l culture medium directly, or after preincubat ing metal ions w i th M R P mixtures at r o o m temperature and under atmospheric oxygen. Tox i c i t y assessments were made by detect ing the co lony forming efficiency o f the fibroblast cells under different experimental condi t ions. 6.0 Study IV 192 6.2 HYPOTHESIS M R P compounds when appl ied to intact cells together w i th polyvalent metal ions cou ld exhibit ant i - o r prooxidant act iv i ty depending on relative component compos i t ion. 6.3 OBJECTIVES • T o assess the in vitro dose response cytotoxic i ty o f specif ic M R P mixtures (i.e. Glu-Lys MRP mixtures 3 and 13; Fru-Lys MRP mixtures 5 and 11) synthesized in Study I and F e 2 + , F e 3 + , and C u 2 + polyvalent metal ions. • T o assess the modulat ion o f F e 2 + , F e 3 + , and C u 2 + ion induced cytotox ic i ty by in t roduc ing the above ment ioned four M R P mixtures to intact cells in vitro, direct ly w i th metal ions, or after a pr ior incubat ion step w i th metal ions conducted under atmospher ic oxygen at r oom temperature. 6.0 Study IV 193 6.4 MATERIALS C 3 H / 1 0 T 1 / 2 mouse embryo fibroblast cells were purchased f rom A m e r i c a n Type Cul ture Co l l ec t i on (Rockv i l l e , M D ) . Du lbecco ' s M o d i f i e d Eag les medium ( D M E ) , fetal ca l f serum, and trypsin were obtained f rom D i f c o Laborator ies (Detroi t , M I ) . Me thy lene blue, t issue culture dishes, f lasks, and tubes were purchased f rom Fisher Scienti f ic C o . (Fair L a w n , N J ) . 6.5 METHODS 6.5.1 Production of MRP Mixtures Simi lar M R P mixtures (filter steril ized) ut i l ized in Study III experiments were used herein. 6.5.2 Assessment of Cytotoxicity of MRP Mixtures and Metal Ions A l l s tock cultures were maintained in 175 c m 2 t issue culture f lasks w i th 50 m L o f culture med ium (450 m L l x D M E , 450 m L deionized disti l led water, 100 m L fetal ca l f serum) in an incubator oven ( M o d e l 3028, F o r m a Scienti f ic, Mar ie t ta , O H ) set at 37 °C w i th 3 % carbondiox ide. M e d i u m in the culture f lasks was changed every 3-4 days. Ce l l s in the f lasks were subcul tured every 7 days when flasks reached confluency. Embryon i c f ibroblast cel ls needed for the experiments were seeded (300 cel ls/mL/plate) in 60 m m culture plates w i th 4 m L o f culture med ium (1 x D M E ) and incubated at 37 °C. M e d i u m in the plates was changed every 3-4 days. In order to study the tox ic i ty o f M R P mixtures or metal ions on f ibroblast cel ls, 0.5 m L o f l x D M E was replaced w i th 0.5 m L o f M R P mixtures (i.e. 10" 3, 10" 2, 10" 1, and 10° % w /v ) or 0.5 m L o f metal ions (e.g. 0 .1 , 10, 50 p M ) . A t the end o f 10 days o f incubat ion, the med ium in the plates were removed by aspirat ion and cells were fixed and stained w i th 0 . 1 % (w/v) methylene blue (1:1 methanol : water) . C o l o n y forming ability o f the cells was determined by count ing the number o f co lonies formed on the petri dish w i th the aid o f an inverted microscope. The results o f the co lon iza t ion study were expressed as an index o f co lonizat ion eff iciency, where: 6.0 Study IV 194 number of colonies formed in the presence of test compound % colonization efficiency = x 100 number of colonies formed in the control (without test compound) 6.5.3 Assessment of the MRP Mixtures Ability to Mitigate Metal Induced Cytotoxicity Exper iments were conducted in a manner similar to the D N A study conducted in Study III, in the presence or absence o f preincubation w i th metal ions and M R P mixtures before exposure to embryonic cells. Three concentrations o f metal ions (i.e. 0 .1 , 1, 10 p M ) and two concentrat ions o f M R P mixtures (i.e. 0 .001%, 0 .01%, w/v) were selected fo r experimentat ion. a) Studies conducted with preincubation M R P mixtures (0.5 m L ) and metal ions (0.5 m L ) were preincubated at r o o m temperature for 30 minutes in 3 m L o f l x D M E before adding the embryonic cells. Immediately after adding cel ls, the plates were incubated at 37 °C for a predetermined t ime per iod (10 days). M e d i a in the plates were changed every 3-4 days wi th new medium containing similar concentrat ions o f M R P and metal ions. b) Studies conducted without preincubation These series o f experiments were conducted similar to the one descr ibed above w i th the except ion that fi lter steri l ized M R P mixtures and metal ions were direct ly m ixed w i th cel ls in the tissue culture plates wi thout a preincubation treatment and incubated fo r a further 10 days at 37° C . A s descr ibed above, media in the culture plates were changed every 3-4 days. 6.6 RESULTS 6.6.1 Cu2+, Fe2+, and Fe 3 + Catalyzed Cytotoxicity A dose response effect o f metal ions on cytotoxic i ty is presented in F i g . 6.1. A 50 u M concentrat ion o f C u 2 + and F e 2 + ions decreased the colony forming abil i ty o f cel ls by approximately 6.0 Study IV (o/0) XDUSIDIJja UOIJBZIIIOI03 bb 6.0 Study IV 196 5 0 % . Compared to C u 2 + and F e 2 + , F e 3 + was the least tox ic to the cells. F o r example, the cytotox ic i ty caused by 10 p M C u 2 + or F e 2 + was equivalent to the cyto tox ic i ty caused by 50 p M concentrat ion level o f F e 3 + . 6.6.2 Cytotoxicity of MRP Mixtures D o s e response curves for the four different M R P mixtures tested herein are presented in F i g . 6.2. A l l four M R P mixtures exhibited a dose dependent cytotox ic i ty a l though there were no signif icant (p>0.05) differences among the four M R P types at a g iven concentrat ion level. Therefore, a c o m m o n concentrat ion level o f M R P that caused least damage to co lony forming abil i ty o f cel ls was selected at 0 . 0 0 1 % (w/v) for all four M R P mixture types under study. 6.6.3 Assessment of Metal Induced Cytotoxicity in the Presence of Model MRP Mixtures A N O V A appl ied to the study conducted wi th C u 2 + indicated the existence o f significant interactions among the fo l low ing: (i) incubat ion; concentrat ion o f C u 2 + ; and the level o f M R P used (p=0.003); (ii) incubat ion; concentrat ion o f M R P ; and the type o f M R P used (p=0.02); and (ii i) concentrat ion and type o f M R P used (p=0.01). The study conducted w i th F e 2 + displayed signif icant (p<0.05) interactions only between: (i) incubat ion and level o f M R P (p=0.01) used; (ii) level o f M R P and type o f M R P (p=0.05) used. Fer r ic ions only showed signif icant interactions among: incubat ion; level o f M R P used; and concentrat ion o f Fe 3 + ( p=0 .03 ) . A. Presence of Cu2+ and MRP mixtures I. M o d u l a t i o n o f C u 2 + i n d u c e d cytotoxic i ty by 0 . 0 0 1 % (w/v) M R P mixtures The results o f the colonizat ion study conducted w i th f ibroblast cel ls in the presence o f C u 2 + preincubated w i th M R P mixtures at room temperature as we l l as dur ing direct appl icat ion wi thout a pr ior incubat ion step are presented in F i g . 6.3a. Add i t i on o f both M R P mixture types promoted co lony forming eff iciency o f the fibroblast cells regardless o f a pr ior incubat ion step 6.0 Study IV 197 Concentration of MRP Mixture (%,w/v) F i g . 6.2: Co lon iza t ion eff iciency o f mouse embryo fibroblast cells in the presence o f model M R P mixtures. Va lues represent mean ± S . D . • = G / M - I ^ A ^ n i i x t u r e 5 , • = Glu-Lys MRP mixture 13, A = Fru-Lys MRP mixture 5, • = Fru-Lys MRP mixture /1. 6.0 Study IV 198 i_mniiiiiiiiiiiiin * v * V * \ © 3 o o o o o o in -J ta S3 s CQ CU i« H o o o © o o o o o o o o o (%) foirapyja uopBzrcoi03 Cu II 112 a oo ti CO CU w Cu CU oo .ti ~ 3 o 3 O JS 3 •*-» X 6 + + 3 II • 3 cu t-. 3 > O JS o © > II . cu 6.0 Study IV 199 per formed w i th C u 2 + . Th is effect was significant (p<0.05) for Glu-Lys MRP mix ture 3 and Fru-Lys MRP mixture 11, conducted using a lower concentrat ion o f C u 2 + . W h e n the concentrat ion o f C u 2 + was increased up to 50 u M , an increase in cytotoxic i ty was noted. A t this concentrat ion o f C u 2 + , direct addi t ion o f M R P mixtures w i th C u 2 + neither enhanced nor mit igated the toxic i ty , but the addi t ion o f M R P mixtures preincubated wi th C u 2 + at r o o m temperature for 30 minutes enhanced cytotox ic i ty and decreased the co lony forming eff iciency o f f ibroblast cel ls. II. The presence o f C u 2 + a n d 0 . 0 1 % (w/v) M R P mixtures The results o f the study conducted wi th 0 . 0 1 % (w/v) o f M R P together w i th C u 2 + is g iven in F i g . 6.3b. D i rec t appl icat ion o f both Glu-Lys M R P mixtures, and Fru-Lys MRP mixture 11, at the above concentrat ion together w i th C u 2 + , effectively lowered the cyto tox ic i ty caused by 0.1 and 10 u M C u 2 + concentrations. Howeve r , addit ion o f preincubated mixtures o f Fru-Lys MRP mixtures w i th 50 p M C u 2 + , significantly (p<0.05) lowered the co lon iza t ion eff ic iency o f cells compared to tox ic i ty caused by C u 2 + alone. Ano ther important observat ion made in this study at both high and l o w M R P mixture concentrat ions employed was the significant (p<0.05) increase in the co lon iza t ion eff ic iency o f the f ibroblast cells both in the presence o f C u 2 + at 0.1 and 10 u,M levels and in the presence o f M R P mixtures. B. Presence of Fe3+ and MRP mixtures I. M o d u l a t i o n o f F e 3 + induced cytotoxic i ty by 0 . 0 0 1 % (w/v) M R P mixtures The results o f the cytotoxic i ty studies conducted w i th F e 3 + ions and 0 . 0 0 1 % (w/v) M R P mixtures are presented in F i g . 6.4a. App l ica t ion o f both 0.1 and 10 u M F e 3 + to cells alone increased co lon izat ion eff iciency o f the cells when compared to M R P mixtures appl ied alone. Therefore, even though direct appl icat ion as wel l as the appl icat ion o f preincubated samples o f 6.0 Study IV 200 o 2 ^ + + o o o C CU E 03 CU U H 3 O <L> u. 3 -*-» X e + + 3 • 3 o <0 + <u u. 3 •*-» X > (%) A D u a p i j p U O I ; B Z I U O I O 3 cd vo bbM3 73 J K E O > 0 ^ — i 43 O +S I 6.0 Study IV 201 « '—F" o 6K o o o IT) ca cu u H © IT) O ro rH J 0 i-H © o o o o o 00 o o o ( % ) fompijp u o p B z i u o i o 3 CL) CL) UH . C L ) VI 1 <n . o C D C D N o CO cd <3 o O o o 'S CO cd C D II co CD „ • - -2 3 & E * > O 3 CD U H P H D O CD U H D .*-» X e U H II • C D U H II CD U H 3 > ° ; P H O J= Q .-s o £ » V 6.0 StudyIV 202 M R P mixtures w i th F e 3 + to fibroblast cells resulted in signif icantly (p<0.05) higher cytotoxic i ty , w i th respect ive to the cytotoxic i ty resulted from F e 3 + alone, the presence o f 50 u M F e 3 + only p roduced a significant increase in cel l cytotoxic i ty relative to the tox ic i ty caused by M R P mixtures alone. H o w e v e r , in all the cases, appl icat ion o f preincubated samples o f Fru-Lys MRP mixtures w i th F e 3 + to cells resulted in greater cytotoxic i ty when compared to cytotox ic i ty induced by Glu-Lys MRP mixtures. II. The presence o f F e 3 + ions together wi th 0 . 0 1 % M R P mixtures T h e results o f the study conducted wi th F e 3 + together w i th 0 . 0 1 % M R P mixtures are presented in F i g . 6.4b. A s the results demonstrate, all four M R P mixtures signif icantly (p<0.05) increased cytotox ic i ty dur ing direct appl icat ion whi le appl icat ion o f preincubated sample combinat ions o f both Glu-Lys and Fru-Lys MRP mixtures us ing both 0.1 and 10 u M concentrat ions o f F e 3 + , decreased the cytotoxic i ty to a level lower than that caused by direct appl icat ion o f M R P mixtures. The appl icat ion o f a pr ior incubat ion step did not completely enhance the co lony forming efficiency o f cells to a similar level observed w i th F e 3 + alone especial ly w i th Fru-Lys MRP mixtures. In the presence o f 50 u M F e 3 + , the co lony forming eff iciency o f cel ls increased only wi th Glu-Lys MRP mixtures when appl ied after preincubat ing w i th F e 3 + ions. C. Presence of Fe2+ and MRP mixtures I. M o d u l a t i o n o f F e 2 + induced cytotoxic i ty by 0 . 0 0 1 % (w/v) M R P mixtures M o d u l a t i o n o f F e 2 + i n d u c e d cytotoxic i ty by 0 . 0 0 1 % (w/v) M R P mixtures is shown in F i g . 6.5a. Glu-Lys MRP mixtures at this concentrat ion signif icantly (p<0.05) decreased both the 0.1 and 10 u M F e 2 + induced cytotoxic i ty, irrespective o f a pr ior incubat ion step. In contrast, the appl icat ion o f Fru-Lys MRP mixtures d id not produce significant improvement in co lony forming 6.0 Study IV 203 * i iron Hi TtTnTnm i un 111 i l l M1 M111 o O r-A to O o o IT/ ta o o fl 0> H (%) Abiraptya normzrao^ CO CD ,SP"CD 3 U H 3 O J3 3 •4—" X £ + + CD U H CD U H CD «_ 3 Tx > o o" II P H BD -5 6.0 Study IV 204 in limn mi miiiHiiinnnTT iniiiiiiiiiiiiiiiiiiii iiiiiiiiiiiiiii iiiiiiiiiiirnT o o fa o eu E CQ CU u © H V3 2 « o 6 at* o c 4) CD c <D 'o ctt CD cd CD .CD m VI cd CO + •2 H CU CU 1° ^ cd O C M O o o © NO © © cd in E £ > O ^ EL B3 6.0 StudyIV 205 abil i ty o f cel ls in the presence o f 0.1 and 10 u M F e 2 + , both dur ing direct appl icat ion as we l l as appl icat ion after a pr ior preincubation step. In addit ion, all M R P mixtures except Glu-Lys MRP mixture 3 constantly lowered the co lony forming eff iciency o f the f ibroblast cel ls in the presence o f 50 p M F e 2 + , even after a pr ior incubation step. II. In the presence o f F e 2 + and 0 . 0 1 % M R P mixtures Co lon i za t i on eff iciency o f f ibroblast cells after direct addi t ion o f a higher concentrat ion o f M R P mixtures (0 .01%, w/v ) , together w i th F e 2 + is i l lustrated in F i g . 6.5b. The co lony forming abil i ty o f cel ls in the presence o f both Fru-Lys and Glu-Lys M R P mixtures together w i th F e 2 + enhanced the cytotoxic i ty dur ing direct appl icat ion, even though the appl icat ion o f preincubated samples o f M R P mixtures w i th F e 2 + significantly decreased the cytotoxic i ty . 6.7 D I S C U S S I O N 6.7.1 M R P I n d u c e d C y t o t o x i c i t y A l l M R P mixtures used in the present study exhibited dose dependent cytotox ic i ty in mouse embryonic f ibroblast cel l culture system thereby support ing the f indings w i th D N A in previous experiments o f this thesis. The induct ion o f cy to tox ic and clastogenic activit ies by r ibose- lysine M R P mixtures was also reported by Vagnare l l i et al. (1991). M R P complexes der ived by react ing r ibose w i th different amino acids were reported to possess different mutagenic activit ies in the A m e s test toward Salmonella typhimurium T A 100 strain ( C u z z o n i et al, 1988; 1989). Strain specif ic i ty o f some simple browning model systems in A m e s test (Shinohara et al, 1980) indicated that b rown ing compounds have a different chemical structure in compar ison to heterocycl ic amines. 6.0 StudyIV 206 HllNlllllllllllllllllll Hiiiiiiiiniiiiiiiiiiiiiiiiiiiiiii * HIIIIIIII IIIIIIUUUUU r -* M B * r -Niiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiini * h-en * I-o to o o o V 3 o o o i-l to S3 cu £ •*-» a cu t« H 3 o o 2 o o o o o o o o o Tf f N © OO V© <N (o/c) X D u a p i i j a U O I ; K Z I U O | 0 3 cu o 2 S J3 3. II W « t - J3 O U H 3 .-a i—i ^ " - P C ^ <n ° • S o X CO i n C vo .2 U H U H 0, ^ - « £ H3 O U H £ O * || || 8 11 E 3 «o ° - i DD H 13 J & > o 6.0 StudyIV 207 6.7.2 Cytotoxicity in the Presence of Metal Ions and MRP Mixtures The presence o f C u 2 + together w i th M R P mixtures enhanced co lon iza t ion abil i ty o f cells in this study regardless o f the preincubation step. Hypothet ical ly , M R P mixtures should increase cyto tox ic i ty o f C u 2 + after a preincubation step since reducing agents are expected to convert non tox ic C u 2 + to tox ic C u + (Study III). The lack o f such results in this study, in part icular w i th Glu-Lys MRP mixtures, indicates the possible role o f M R P mixtures to act as antioxidant metal chelators. Al ternat ively, the increased toxic i ty observed after direct appl icat ion o f F e 3 + and M R P mixtures to cel ls, and the decreased toxic i ty observed by apply ing the same compounds after a preincubat ion step, at both concentrat ion levels o f M R P mixtures tested herein, cou ld be attributed to the characterist ic reducing power and relatively lower chelat ing act ivi ty, o f these M R P mixtures, toward F e 3 + when compared to C u 2 + ions. In the case w i th F e 2 + , bo th Glu-Lys and Fru-Lys MRP mixtures at 0 . 0 0 1 % (w/v) exhibited signif icantly higher co lony fo rming activity dur ing direct appl icat ion, even though F e 2 + was a strong cytotox ic compound . Th is observat ion further proves that M R P mixtures have the ability to chelate F e 2 + ions at that concentrat ion. Al ternat ive ly , increased co lony forming efficiency observed after a preincubat ion step, in part cou ld be attributed to the possible oxidat ion o f F e 2 + to F e 3 + ions by autooxidat ion reactions occur r ing dur ing preincubat ion. Overa l l , the cel l culture experiments conducted in this study w i th M R P mixtures showed a l ower degree o f tox ic i ty compared to D N A systems used in earlier experiments. Th is in part may be a result o f cel lular enzymes that deactivate the toxic i ty o f M R P mixtures (K i t ts et al, 1993a; P o w r i e et al, 1981) wi th in the cells or that the cells were not permeable to M R P mixtures at that molecu lar weight range. 6.0 StudyIV 208 6.8 C O N C L U S I O N A cel l culture system comprised o f C 3 H 1 0 T 1 / 2 mouse embryo f ibroblast cells were employed in this study in order to determine the modulat ion o f metal induced cytotox ic i ty by mode l MPvP mixtures. A n experimental protocol similar to that used in the D N A study was employed w i th the except ion that M R P mixtures were not preincubated in an atmosphere o f argon. Resu l ts showed that all four Glu-Lys and Fru-Lys MRP mixture types at both 0 . 0 0 1 % and 0 . 0 1 % (w/v) concentrat ions, improved colonizat ion abil ity o f the cells regardless o f both the C u 2 + concentrat ion used, and existence o f a pr ior incubation step. W h i l e the appl icat ion o f F e 3 + together w i th 0 . 0 0 1 % or 0 . 0 1 % (w/v) M R P mixtures whi le addit ively enhanced cytotox ic i ty , the direct appl icat ion o f F e 2 + d id not induce this effect. The results also indicated that the tox ic i ty caused by both F e 3 + and F e 2 + ions could be eliminated completely by in t roduc ing a preincubat ion step conducted at r o o m temperature. A m o n g these observat ions, the increase in co lon izat ion eff ic iency o f M R P mixtures in the presence o f F e 3 + after preincubat ion, are l ike ly due to the chelat ing act iv i ty o f M R P mixtures. This is concluded since preincubat ion o f F e 3 + w i th a reducing agent l ike M R P should increase cytotoxic i ty as a result o f reducing F e 3 + to tox ic F e 2 + ions. Al ternat ive ly , the increase in co lonizat ion efficiency o f cells after preincubat ing w i th F e 2 + can also be attributed to the autoxidat ion o f F e 2 + to F e 3 + ions at r o o m temperature. Thus , both the chelat ion and reducing activit ies o f M R P have again been shown to be important in determining its abi l i ty to modulate metal catalyzed cytotoxici ty. F o r example, M R P mixtures w i th strong chelat ion act iv i ty and less reducing power should effectively improve the co lon iza t ion eff iciency o f cel ls by retarding the metal induced toxici ty. In this regard, Glu-Lys MRP mixtures are more effective b ioact ive compounds when compared to Fru-Lys MRP mixtures. M o r e o v e r , results 6.0 StudyIV zuy using this t issue culture technique suggest that the M R P compounds w i th genotox ic potential may not consistantly exhibit cy to tox ic effects under certain circumstances. 7.0 Study V 210 7.0 Study V Modulation of Metal Induced DNA Nicking and Cytotoxicity by Coffee Maillard Reaction Products 7.1 I N T R O D U C T I O N Coffee, a popular beverage around the wor ld, is made by drying harvested coffee beans which are then roasted, milled, and finally extracted with hot water. Coffee is available to the consumer in both brewed or instant form. Estimates o f coffee consumption in the Uni ted States have indicated that an average person consumes 1.9 cups o f coffee per day, o f which 1.33 cups are f rom ground roasted coffee and 0.56 cups are derived from instant coffee ( ICO, 1982). Dur ing roasting, coffee beans are exposed to very high temperatures (> 3 0 0 0 C ) to obtain the desirable colour, f lavour, and aroma o f the final coffee brew. Careful control o f the roasting step is a primary concern in coffee processing since amino acids and proteins present in the green coffee beans are subjected to non-enzymatic browning reactions involving available reducing sugars. Therefore, coffee wi th b rown pigments is considered to be a food system containing naturally derived food M R P mixtures. Dur ing the past decade, the effect o f coffee on nutritional factors has been studied extensively since iron deficiency anaemia is often prevalent in areas where coffee is commonly consumed. A n inhibitory effect o f coffee on non-heme iron absorption in human subjects has been reported by M o r c k et al. (1983). Ev idence for the affinity o f coffee pigments to bind copper and i ron in vitro was demonstrated by H o m m a et al. (1986) and in vivo by Gregor and Emery (1987). H o m m a et al. (1990) reported a dissociation constant value o f 3.4 x 10"5 ( p M ) for the zinc chelating activity o f an instant coffee component having a molecular weight o f 3,000 daltons. Asakura et al. (1990) showed that the 7.0 Study V 211 l igands present in coffee which bind zinc are acidic in nature and have a molecular size o f less than 5,000 daltons. Coffee has also been extensively evaluated for its possible genotoxicity. In vitro bacterial and mammalian cell culture tests have established that coffee can exert a weak mutagenic effect (Friedrich et al, 1985; Aesbacher and Wurzner, 1980) and can induce chromosomal damage to cultured human lymphocytes (Aesbacher et al, 1985). However , the genotoxicity o f coffee observed wi th in vitro test systems has been shown to be either lowered or abolished by the addition o f mammalian liver enzymes (Aesbacher et al, 1985; Nagao et al, 1979). In vivo experiments performed wi th rodents have also shown no significant increase in the incidence o f micronuclei or sister chromatid exchanges fol lowing the oral administration o f coffee (Shimizu and Yano , 1987). It therefore appears that coffee alone may not be genotoxic to organisms that have the enzyme systems needed for detoxif ication o f xenobiotics. Several reports on the modulation o f chemical carcinogenesis and mutagenesis by coffee have also been reported (Stich et al, 1982; Wattenberg and L a m , 1984). Coffee has also been referred to as a chemopreventative beverage due to its bioactive neutralizing effect in Drosophila melanogaster on known environmental mutagens (Abraham and Graf, 1996). Ant igenotoxic effects o f coffee has also been shown in mice exposed to similar mutagens (Abraham ,1996). Since coffee constituents chelate metal ions, the mitigation o f the Fenton reaction by chelation o f metal ions could be involved in the bioactivity against potential mutagenesis. The previous four chapters (Study I, II HI, and IV ) in this thesis were focused on studying the effect o f model M R P mixtures on D N A nicking and cell colonization efficiency in the presence o f several metal ions. The objective o f the present study was to determine the behaviour o f a food derived M R P mixture (i.e. coffee M R P pigments extracted from three coffee preparation processes) in modulating D N A nicking and cytotoxicity. 7.0 Study V 212 7.2 HYPOTHESIS F o o d derived M R P mixtures behave similarly to model M R P mixtures in exhibiting antioxidant or prooxidant activity. The expression o f any such prooxidant activity may lead to genotoxic and cytotoxic activities. 7.3 OBJECTIVES • T o evaluate the C u 2 + chelation activity, reducing activity, and elementary composit ion o f brewed, boi led, and instant coffee M R P s in characterizing anti-/prooxidant activity. • T o determine the potential D N A nicking effect o f three coffee M R P types in the presence o f C u 2 + , F e 2 + , and F e 3 + ions during direct application or application after preincubating the coffee M R P s and metal ions under atmospheric oxygen, and in argon, at room temperature. • T o assess the colony forming efficiency o f C 3 H 1 0 T 1 / 2 mouse embryo fibroblast cells from three coffee M R P types and C u 2 + , F e 2 + , and F e 3 + ions during direct application to cells or after preincubating the coffee M R P compounds with metal ions at room temperature under atmospheric pressure. 7.0 Study V 213 7.4 MATERIALS Nescafe® regular roasted and instant coffee types were purchased from Canada Safeway L td . (Vancouver, B . C ) . Dialysis tubing and Whatman # 4 filter papers were purchased from Spectrum Scientif ic Company (Houston, T X ) and Fisher Scientific C o . (Fair L a w n , N J ) , respectively. A l l other materials used in this study are described elsewhere in this thesis. 7.5 METHODS 7.5.1 Isolation of Coffee MRPs Three coffee M R P types referred to as brewed (Br) , boiled (Bo) , and instant (I) were extracted and used throughout the experiments. Extractions were conducted three times. B r e w e d coffee M R P s were prepared using a home coffee maker (Phillips Cafe El i te, Hol land) set at medium brew, by adding 20 g o f roasted ground coffee and 250 m L o f boil ing water. The filtrate was collected and centrifuged at 750 x g (g = gravitational force) for 45 minutes at room temperature using a I E C Centra - 7 R (International Equipment C o . , U S A ) centrifuge and dialysed (molecular weight cut o f f 3.5 k D ) against several volumes o f deionized distilled water. The non-dialysable fraction was collected and lyophil ized. F o r the preparation o f boiled coffee M R P s , 20 g o f the same roasted coffee was boi led in 250 m L o f deionized distilled water for 1 hour in a covered beaker using a laboratory hot plate (Corning), set at posit ion 8. The resulting coffee brew was filtered through Whatman # 4 filter paper and the filtrate was centrifuged (750 x g for 45 minutes), dialysed, and lyophil ized as described above. Instant coffee M R P s were extracted according to the same procedure, using 20 g o f instant coffee powder and 250 m L o f water. 7.5.2 Gel Filtration and Elemental Analysis of Coffee MRPs These procedures are described in Study IU (5.5.2) and Study I (3.5.5), respectively. 7.0 Study V 214 7.5.3 Copper Binding Activity of Coffee MRPs This procedure is described in Study in (5.5.4). 7.5.4 DNA Nicking Studies and Tissue Culture Experiments These procedures are described in Study IV and V, respectively. 7.6 RESULTS 7.6.1 PART I - Chemical Characteristics of Coffee MRPs Gel filtration studies of coffee constituents indicated that coffee MRP mixtures were composed of a mixture of compounds with a variety of molecular weights. Coffee MRPs having a molecular weight of 5 - 6 kD [yield = 22%, (w/v)] were collected by gel filtration chromatography and used throughout the experiments. This specific molecular weight range was chosen on the basis that they predominated the coffee MRP pigments present in coffee brew and also corresponded to the molecular weight of MRP mixtures collected from previous model MRP mixtures. The empirical formulae calculated for the 5 - 6 kD molecular weight coffee MRP mixtures from elementary analysis data are given in Table 7.1. All three sources of coffee MRP mixtures in general possessed a greater percentage of carbon and a relatively smaller percentage of nitrogen compared to model MRPs. However, among the three coffee MRP mixture types, there were no significant differences in C : N or C:0 ratios. The dissociation constants (Kd) calculated for three coffee MRP mixtures are given in Table 7.2. Brewed coffee MRP pigments exhibited the highest Kd value and the highest number of association sites (n) although there were no significant differences between the three coffee MRP types. The reducing activity of the three coffee MRP mixtures, exhibited significantly (p<0.05) less reducing activity when compared to ascorbic acid (Fig. 7.1). Instant coffee MRP mixtures exhibited 7.0 Study V 2 1 5 Table 7.1: Elementary composi t ion o f coffee M R P pigments. Sample % C % H % N % O 1 Empi r ica l fo rmulae 2 C : 0 3 C : N 4 B r e w e d (Br ) 42.11 6.52 2.02 49.35 C 24.30 H 45.18 NO 2 1 . 3 7 1:1.137 1 :0 .042 B o i l e d ( B o ) 44.93 6.66 2.22 46.19 C 23.61H42.00 N O ,8.20 1:1.297 1 :0 .050 Instant(I) 43.33 6.38 1.89 48.40 C 26.74H 47.26 N O 22.40 1:1.194 1:1.037 1 = Calcu la ted f rom C , H , and O percentages 2 = Calcu la ted as N = 1 3 = C a r b o n to oxygen ratio 4 = Ca rbon to ni t rogen ratio 7.0 Study V 216 Tab le 7.2: C o p p e r chelating activi ty o f brewed (Br ) , boi led ( B o ) , and instant (I) coffee M R P pigments. M R P B o u n d C u 2 + K d n3 Sample ( u M C u 2 + / mg cof fee) 1 ( u M ) 2 B r e w e d 31.50 4.3 x 10" 5 20.12 Bo i l ed 26.85 3.8 x 10" 5 18.48 Instant 26.01 3.6 x 10" 5 18.31 1 = measured by tetra methyl murexide method 2 = dissociat ion constants 3 = number o f associat ion sites 7.0 Study V 217 CN 3 3 u e q j n s q y 7.0 Study V 218 the highest reducing activity among the three sources o f coffee M R P mixtures tested herein, although this difference was not significant (p>0.05). 7.6.2 PART H - DNA Nicking Assays Conducted with Coffee MRPs 7.6.2.1 Dose Response DNA Nicking Effect of Coffee MRPs and Fe2+ Ions D o s e dependent D N A nicking activity o f F e 2 + ions were similar to that reported in F ig . 5.2 in Study JJI. The dose response behaviour o f three coffee M R P s examined at four different p H values are shown in Figs. 7.2a and b, respectively. A s the results demonstrate, coffee M R P s up to a concentration o f 0 .01% (w/v), gradually increased the degree o f supercoiled D N A breakage and above that concentration the degree o f breakage did not change. The p H o f the medium did not seem to significantly (p<0.05) affect the D N A nicking. 7.6.2.2 DNA Nicking Effect of Coffee MRPs in the Presence of Fe 2 + The results o f the D N A study conducted with F e 2 + , incoporating a lower concentration o f three coffee M R P s (i.e. brewed, boiled, and instant) at four different p H values are presented in Figs. 7.3 a and b. Ferrous ion alone caused a remarkably high level o f D N A nicking at both 7.5 and 4.0 p H values compared to coffee M R P s alone. The application o f coffee M R P s to D N A together wi th F e 2 + directly did not exhibit significant protective effect on the F e 2 + caused D N A breakage at all four p H values. W h e n coffee M R P s were applied to D N A after preincubating with F e 2 + under atmospheric oxygen, a significant (p<0.05) protection was observed only with brewed coffee M R P at p H 7.5. Only M R P s derived f rom boiled coffee produced a significantly (p<0.05) l ow D N A breakage relative to F e 2 + alone, when p H was lowered to 4.0. A different response in lowering the D N A breakage caused by F e 2 + was observed when coffee M R P s were preincubated with F e 2 + under an atmosphere o f argon, compared to atmospheric oxygen. F o r example, both boiled and instant coffee M R P sources failed to decrease D N A breakage after argon pre-treatment wi th F e 2 + at p H 7.5. Interestingly, the boiled coffee M R P s 7.0 Study V 2 1 9 100 80 4 60 40 S 03 £ cu PS < <U 0 U u 1 100 c cu u cu 0 1E-04 0.001 0.01 0.1 Concentration (%, w/v) 80 60 4 40 0 1E-04 0.001 0.01 0.1 Concentration (%, w/v) F i g . 7 .2a : , D o s e response o f coffee M R P concentrat ions on D N A n ick ing activity. ( A ) = p H 7.5, (B) = p H 4.0. Va lues represent mean ± S . D . (n = 3). S . D < 1% not shown. • = B r e w e d , A = B o i l e d , • = Instant. 7.0 Study V 220 Concentration (%, w/v) F i g . 7.2b: D o s e response o f coffee M R P concentrat ions on D N A n ick ing activity. ( A ) = p H 3.2, (B) = p H 2.6. Va lues represent mean + S . D . (n = 3). S . D < 1% not shown. • = B r e w e d , A = B o i l e d , • = Instant. 7.0 Study V Br Bo I Type of coffee F i g . 7.3a: Ef fect o f coffee M R P (0 .001%, w /v ) on F e 2 + catalyzed D N A n ick ing activity. ( A ) = p H 7.5, (B) = p H 4.0. B r = B r e w e d coffee; B o = B o i l e d coffee; I = Instant coffee. Va lues represent mean ± S . D . *= Signif icantly different (p<0.05) w i th respect to n ick ing caused by F e 2 + . 10 u M concentrat ion o f F e 2 + was used at p H 7.5; 5 u M concentrat ion o f F e 2 + was used at p H 4.0. • = C o n t r o l , O = 0 . 0 0 1 % (w/v) coffee M R P , • = F e 2 + , EQ = F e 2 + + Co f fee M R P (wi thout pre-incubation), M = F e 2 + + Cof fee M R P (p re incuba ted in atmospher ic oxygen), H = F e 2 + + Cof fee M R P (pre-incubated in argon). 7.0 Study V 222 Fig. 7.3b: Effect of coffee MRP (0.001%, w/v) on Fe 2 + a catalyzed DNA nicking activity. (A) = pH3.2, (B) = pH2.6. Br = Brewed coffee; Bo = Boiled coffee; I = Instant coffee. Values represent mean ± S.D. *= Significantly different (p<0.05) with respect to nicking caused by Fe2+. 5 pM concentration of Fe2+ was used at both pH values. • = Control, ^ =0.001% (w/v) coffee MRP, • =Fe2+, Dfl =Fe2+ + Coffee MRP (without pre-incubation), S = Fe2 + + Coffee MRP (pre-incubated in atmospheric oxygen), H = Fe 2 + + Coffee MRP (pre-incubated in argon). ( 7.0 Study V 223 retained the protective effect under both oxygen and argon pre-incubations, but only at a p H o f 7.5. A similar result was also observed for boiled coffee M R P at p H 4.0 under preincubations in argon atmospheres. These results were specific to low concentrations o f coffee M R P s (0 .001%, w/v) . When coffee M R P concentrations were increased up to 0 .01% (w/v), all protective properties o f coffee M R P s relative to F e 2 + were lost (Figs. 7.4 a and b), regardless o f p H or preincubation conditions conducted in atmospheres o f argon or oxygen. 7.6.3 PART m - Colony Forming Efficiency of C3H10T1/2 Mouse Embryo Fibroblast Cells in the Presence of Polyvalent Metal Ions and Coffee MRPs The dose response effect o f three coffee M R P sources on fibroblast cells is presented in F ig . 7.5. A decrease in colonization efficiency was only observed when the concentration o f coffee M R P s applied reached 0 . 1 % (w/v). A t this specific concentration level, the colony forming ability o f cells was decreased by 80%. 7.6.3.1 Colonization Efficiency of Cells in the Presence of Fe2+ and Coffee MRPs Colonizat ion efficiency o f fibroblast cells in the presence o f three F e 2 + ion concentrations (0.1, 10, and 50 p M ) and two coffee M R P concentrations (0.001, 0 .01%, w/v) are presented in Figs. 7.6 a and b, respectively. A l l three coffee M R P sources when applied directly to cells together wi th F e 2 + , significantly increased the colonization efficiency o f cells, at both 0.1 and 10 p M F e 2 + compared to F e 2 + alone. Increasing F e 2 + concentrations up to 50 p M , significantly (p<0.05) lowered the colonization efficiency o f the fibroblast cells regardless o f the coffee M R P source used. A l l three coffee M R P s , when applied to cells fol lowing prior incubation with F e 2 + , always enhanced the colonizat ion efficiency o f cells when compared to the control wi th F e 2 + alone, regardless o f the F e 2 + concentration used. Similar results were observed when cells were exposed to a higher concentration o f coffee M R P (0 .01%, w/v) together wi th Fe 2 + di rect ly . A n exception to this was for instant coffee wi th 50 p M 7.0 Study V Type of coffee F i g . 7.4a: Ef fect o f coffee M R P (0 .01%, w/v ) on F e 2 + catalyzed D N A n ick ing act ivi ty. ( A ) = p H 7 . 5 , (B) = p H 4 . 0 . B r = B r e w e d coffee; B o = B o i l e d coffee; I = Instant coffee. Va lues represent mean ± S . D . *= Signif icant ly different (p<0.05) wi th respect to n ick ing caused by F e 2 + . 10 u M concentrat ion o f F e 2 + was used at p H 7.5; 5 u M concentrat ion o f F e 2 + was used at p H 4.0. • = Con t ro l , • = 0 . 0 1 % (w/v) coffee M R P , • = F e 2 + , DO = F e 2 + + Co f fee M R P (without pre-incubation), HI = F e 2 + + Cof fee M R P (pre-incubated in atmospher ic oxygen), H = F e 2 + + Cof fee M R P (pre-incubated in argon). 7.0 Study V 225 Br Bo I Type of coffee F i g . 7.4b: Ef fect o f coffee M R P (0 .01%, w/v) on F e 2 + a catalyzed D N A n ick ing act iv i ty. ( A ) = p H 3 . 2 , (B) = p H 2 . 6 . B r = B r e w e d coffee; B o = B o i l e d coffee; I = Instant coffee. Va lues represent mean ± S . D . *= Signif icant ly different (p<0.05) wi th respect to n ick ing caused by F e 2 + . 5 p M concentrat ion o f F e 2 + was used at both p H values. • = C o n t r o l , m = 0 . 0 1 % (w/v) coffee M R P ; • = F e 2 + , DD = F e 2 + + Co f f ee M R P (wi thout pre-incubation), S = F e 2 + + Cof fee M R P (pre- incubated in atmospher ic oxygen) , H = F e 2 + + Cof fee M R P (pre-incubated in argon). 7.0 Study V 226 (o / o) ADirapij ja U O I ; B Z I U O I O 3 7.0 Study V 227 w „ ft, t — H H H H 8 J 0 + I 3J0 + j a a=io + J a o o CN o CO o C M O OO (%) Aouapuia uoueziuoioo .8 it + flN OQ + 1 + —*-CM ~ 2' - * .8 •5 vp _ 0 s -^C; — s o ^ £ o 2i cs S ^ o £ © 2- <=> v » 2 w vo U H + + + «H l-C O 03 03 CQ CQ H H H H nn 7.0 Study V 228 + I o t-n +1 <L> ° 8 3-8 0 s -CQ 03 ~5 < O + + O • 2? « ffl PQ CQ M w II (%) Aousnyja U O I J B Z I U O I O O 7.0 Study V 229 of Fe 2 +. At this specific concentration of coffee MRP, instant coffee MRP did not exhibit a significant increase in cell colonization efficiency compared to Fe 2 + alone even after a preincubation step. Another interesting observation made herein, was the increase in colonization efficiency of cells when cells were exposed to coffee MRPs and Fe 2 + together, when compared to each MRP or Fe 3 + individually. 7.6.3.2 Colonization Efficiency of Cells in the Presence of Fe34" and Coffee MRPs Colonization efficiency of mouse embryo fibroblast cells in the presence of 0.001% (w/v) and 0.01% (w/v) coffee MRPs together with three Fe 3 + concentrations (0.1, 10, and 50 uM) are presented in Figs. 7.7 a and b, respectively. The presence of Fe 3 + alone in the cell culture media at 0.1 and 10 p M caused colonization efficiencies similar to the presence of coffee MRPs alone. However, an increase in Fe 3 + concentration up to 50 pM, dramatically lowered the colony forming efficiency of cells compared to coffee MRPs alone. Application of 0.001% (w/v) coffee MRPs together with Fe 3 + both at 0.1 and 10 p M Fe 3 + had no significant effect (p>0.05) on colonization efficiency of cells regardless of the preincubation step. However, an additive decrease in colony forming efficiency was always noted when all three coffee MRP sources (0.001%, w/v) were present together with the highest concentration of Fe 3 + (50 pM). At this specific Fe3+concentration, colony forming efficiency of cells was decreased regardless of the prior incubation step. Similar observations were made when coffee MRP samples were tested at a fixed concentration of 0.01% (w/v), except for boiled coffee MRP together with 10 p M Fe 3 + during direct application and brewed coffee MRP with 0.1 p M Fe 3 + after preincubation. These two particular treatments significantly enhanced the colony forming efficiency of cells compared to Fe 2 + alone. 7.0 Study V 230 •a T3 •3 m + + _ £ £ "« * £ - . « -+ + + + »J0S + J f l M O T + -»H »J0 + J f l o ON o vo o (o / o) Xonapijja uopuzraoif^  +1 a «* e 5? iC • V—' ° o + + t - i W 03 03 1-o >^ qa u C l p ~ | o o o ° « v ? o N HH II CD 7.0 Study V 231 I—nm 9AO + <>a w o + -«a o © © © "> CD on CD CD w • I—, \ 0 I—t PQ + + i 8«§ i " O o _ QX Q X <—I o o + + CQ CQ - i « ( o / o ) X D u a p i j j a uopBzino[03 7.0 Study V 232 7.6.3.3 Colonization Efficiency of Fibroblast Cells in the Presence of Cu 2 + and Coffee MRPs Results of the study conducted with Cu 2 + and coffee MRPs fixed at a concentration of 0.001% (w/v) is illustrated in Fig.7.8a. At all three test concentrations of Cu 2 + , coffee MRP mixtures significantly (p<0.05) enhanced the colony forming efficiency of fibroblast cells. This efficiency was further enhanced by applying coffee MRP mixtures preincubated at room temperature with Cu 2 + . In contrast to the findings observed with Fe 2 +, the employment of coffee MRP mixtures with 50 u M Cu 2 + did not induce cytotoxicity. Similar results were also observed for 0.01% (w/v) of coffee MRP together with Cu 2 + ions (Fig. 7.8b). 7.7 DISCUSSION The Maillard reaction is largely responsible for the roasted, toasted, or caramel like aromas, as well as the development of brown colour in protein and carbohydrate rich foods, following a thermal treatment (Nursten, 1986). Due to the inherent complexity of food systems, such as coffee, the majority of the work on M R has been accomplished in reference to simple model systems of amino acids and reducing sugars. Model systems, however, do not necessarily behave similarly to food derived systems. Therefore, it is important to study characteristics of both model and food MRP systems. 7.7.1 Chemical Characteristics of Fractionated Coffee MRPs All coffee MRPs tested in this study demonstrated an extremely strong attraction for copper ions. These measured copper chelating activities were similar to the previous reported values by Homma et al. (1986) although the pH of the present study (i.e., 7.5) exceeded that used by Homma and his co-workers. In this experiment higher number of carbon atoms for coffee MRP emperical formulae indicate the existance of C-20 to C-23 MRP polymers in the system. Despite the somewhat similar molecular weights of fractionated MRPs from both model and coffee browning starting 7.0 Study V 233 s •—•iminiiiii M1111111 II 1111111 n r r i r r •—iiimiiitiiiniiitiiiiiiiiiiiiniiiiiniiii i • • i n i u i rTTnnTnnH O O 00 -f-o (ll)nO0 + I (ii)noo+oa (n)noo+-«a x ° * -CD 5 1 CD o «t! . N C o CD Sj u u o o o CJ CJ " i § £ -° T 3 > x? > ^ "-- -s x= ox c j , c j .2 — x ° X ? 0 S©X 3 CD .-, II mi (%) Aouapuja uouezmoioo 7.0 Study V 234 (ii)«ooi+ja di)no ro+J8 (ii)noo+-«a X l i s v ? s: s? s 2 0 .8 + o (%) A D u a p i j p U O I ; B Z I U O | 0 3 at 7.0 Study V 235 material, the larger polymers characterizing coffee M R P wou ld be expected to possess a greater number o f copper binding sites and therefore to chelate metal ions more effectively. This wou ld explain the observation that coffee M R P mixtures were more effective chelating agents compared to model M R P mixtures. These findings therefore strongly suggest the possible existance o f important structural differences between food derived M R P s and model M R P mixtures which are derived by heating simple reducing sugars with amino acids. 7.7.2 DNA Nicking Patterns Observed with Coffee MRPs in the Presence of Polyvalent Metal Ions D N A nicking assays in this study were performed with the objective o f assessing the conditions for modulat ion o f metal catalyzed Fenton reaction by coffee M R P s . The basis for this study is the fact that oxidative damage to biological materials occurs through the metal driven Haber-Weiss reaction (Gra f et al, 1984) as given by the fol lowing scheme: 0 2 + F e 3 + • F e 2 + + 0 * 2 [1] 0*2 + H 2 0 2 + F e 2 + • OFT + O F T + 0 2 [2] H 2 0 2 + F e 2 + • F e 3 + + OFT + O F T [3] The Haber-Weiss cycle is initiated by numerous reducing substances, including ascorbic acid, a -tocopherol , and catechol, to name a few (Mahoney and Graf, 1986). In addition, chelating agents such as phytate and E D T A were shown in Study I V to form ligands in a manner such that both reactions [1] and [2], or [1] and [3] in the Haber-Weiss cycle are prohibited (Floyd, 1983; Mahoney and Graf, 1986). A s shown earlier, coffee M R P s strongly chelate metal ions and one important component for this metal binding activity is the melanoidin fraction present in coffee, irrespectively o f the method o f processing. Moreover , the results o f this study further showed that coffee M R P s possess a measurable amount o f reducing activity as wel l . Hence, studying the modulation o f Haber-Weiss cycle by coffee 7.0 Study V 236 M R P compounds that possess both metal chelating and reducing activity is relevant for evaluating the beneficialmarmful effects o f coffee M R P s on D N A and on intact cell systems. The general trend o f the results f rom the D N A study suggested that coffee M R P s do not possess significant positive effects in decreasing Fe 2 + - induced D N A nicking. However , the specific D N A protecting effects observed with brewed and boiled coffee M R P s during direct application, at specific p H values indicated that this protection was partly due to the chelating ability o f coffee M R P s . This possibility was confirmed by the preincubation studies conducted in argon, wh ich also exhibited a similar decrease in D N A nicking. A rgon , which is known to be an inert gas, does not stimulate metal catalyzed Harber-Weiss reactions since oxygen is a prerequisite for this reaction to occur. Therefore, a decrease in D N A nicking observed with prior incubation o f coffee M R P s wi th metal ions in argon can only be associated with the chelation activity o f metal ion by coffee M R P s . However , different degrees o f protection were observed with specific coffee M R P sources at certain p H values, which may be explained by the different solubilities or surface charge o f these specific M R P sources at different p H conditions. The net surface charge o f a compound is dependent on its iso-electric point. Therefore, depending on the p H o f the medium, the variable charge o f different coffee M R P compounds could be expected and therefore contribute to varying metal binding affinity o f the compound under different conditions. 7.7.3 In Vitro Colonization Efficiencies of Fibroblast Cells in the Presence of Polyvalent Metal Ions The overall activity o f coffee M R P s in modulating cytotoxicity, induced by copper and iron metal ions, seems to be high compared to their effectiveness in D N A test systems. A s discussed above, on the basis o f mechanism o f action, coffee M R P s can modulate metal induced cytotoxicity by acting as a chelating agent. O n the other hand, coffee M R P s may enhance the cytotoxicity due to its reducing 7.0 Study V 237 power which may drive the Fenton reaction. A s such, the margin that defines whether a certain coffee M R P compound is an antioxidant or a prooxidant depends totally on the balance between reducing and the chelating potential o f the particular coffee M R P compounds. This effect was clearly demonstrated in the cell culture experiments conducted with F e 3 + ions. A t a higher coffee M R P concentration, direct application o f F e 3 + to cells together with coffee M R P s produced an increase in colonizat ion efficiency o f cells whi le application after a preincubation, greatly lowered the colonizat ion efficiency o f cells compared to other results obtained with a lower concentration o f coffee M R P s . In this study, whi le the increase in colony forming ability o f cells in the presence o f higher concentrations o f coffee M R P during direct application can be attributed to the greater number o f binding sites present in coffee M R P s , the decrease in colony forming ability o f cells after a preincubation wi th coffee M R P is l ikely attributed to the increased reducing potential o f coffee M R P s at higher concentration levels. In addition, cell culture studies further demonstrated that coffee M R P s also have the ability to chelate F e 2 + effectively since direct application studies performed wi th coffee M R P s in the presence o f F e 2 + significantly decreased the cytotoxicity induced by F e 2 + ions. Moreover , the further increase in colony forming efficiency noted after preincubation o f F e 2 + wi th coffee M R P s suggests that either these metal bound M R P s have different solubilities or have different oxidation reduction potentials compared to non-metal bound coffee M R P s . Coffee brew M R P pigments decreased cytotoxicity induced by C u 2 + ions both during direct application and after a preincubation step, except at the 50 p M concentration. Hypothetical ly, coffee M R P mixtures should have exerted a decrease in the colony forming efficiency o f cells after preincubating wi th C u 2 + since reducing agents are expected to convert non-toxic C u 2 + to cytotoxic C u + (Kawakish i et al, 1990). However , the fact that this did not happen indicates that coffee M R P s act as a metal chelating antioxidant to a greater extent than reducing agents. These results further 7.0 Study V 238 demonstrate that when coffee M R P s become oversaturated with bound metal ions, a greater proport ion o f metal ions are free in solution and are available to attack D N A , thus inducing D N A stand scissions. The anti-genotoxic effects o f coffee have been observed against several potent genotoxins and carcinogens in bacterial assays as mentioned in the introduction section o f this study. R o s i n et al. (1982) reported that glucose-lysine Mai l lard compounds in the absence o f metal ions induced a gene conversion frequency o f 9.3 convertants/10 5 survivors, compared to 1.2 convertants/10 5 survivors in control samples without Mai l lard compounds. The presence o f F e 3 + suppressed the convertogenic activity o f Mai l la rd products completely while C u 2 + reduced it to a lesser extent. Since coffee is a more complex mixture o f compounds than model M R P mixtures, the precise mechanism(s) by wh ich coffee M R P pigments exerts anti-genotoxic effects are less definitive. There are indications that phenolic constituents present in coffee (e.g. chlorogenic acid, caffeic acid, ellagic acid, premelanoidins, etc.) can inhibit the genotoxic and carcinogenic activities o f these compounds (Aesbacher and Jaccaud, 1990). The present study further confirmed the above suggestions by demonstrating the ability o f coffee M R P s to mitigate metal ions. 7.8 CONCLUSION Results o f this chapter demonstrate for the first time that the modulat ion o f D N A strand scissions and cytotoxicity f rom commonly found coffee derived M R P mixtures is mediated by the characteristic balance between reducing power and metal chelating activity. This in turn has a primary influence on whether the compound has antioxidant or prooxidant potential. In general, coffee M R P compounds due to their high chelating activity mitigated both C u 2 + and F e 2 + induced cytotoxicity, during direct application or after a preincubation step. The direct application o f l ow concentrations o f coffee M R P mixtures with F e 3 + neither increased nor decreased cytotoxicity. 7.0 Study V 239 However , application o f coffee M R P mixtures with F e 3 + after a preincubation step slightly lowered the cytotoxicity. This cytotoxicity lowering effect o f coffee M R P s wi th F e 3 + was dependent on the concentration o f coffee M R P s used. Thus, the results suggested that the cytoxicity o f transition metals in the presence o f coffee M R P constituents is dose dependent and greatly influenced by both the chelating activity as wel l as characteristic reducing power o f the derived food M R P compounds. 240 8.0 General Conclusion N u m e r o u s studies in the literature report that the extent o f M a i l l a r d b rown ing react ion is largely dependent on the type o f reactants and react ion condi t ions used and that those MKT der ived compounds have the abil i ty to act as metal chelat ing and ant ioxidant compounds. H o w e v e r , very little attention has been given towards identi fying the inf luence o f react ion condi t ions in modulat ing the physico-chemical characterist ics o f these Ma i l l a rd reaction compounds and also their eff icacy in modulat ing metal ion dr iven Habe r -We i ss react ion under in vitro condi t ions. T h e f ive studies (Study I through V ) reported herein evaluated the in vitro metal chelat ing and ant ioxidant activit ies o f both model systems and coffee der ived M a i l l a r d react ion products, under a var iety o f condit ions in the presence or absence o f polyvalent metal ions. T h e init ial study invo lved the synthesis and characterization o f model M R P mixtures for their phys ico-chemical characterist ics. The results o f this study strongly indicated that react ion temperature, durat ion o f react ion, p H , water activity, as we l l as the type o f sugar, are all contr ibut ing factors in determining the y ie ld , elementary composi t ion as we l l as the phys ico-chemica l , cy to tox ic and D N A n ick ing characteristics o f model M R P mixtures. In general , al l M R P mixtures possessed detectable metal b inding and antioxidant characteristics, even though oxygen electrode studies conducted w i th copper ions, as a promoter o f l ip id ox idat ion react ions, indicated some ability to also exhibit prooxidant activit ies. Fract ionat ion o f crude M R P mixtures by chelation chromatography indicated the presence o f c o m m o n molecular weight metal chelat ing components hav ing molecular weights o f approximately 5.7 and 12.3 k D , wh i ch was conf i rmed by M A L D I -9.0 General Conclusion 241 M S . Th is f inding suggested the presence o f different structured compounds having similar molecu lar weights in M R P mixtures synthesized under vary ing condi t ions and possessing var ious metal chelat ing and antioxidant activit ies. In general, Glu-Lys M R P mixtures possessed stronger chelat ing and antioxidant activit ies and less D N A n ick ing effects compared to the Fru-Lys counterparts. N e x t three studies involved the investigation o f antioxidant act iv i ty o f synthesized M R P mixtures in mode l l ip id and non- l ip id systems in the presence o f metal ions. T h e l ip id model systems employed herein were a l inoleic acid emulsion and a prototype cook ie dough food system. The non- l ip id model systems were a bacteriophage P M 2 D N A and C 3 H 1 0 T 1 / 2 mouse embryo f ibroblast cel l culture system. C o o k i e dough and l inoleic acid emulsion experiments in Study II indicated that Fru-Lys M R P mixtures, regardless o f the presence o f metal ions, exhibited proox idant act iv i ty when appl ied to l ip id models at higher concentrations. In contrast, Glu-Lys M R P mixtures only exhibi ted antioxidant act ivi ty under similar experimental condit ions. Th is is mainly considered a result o f stronger chelat ing and weaker reducing activit ies o f the studied Glu-Lys M R P mixtures. B o t h M R P types, however , possessed relatively weak antioxidant activit ies compared to a -tocophero l . Y e t , these M R P mixtures were capable o f mit igat ing the metal induced prooxidant act iv i ty o f a - tocophero l . These f indings demonstrated the usefulness o f mode l M R P mixtures together w i th oc-tocopherol under certain experimental condit ions. Genotox ic i t y o f M R P mixtures when analyzed in the presence o f polyvalent metal ions us ing P M 2 phage D N A , indicated that even very minute concentrat ions o f F e 2 + was sufficient to enhance degree o f D N A nick ing, wi th respect to M R P mixtures alone. H o w e v e r , w i th respect to F e 2 + wh i le specif ic Glu-Lys M R P mixtures were noted to decrease D N A breakage, other specif ic 9.0 General Conclusion 242 Fru-Lys M R P mixtures actually enhanced the breakage relative to F e + alone. T h e addi t ion o f M R P mixtures after preincubation wi th F e 2 + under atmospheric oxygen, however , signif icantly lowered D N A n ick ing regardless o f the M R P type and concentrat ion o f metal ion used. Simi lar preincubat ions conducted in argon revealed that the protect ive effect o f M R P predominant ly arose as a result o f chelat ion o f metal ions by Glu-Lys M R P mixtures o r as a result o f ox id iz ing F e 2 + to F e 3 + by copper bound Fru-Lys M R P mixtures. The same M R P mixtures when applied to C 3 H / 1 0 T 1 / 2 mouse embryo f ibroblast cell culture systems directly, wi thout a pr ior incubation step, indicated no tox ic effects in the presence o f C u 2 + and F e 2 + ions. App l ica t ion o f preincubated M R P mixtures w i th F e 3 + , however , signif icantly enhanced the cytotoxic i ty compared to cytotoxic i ty caused by F e 3 + a lone, whereas the appl icat ion o f preincubated M R P mixtures w i th F e 2 + and C u 2 + signif icantly lowered cytotox ic i ty compared to F e 2 + and C u 2 + alone. Mo reove r , addi t ion o f higher concentrat ions o f M R P mixtures together w i th C u 2 + decreased the cytotoxic i ty both dur ing direct appl icat ion as we l l as appl icat ion after a pr ior incubation step. The f indings o f this study indicate that M R P mixtures mit igated the copper induced cytotoxic i ty more effectively than i ron induced cytotoxic i ty . T h e f inal study o f this thesis was dedicated towards invest igat ing the phys ico-chemica l , propert ies, as we l l as metal catalyzed D N A nick ing and cytotox ic propert ies o f coffee M R P mixtures. Non-d ia lysab le coffee M R P s extracted f rom three coffee b rew types; i) home brewed us ing a house-hold coffee maker (B r ) ; ii) boi led ( B o ) ; and ii i) instant (I) coffee b rew were used as the food der ived M R P mixtures in this regard. A l l coffee brew M R P s had remarkably high copper chelat ing and l o w reducing activit ies compared to model M R P mixtures. Therefore, in both D N A n ick ing and cy to tox ic studies, coffee M R P s exhibited less tox ic effects compared to mode l M R P 9.0 General Conclusion 243 mixtures. Speci f ical ly , in cytotoxic i ty studies, coffee M R P s together w i th the transi t ion metal ions studied, p romoted the cel l co lonizat ion efficiency o f fibroblast cells in the presence o f C u 2 + and F e 2 + ions. 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Fig. 10.1: Time temperature curves of Glu-Lys and Fru-Lys MRP synthesis experiment numbers 5 (A) and 11 (B). —•— Glu-Lys -o— Fru-Lys —a— Ambient temperature 

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