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UBC Theses and Dissertations

A study of the measurement and degradation of folic acid Ruddick, Jane Elizabeth 1978

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A STUDY OF THE  MEASUREMENT  AND DEGRADATION OF F O L I C A C I D  by  JANE E L I Z A B E T H RUDDICK B.Sc,Hons. U n i v e r s i t y M.Sc,  o f L o n d o n , 1972  University of British  Columbia,  1974  A THESIS SUBMITTED I N P A R T I A L FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE  STUDIES  Department o f Food S c i e n c e  We a c c e p t t h i s  t h e s i s as conforming  to the required  standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA A p r i l 1978 "cv)  J a n e E l i z a b e t h R u d d i c k , 1978  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of B r i t i s h 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 representatives.  It  is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department pf  ^\  FOOD SCIENCE  The University of B r i t i s h Columbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  MAY 31st 1978.  - ii  -  ABSTRACT  F o l i c a c i d i s p r e s e n t i n foods i n many d i f f e r e n t  forms.  C o n c e n t r a t i o n s o f N ^ - m e t h y l t e t r a h y d r o f o l i c a c i d (one form o f f o l a t e ) were determined  by a r a d i o a s s a y and t h e L a c t o b a c i l l u s c a s e i  b i o l o g i c a l assay methods.  micro-  The c o e f f i c i e n t o f d e t e r m i n a t i o n f o r samples  a n a l y z e d by b o t h methods was 0.86 b u t t h e r a d i o a s s a y method was found t o be f a s t e r , s i m p l e r and more a c c u r a t e than t h e m i c r o b i o l o g i c a l method. The m i l k f o l a t e b i n d e r and L. c a s e i  were found t o r e a c t d i f f e r e n t l y  t o p t e r o y l g l u t a m i c a c i d (another form o f f o l a t e ) compared t o N"*methyltetrahydrofolic acid.  The two methods o f assay were found t o be  s u i t a b l e o n l y f o r measuring t h e form o f f o l a t e used t o c o n s t r u c t t h e standard curve f o r t h e assay. used t o  I t was found t h a t n e i t h e r method c o u l d be  q u a n t i f y - f o l a t e l e v e l s i n foods a c c u r a t e l y .  Thus, a t b e s t ,  p r e s e n t l y a v a i l a b l e d a t a on food f o l a t e l e v e l s c a n o n l y be compared t o o t h e r v a l u e s o b t a i n e d by an i d e n t i c a l method.  Also folate concentration  v a l u e s p r e s e n t l y a v a i l a b l e may n o t n e c e s s a r i l y be a r e f l e c t i o n o f t h e i r n u t r i t i o n a l s i g n i f i c a n c e t o man. The r a d i o a s s a y method was used f o r t h e measurement o f N"*m e t h y l t e t r a h y d r o f o l i c a c i d d e g r a d a t i o n under v a r i o u s c o n d i t i o n s . I t was found t h a t N ^ - m e t h y l t e t r a h y d r o f o l i c a c i d d e g r a d a t i o n i n t h e presence of an u n l i m i t e d oxygen s u p p l y c o u l d be d e s c r i b e d as a pseudo f i r s t reaction. temperature  order  Rate c o n s t a n t s f o r t h e r e a c t i o n were found t o i n c r e a s e as t h e o f t h e r e a c t i o n i n c r e a s e d and were d e s c r i b e d i n terms o f t h e  Arrhenius equation.  - i i i  The  -  o v e r a l l r e a c t i o n a p p e a r e d t o be  a l i m i t e d oxygen supply. system  delayed  The  second  presence  order i n the presence  of mercaptoethanol  of  i n the  assay  the beginning of the N ^ - m e t h y l t e t r a h y d r o f o l i c a c i d  degradation reaction.  The  presence  of a s c o r b i c a c i d  i n the  N^-  m e t h y l t e t r a h y d r o f o l i c a c i d / b u f f e r s o l u t i o n a l s o delayed the s t a r t the r e a c t i o n . presence  The  o f t h e two The  concluded  r e a c t i o n r a t e c o n s t a n t s were not a l t e r e d by reducing agents,  an o x i d a t i o n r e a c t i o n .  The  degradation product  i d e n t i f i e d as N ^ - m e t h y l d i h y d r o f o l i c a c i d by u l t r a v i o l e t The  processing. and  i n foods c o n t a i n i n g f o l a t e which  Experiments  d e l a y e d by  was  of  are subjected to  heat  w i t h food m a t e r i a l s containing ascorbic acid  exposed to h i g h temperatures  f o l a t e was  therefore  spectroscopy.  r e s u l t s of t h i s research i m p l i c a t e the importance  reducing agents  the  however.  d e g r a d a t i o n o f N ^ - m e t h y l t e t r a h y d r o f o l i c a c i d was  t o be  of  indicated  the presence  t h a t the d e g r a d a t i o n of  of the reducing  agent.  free  - iv-  TABLE OF CONTENTS  ABSTRACT  i i  TABLE OF CONTENTS  iv  LIST OF TABLES  viii  LIST OF FIGURES  ix  ACKNOWLEDGEMENTS  xii  INTRODUCTION CHAPTER I.  Page  1 REVIEW OF LITERATURE  _. . 4  DEFINITION  4  BIOCHEMICALLY ACTIVE FOLATES - THEIR DISTRIBUTION AND AVAILABILITY IN FOODS  6  METHODS OF FOLATE MEASUREMENT.  7  B i o l o g i c a l Methoda  8  Microbiological Methods  8  Biochemical, Chemical and Instrumental Methods  9  LOSSES OF FOLIC ACID IN FOOD PROCESSING . . . . 11 CHAPTER I I .  A COMPARISON OF MICROBIOLOGICAL ASSAY AND RADIOASSAY METHODS FOR MEASURING FOLATE . . . . 13 INTRODUCTION  13  MATERIALS AND METHODS Preparation of N~*- Methyltetrahydrof o l i c Acid Standards  14  Preparation of Unknown Concentrations of N CH FH. 3 4 Preparation of Food Extracts 5  0  14 16 16  Preparation of Pteroylglutamic Acid  S t a n d a r d s . .17  Maintenance o f S t o c k C u l t u r e and P r e p a r a t i o n of Inoculum . . .  17  M i c r o b i o l o g i c a l Assay Procedure  18  Radioassay Procedure  19  Experimental Procedure  20  N CH FH^ Recovery  21  5  3  Study  RESULTS  23  N CH„FH, S t a n d a r d Curves j 4 - M i c r o b i o l o g i c a l Assay  23  5  23  -Radioassay  23  Comparison o f F o l a t e C o n c e n t r a t i o n s U s i n g Both Methods Comparison o f F r e e and T o t a l F o l a t e L e v e l s of Baby Foods O b t a i n e d U s i n g B o t h Methods. Comparison Curves N CH FH 5  3  CHAPTER I I I .  4  25  . . 25  o f N^CH^FH^ a n d P t e G l u S t a n d a r d  Recovery  29 Study  ?9  DISCUSSION  3  CONCLUSIONS  39 i  K I N E T I C STUDIES INTRODUCTION  5  C 1  4  1  METHODS  4  3  P r e p a r a t i o n o f N^CH^FH^ S o l u t i o n s  4  3  4  3  K i n e t i c S t u d i e s U s i n g T h e r m a l D e a t h Time Pouches K i n e t i c S t u d i e s U s i n g U n l i m i t e d Oxygen Supply.  .  K i n e t i c S t u d i e s U s i n g U n l i m i t e d Oxygen S u p p l y and a M e r c a p t o e t h a n o l - F r e e A s s a y S y s t e m . . .  .47  Kinetic  4 3  S t u d i e s U s i n g L i m i t e d O x y g e n S u p p l y . . 47  - v i-  THEORETICAL CONSIDERATIONS.  48  First  48  Order  Reactions  The A r r h e n i u s E q u a t i o n Second Order  • -50  Reactions  50  RESULTS  52  K i n e t i c S t u d i e s U s i n g Thermal Death Time Pouches  52  Kinetic  S t u d i e s U s i n g U n l i m i t e d O x y g e n S u p p l y . .54  T h e H o r i z o n t a l X-Y The S l o p e s Y-Z  54 . .57  K i n e t i c S t u d i e s U s i n g U n l i m i t e d Oxygen S u p p l y and a M e r c a p t o e t h a n o l - F r e e A s s a y System . . . . Kinetic  CHAPTER I V .  62  S t u d i e s U s i n g a L i m i t e d O x y g e n S u p p l y . .62  DISCUSSION  7  1  CONCLUSIONS  7  3  I D E N T I F I C A T I O N OF THE DEGRADATION PRODUCT AND THE EFFECT OF ASCORBIC A C I D ON RATE OF DEGRADATION OF N -METHYLTETRAHYDROFOLIC A C I D 75 5  INTRODUCTION  7  5  I  METHODS.  6  Ultraviolet  Spectroscopy  The E f f e c t o f A s c o r b i c A c i d o n t h e D e g r a d a t i o n o f N CH.FH. i n a F o o d 3 4 5  7  ^  7  7  7  7  7  7  7  8  DISCUSSION  8  5  CONCLUSIONS-  8  7  RESULTS Ultraviolet  Spectroscopy  The E f f e c t o f A s c o r b i c A c i d on t h e D e g r a d a t i o n of N CH FH. i n a Food 5  0  - v i i -  REFERENCES  88  APPENDICES  95  A.  Replacement o f T r i s - N a C l B u f f e r w i t h Phosphate B u f f e r i n t h e R a d i o a s s a y Method  B.  Method  o f Quench C o r r e c t i o n  C.  D e t e r m i n a t i o n o f H e a t i n g and C o o l i n g Times f o r Thermal D e a t h Time Pouches  ...  .95 97  Lag 101  - viii  Table I  -  L I S T OF TABLES  Page  N^CH^FH^ c o n c e n t r a t i o n s u s e d i n p r e p a r a t i o n o f standard curves  II  15  V o l u m e s o f s o l u t i o n s u s e d i n N^CH^FH^ r e c o v e r y study  . .22  III  F r e e and t o t a l f o l a t e a c t i v i t y  o f baby foods.  IV  P a i r e d comparison Student's t — t e s t v a l u e s o f  30  P t e G l u a n d N^CH^FH^ s t a n d a r d c u r v e s o b t a i n e d b y m i c r o b i o l o g i c a l and r a d i o a s s a y t e c h n i q u e s  33  V  Recovery  34  VI  Pseudo f i r s t  VII  of N CH FH 5  3  4  from m i l k e x t r a c t  order constants  ( k ' ) o f N CH„FH, 3 4 d e g r a d a t i o n i n an abundant s u p p l y o f oxygen  Pseudo f i r s t  5  order rate constants  ( k ' ) o f N^CH„FH. 3 4 d e g r a d a t i o n i n an abundant s u p p l y o f oxygen  measured by a m e r c a p t o e t h a n o l - f r e e  VIII  Second o r d e r r a t e c o n s t a n t s degradation i n a limited  assay  system  65  ( k ) f o r N^CH^FH^  s u p p l y o f o x y g e n a t 20°C. . . .70  IX  Effect  A-l  3 H P t e G l u bound i n r a d i o a s s a y method u s i n g two  B-l  59  o f h e a t i n g o n N^CH^FH^ s t a b i l i t y  81  b u f f e r system  96  Composition  99  o f quenched samples  - i x-  L I S T OF FIGURES  Figure  Page  1  Structure of N^-methyltetrahydrofolic acid  2  Basic structure of a l l folate  compounds-  pteroylglutamic acid  3  Regression standard  5  a n a l y s i s p e r f o r m e d o n N^CH^FH^  curves  o b t a i n e d b y L_. c a s e i  m i c r o b i o l o g i c a l assay method  4  Regression standard  5  24  a n a l y s i s p e r f o r m e d o n N^CH^FH^  curves  Regression  3  o b t a i n e d by r a d i o a s s a y method  26  a n a l y s i s o f r a d i o a s s a y o n L_. c a s e i  m i c r o b i o l o g i c a l assay method o f m e a s u r i n g N CH„FH, c o n c e n t r a t i o n 3 4  27  5  6  Regression  a n a l y s e s p e r f o r m e d on P t e G l u and  N^CH^FH^ s t a n d a r d  curves  o b t a i n e d b y L_. c a s e i  m i c r o b i o l o g i c a l a s s a y method  7  Regression  31  a n a l y s e s performed on P t e G l u and  N^CH^FH^ s t a n d a r d  curves  obtained by  r a d i o a s s a y method  32  8  Thermal death  9  Apparatus used f o r k i n e t i c  10  Degradation i n thermal  time pouch w i t h weight  studies .  o f N CH FH^ h e a t e d 5  death  attached  time pouches  45  46  t o 100°C 53  - x -  11  Degradation in  12  o f N^CH^FH^ h e a t e d  to various  temperatures  an u n l i m i t e d oxygen s u p p l y  Influence of temperature  55  on l e n g t h o f t i m e t o  b e g i n n i n g o f N^CH^FH^ d e g r a d a t i o n r e a c t i o n i n t h e presence  13  o f an u n l i m i t e d oxygen supply  Regression analyses performed in  F i g u r e 11 a c c o r d i n g t o pseudo f i r s t  reaction equation  14  15  58  (k') w i t h  60  (8)  Degradation  61  o f N^CH^FH^ h e a t e d  to various  an u n l i m i t e d oxygen s u p p l y and assayed  mercaptoethanol-free  Regression analyses performed  equation  temperatures using a  system  63  on d a t a p r e s e n t e d i n  F i g u r e 16 a c c o r d i n g t o p s e u d o f i r s t  18  temperature  P l o t o f d a t a i n F i g u r e 14 a c c o r d i n g t o A r r h e n i u s  in  17  order  reaction  equation  16  presented  (6)  V a r i a t i o n of r a t e constant of  on d a t a  56  order  reaction  (6)  64  Influence of dissolved  oxygen content  o n N^CH^FH^  d e g r a d a t i o n a t 20 C  19  Change i n o x y g e n c o n c e n t r a t i o n a t 20°C i n t h e presence  20  66  o f N CH„FH. 3 4  Regression analyses performed presented  68  5  on p a r t o f t h e d a t a  i n F i g u r e 18 a c c o r d i n g t o s e c o n d  reaction equation  (16)  order 69  - x i -  21  Decrease i n u l t r a v i o l e t a b s o r p t i o n w i t h l e n g t h o f t i m e ( i n m i n u t e s ) o f h e a t i n g N^CH^FH^ samples i n phosphate b u f f e r t o 70°C  79  22  U l t r a v i o l e t spectrum o f N^. m e t h y l d i h y d r o f o l i c a c i d . . 82  23  The i n f l u e n c e o f a s c o r b i c a c i d on t h e d e g r a d a t i o n o f f r e e and t o t a l f o l a t e i n a f o o d h e a t e d t o 100°C i n t h e presence o f an u n l i m i t e d oxygen s u p p l y  Bl  Quench c o r r e c t i o n c u r v e . . . .  84  100  - x i i -  ACKNOWLEDGEMENT S  I w i s h t o e x p r e s s my g r a t i t u d e t o  - my r e s e a r c h s u p e r v i s o r s , D r . J . F . R i c h a r d s a n d Dr. J . Vanderstoep  for their  encouragement throughout  guidance, support and  t h e course o f t h i s  - t h e members o f my c o m m i t t e e ,  study;  D r . M.A. T u n g , P r o f e s s o r  B. M a r c h , D r . S. N a k a i a n d D r . J . L e i c h t e r f o r t h e i r helpful  - John  s u g g e s t i o n s and f o r t h e r e a d i n g o f t h i s  thesis;  f o r g i v i n g me t h e i n c e n t i v e t o b e g i n a n d t h e  courage  t o carry out t h i s research.  -  1  -  INTRODUCTION  The  f o l a t e s a r e a g r o u p o f w a t e r s o l u b l e compounds w i t h i n  the v i t a m i n B complex. important  T h e y a r e p h y s i o l o g i c a l l y and  e s p e c i a l l y to higher  a n i m a l s and  r o l e i n a m i n o a c i d m e t a b o l i s m and pyrimidine  nucleotides.  i n the  or e l e v a t e d  change i n t h e b l o o d  Folate d e f i c i e n c y i s probably  v i t a m i n d e f i c i e n c y i n man I t has  s y n t h e s i s of purine  absorption  requirement produces a c h a r a c t e r i s t i c anemia.  and  i s common t h e w o r l d  b e e n s u g g e s t e d t h a t one  d e s t r u c t i o n of f o l a t e s d u r i n g  of food  1968b).  it  becomes i m p o r t a n t  during  processing.  better understanding of the v a r i o u s  Herbert,  to d i e t a r y metabolic known  the most  as  prevalent  over.  the processing  and  cooking  Because of t h e i r e s s e n t i a l r o l e i n n u t r i t i o n  t o a t t e m p t t o e n s u r e maximum r e t e n t i o n o f f o l a t e s Such measures can  o n l y be  achieved  as a r e s u l t of  a  of the r e s p e c t i v e r e s i s t a n c e to d e s t r u c t i v e processes  of the l i t e r a t u r e  h a v e b e e n c o n d u c t e d on  1966;  and  f o l a t e compounds.  A search  e t a l . , 1943;  important  of the major causes of f o l a t e  d e f i c i e n c y i s the (Herbert,  p l a y i n g an  A d e f i c i e n c y o f t h i s v i t a m i n due  inadequacy, f a i l u r e of alimentary  megaloblastic  t o man,  biochemically  some  l o s s e s of f o l a t e w i t h i n food m a t e r i a l s  S c h w e i g e r t e t a l . , 1946; 1967;  indicates that while  1968b; F o r d  H a n n i n g and  e t a l . , 1968;  studies (Cheldin  M i t t s , 1949;  Schroeder, 1971),  Ghitis, there  i s a complete l a c k of i n f o r m a t i o n p e r t a i n i n g t o the k i n e t i c s of f o l a t e breakdown w i t h i n a simple  system.  ( T h i s may  be  due  to both  the  - 2 -  complexity simple step  o f t h e v i t a m i n and t h e l a c k o f a r a p i d , a c c u r a t e and  assay method).  Kinetic  i n f o r m a t i o n such as t h i s i s t h e f i r s t  t o c o r r e l a t i n g l o s s e s o f the v i t a m i n t o food  various processes Since the k i n e t i c  and s t o r a g e  composition  and t o  techniques.  t h e f o l a t e v i t a m i n i s so complex, t h e f o r m chosen f o r  s t u d i e s was N ^ - m e t h y l t e t r a h y d r o f o l i c a c i d  s t r u c t u r e o f t h i s form o f f o l a t e i s i l l u s t r a t e d  (N^CH^FH^).  i n F i g u r e 1.  The  The e f f e c t  o f h e a t i n g N^CH^FH^ i n p h o s p h a t e b u f f e r t o v a r i o u s t e m p e r a t u r e s a n d a t v a r i o u s o x y g e n c o n c e n t r a t i o n s was e v a l u a t e d . reducing  a g e n t s on t h e r a t e o f d e g r a d a t i o n  The e f f e c t o f v a r i o u s  o f N^CH^FH, was a l s o  studied.  3 4 Following i n i t i a l  comparative studies w i t h the L a c t o b a c i l l u s c a s e i  m i c r o b i o l o g i c a l assay method, a r a d i o a s s a y N CH„FH, 5  levels.  s y s t e m was c h o s e n f o r m e a s u r i n g  Figure  1.  S t r u c t u r e of N  methyltetrahydrofolic acid.  - 4 -  CHAPTER 1.  REVIEW OF  LITERATURE  DEFINITION  Folic several  a c i d has  been the  s p e c i e s o f a n i m a l s and  confusing roster  eventually  1935,  n e c e s s a r y i n the  c a l l e d " V i t a m i n M"  (Day  anemia i n c h i c k s  due  several  1975).  pteroic  acid  f o r m s and  by  the  acid"  Generically,  t h e i r reduced or  family  an  and  unknown  named  factor  "the  (Stokstad  " f o l a t e " be  of  family  compounds, o r either  L a t i n term f o r  of  the monoglutamate, the analogues.  and  used i n the  free  meanings related  Specifically, i t is  acid.  I t has  T h e n e n , 1972)  these, with  conjugated with  acid.  polyglutamate  been proposed  that  g e n e r a l s e n s e f o r any  a m i x t u r e of or  closely  leaf  the  compounds, f o l i c  a c c e p t e d t o h a v e two  compound, p t e r o y l g l u t a m i c  a c i d " and  shown i n F i g u r e 2,  of  Hogan  Stokstad. reported a  from the  i s presently  substituted  IUPAC-IUB c o m m i s s i o n  of  described.  I n 1939,  i t covers a broad range of  "folic the  a c i d , was  c a s e i , w h i c h he  acid",  related  compounds w h i c h i n c l u d e  used to d e s c r i b e the  a  similar  o t h e r f a c t o r s w e r e shown t o b e l o n g t o  chemically  term " f o l i c  (Malin,  had  M i t c h e l l et_ a l . ( 1 9 4 1 ) i s o l a t e d a c r y s t a l l i n e compound  same n u t r i t i o n a l l y and The  same o r  to a deficiency  I n 1943,  from spinach which they c a l l e d " f o l i c T h e s e and  vitamin  i n monkeys,which  et a l . , 1935).  n u t r i t i o n of L a c t o b a c i l l u s  f a c t o r " and  (folium).  the  l e d t o a n e m i a , n o w known t o r e s p o n d t o f o l i c  d e s c r i b e d an  casei  learned that  a nutritional deficiency  f a c t o r w h i c h t h e y t e r m e d " V i t a m i n B".  L_.  Hence, the  involving  studied.  e a r l y as  T h i s f a c t o r was Parrott  microorganisms.  o f names b e f o r e i t was  s u b s t a n c e s were b e i n g As  s u b j e c t o f many i n v e s t i g a t i o n s  two  the  the  terms  member  basic  structure  o r more g l u t a m i c  GLUTAMIC  ACID  2 - A M I N O - 4 - H YDROX YAMINOBENZOIC  6-METHYLPTERIN  1  ACID  o OH  r  CH, i  9  CH, -NH  CH COOH  OH  F i g u r e 2.  Basic  structure  o f a l l f o l a t e compounds-pteroylglutamic  acid.  i  - 6 -  acid  residues.  BIOCHEMICALLY A C T I V E FOLATES - T H E I R D I S T R I B U T I O N AND  The the  forms of f o l i c  acid  A V A I L A B I L I T Y I N FOODS  that are biochemically active d i f f e r  s t r u c t u r e shown i n F i g u r e 2 i n v a r i o u s w a y s .  Firstly,  the  bio-  c h e m i c a l l y a c t i v e forms a r e reduced - i . e . they have a d d i t i o n a l attached to the p t e r i d i n e r i n g .  hydrogens  S e c o n d l y , t h e f o l a t e m o l e c u l e may  an a d d i t i o n a l c a r b o n a t t a c h e d t o t h e p t e r i d i n e r i n g o r t o t h e b e n z o i c a c i d as a f o r m y l o r a m e t h y l group two p o s i t i o n s a s m e t h y l e n e  or methenyl.  from  hold p-amino  o r as a b r i d g e between t h e s e  The  third  important biochemical  c h a n g e i s t h a t some n a t u r a l l y o c c u r r i n g f o r m s a r e c o n j u g a t e d i n p e p t i d e like  l i n k a g e t h r o u g h t h e i r gamma c a r b o x y l  acid  residues. Malin  forms o f f o l i c to  (1975) has  groups  s e p a r a t e t h e many n a t u r a l f o l i c  ( S i l v e r m a n e t a l . , 1961;  Kas  By e m p l o y i n g  folic  tissues.  A t t e m p t s h a v e b e e n made  S h i n e t a l . , 1972a, 1972b;  such procedures, the  distribution  a c i d has been determined f o r c e r t a i n r e p r e s e n t a t i v e foods R e s u l t s show a w i d e d i s t r i b u t i o n o f g e n e r a l t y p e s o f  derivatives.  Rat k i d n e y and l i v e r  and  folate  f o l a t e s a r e a p p r o x i m a t e l y 40 p e r c e n t  methyl d e r i v a t i v e s - m a i n l y pentaglutamates - whereas r e d c e l l s o n l y t h e m e t h y l form p r e s e n t as b o t h penta-and 1972a, 1974).  active  a c i d d e r i v a t i v e s by v a r i o u s  c h r o m a t o g r a p h i c methods  of  o r more g l u t a m i c  s t a t e d t h a t t h e r e a r e more b i o l o g i c a l l y  a c i d t h a n o f a n y known v i t a m i n .  and C e r n a , 1 9 7 6 ) .  t o one  hexaglutamate  M i l k also contains mainly methyl d e r i v a t i v e s ,  contain  (Shin eta l . , 60 p e r c e n t  -  of which are present  as  7 -  the monoglutamate  ( S h i n , et^ _ a l . , 1 9 7 5 ) .  m a t e r i a l v a r i e s i n i t s d i s t r i b u t i o n of f o l a t e d e r i v a t i v e s . c a b b a g e and 1973;  T a m u r a e t a l . , 1976)  15 p e r c e n t  of t h i s form, the r e s t being  derivative  (Shin et a l . , 1975). Much w o r k has  of the f o l i c  et_ a l . , It  Lear,  1969;  1956;  Streiff  R o s e n b e r g and  and  soybeans c o n t a i n  e i t h e r t h e 5-  b e e n p u b l i s h e d on  a c i d d e r i v a t i v e s i n food  but  o r 10-  the a v a i l a b i l i t y  (Swendseid et a l . ,  R o s e n b e r g , 1967;  Godwin, 1971;  conjugates  carboxypeptidase  a r e a c t e d upon by  Spray,  the a v a i l a b i l i t y  et_ a l . ,  of the conjugate  e s t i m a t e b a s e d on  glutamate suggests  their  and  (^glutamyl  t h a t 8 0 % may  be  present  there i s l i t t l e  In  i n various  difference i n  the monoglutamate form of  studies with tritium labelled  Butterworth  ^glutamyl  i n t e s t i n a l enzymes  1977)»  folate.  f o l a t e mono-  absorbed.  FOLATE MEASUREMENT  Methods f o r d e t e r m i n i n g  folic  acid  l e v e l s may  be  grouped  three categories". 1)  B i o l o g i c a l methods  2)  M i c r o b i o l o g i c a l methods  3)  Biochemical,  chemical  and  1952;  1973).  - "conjugase") r e l e a s i n g the monoglutamic f o l a t e .  food m a t e r i a l s (Stokstad  METHODS OF  absorption  1968a;  T a m u r a and - S t o k s t a d ,  t h e a b s e n c e o f c e r t a i n c o n j u g a s e i n h i b i t o r s known t o be  An  and  Herbert,  only  formyl  1947;  i s known w i t h c e r t a i n t y t h a t p r i o r t o a b s o r p t i o n , t h e  peptide  Lettuce  o r a n g e j u i c e c o n t a i n m a i n l y m e t h y l d e r i v a t i v e s ( B a t r a et_ a l . ,  C h a n e t a l . , 1973,  J a n d l and  Plant  i n s t r u m e n t a l methods'  into  - 8 -  Biological  Methods  O ' D e l l and H o g a n ( 1 9 4 3 )  and C a m p b e l l  t h e c h i c k a s s a y method o f f o l a t e measurement. symptoms when f e d c r y s t a l i n e f o l i c form t h e b a s i s o f t h i s method. assays  (Ransome a n d Elvehjem>,  developed  Recovery  deficiency  from  a c i d a n d f o o d s : o f unknown p o t e n c y  Rats have a l s o been used 1943,  A s e n j o , 1948).  advantages  o f a n i m a l assays a r e t h e i r expense  involved.  Freed  supplement  microbiological data.  Microbiological  e t a l . (1944)  and  for biological  The m a i n the time  dis-  factor  (1966) h o w e v e r , a d v o c a t e d t h e u s e o f a n i m a l a s s a y s t o  Methods  T e p l y and E l v e h j e m  (1945)  d e v e l o p e d a method o f f o l a t e  analysis  u s i n g t h e t i t r i m e t r i c measurement o f growth o f L a c t o b a c i l l u s c a s e i . organism  h a s s i n c e become t h e m o s t p o p u l a r a l t h o u g h t h e f o l l o w i n g  or have been, employed:Pedicoccus cerevisiae el: a l . , and  1944).  The  the f l a g e l l a t e ,  Bacillus  (Kavanagh,  ciliated  likely  1963)  Crithidia  1955)  emphasised  (Nathan et a l • ,  (Jukes,  to  1955)  1958).  that the choice of micro-  a s s a y must t a k e i n t o c o n s i d e r a t i o n t h e t y p e o f  responding w e l l  are,  S t r e p t o c o c c i s p e c i e s (Luckey  fasciculata  t o be p r e s e n t i n t h e s a m p l e .  organism  ( B a k e r et_ a l . ,  p r o t o z o a n , Tetrahymena g e l e i i  Cooperman (1967) has o r g a n i s m f o r any  coagulans  This  folates  F o r e x a m p l e , 1/. c a s e i i s t h e o n l y  methyl folates -  (as p r e v i o u s l y  s u c h f o l a t e s a r e v e r y common i n f o o d m a t e r i a l s ) - _ S .  faecalis  stated,  (AOAC  recommended a s s a y m e t h o d ) d o e s n o t r e s p o n d t o N"* m e t h y l f o l a t e s .  However,  -  it  does grow w e l l  metabolically  i n the  are  p o l y g l u t a m a t e form. liberate free  -  presence of  inactive for  Folates  9  frequently  f o u n d i n n a t u r e t o be  T h u s , i t was  f o u n d n e c e s s a r y by  f o l a t e s p r i o r to m i c r o b i o l o g i c a l  was  to prevent f o l i c  l a t e r shown b y  Lactobacillus in  the  folic  Herbert  Conjugase  casei.  T h u s he  standards to obtain  as  in  The  assessment of  ascorbate  include  growth  of  several  are  samples.  be  affected  by  ascorbate  than  p a r t i c u l a r l y w e l l adapted to simultaneous  assays  H o w e v e r , d i f f e r e n t m i c r o o r g a n i s m s show v a r y i n g  r e s p o n s e s t o t h e v a r i o u s f o l a t e compounds. may  to  response.  M i c r o b i o l o g i c a l methods l e s s t i m e f o r p r e p a r a t i o n a n i m a l a s s a y s and  be  the  a growth stimulant  deemed i t n e c e s s a r y t o  a true  al.,1949).  a c i d h a v e b e e n shown t o acid  to has  Dabrowska et  acid destruction.  (1961) t o a c t  the  many w o r k e r s  T o e n n i e s e t a l . (1956) employed a s c o r b i c solvent  is  present i n  assay.  ( B i r d e t a l . , 1945,  Because reduced forms of  extraction  acid which  man.  been employed f o r t h i s purpose  heat l a b i l e ,  pteroic  the  presence or  In a d d i t i o n ,  absence of  several  their  growth  growth  growth  factors.  T h i s i s p a r t i c u l a r l y i m p o r t a n t when m e a s u r i n g f o l a t e l e v e l s i n c o m p l e x food  materials.  B i o c h e m i c a l , C h e m i c a l and  I n s t r u m e n t a l Methods  M o s t b i o c h e m i c a l , , c h e m i c a l and o n l y f o r p h a r m a c e u t i c a l p r e p a r a t i o n s of inadequate to d e t e c t the Details  o f much o f  low  instrumental  folates since  methods a r e  their sensitivity  c o n c e n t r a t i o n s of f o l a t e s o c c u r r i n g  t h i s methodology w i l l  suitable  t h e r e f o r e n o t be  in  discussed  is  foodstuffs. here.  - 10 -  A r e l a t i v e l y new m e t h o d , h o w e v e r , d e v e l o p e d  f o rthe quantitative  e s t i m a t i o n o f s e r u m f o l a t e l e v e l s , h a s b e e n shown t o b e s e n s i t i v e to low l e v e l s of f o l a t e ,  similar  The r e p o r t e d p r e s e n c e  t o t h o s e commonly  found i n foods.  of a folate binding fraction i nmilk  ( G h i t i s , 1967) and t h e l a t e r a s s o c i a t i o n o f t h i s f o l a t e b i n d i n g f r a c t i o n with the lactoglobulin fraction of milk protein  (Ford e t a l . , 1969)  h a s b e e n shown t o p r o v i d e t h e b a s i s f o r t h i s r a p i d a n d s e n s i t i v e method.  A radioligand  b y M e t z et^ al. ( 1 9 6 8 ) . direct  c o m p e t i t i v e i n h i b i t i o n a s s a y was f i r s t  However,  pteroylglutamic acid  reported  Waxman e t a l . ( 1 9 7 1 ) d e s c r i b e d a s e n s i t i v e ,  c o m p e t i t i v e assay u s i n g t r i t i a t e d  3 5 ( H N CH„FH,). i 4  assay  5-methyl t e t r a h y d r o f o l i c  t h e a u t h o r s recommended  3 ( H P t e G l u ) be s u b s t i t u t e d  a s e q u e n t i a l assay system.  Archibald  that  tritiated  3 5 f o r t h e H N CH^FH^ i n  e t a l . (1972), Rothenberg e t a l .  ( 1 9 7 2 ) a n d T a j u d d i n a n d G a r d y n a ( 1 9 7 3 ) e m p l o y e d t h e recommended with slight modifications.  acid  procedure  Waxman a n d S c h r e i b e r ( 1 9 7 3 ) e m p l o y e d a  c r y s t a l l i n e bovine b e t a l a c t o g l o b u l i n as a source o f p r o t e i n b i n d e r w h i l e b i n d e r s o t h e r t h a n b o v i n e m i l k h a v e b e e n u t i l i z e d b y Kamen a n d Caston  (1974). A t t h e p r e s e n t t i m e , t h e r a d i o a s s a y method h a s n o t been u s e d  f o r t h e assay o f f o l a t e l e v e l s i n food m a t e r i a l s although t h e s e n s i t i v i t y o f t h e m e t h o d - 0.62 t o 50 n g / m l o f s a m p l e ( f o r N^CH^FH^) - a p p e a r s t o be a d e q u a t e f o r f o o d f o l a t e  assay.  - 11 -  LOSSES OF F O L I C ACID I N FOOD PROCESSING  The m a j o r i t y o f p u b l i c a t i o n s p e r t a i n i n g t o f o l i c research  involve studies of i t s biochemical  Very l i t t l e  data a r e a v a i l a b l e concerning  on v i t a m i n  content.  and c l i n i c a l  acid aspects.  the e f f e c t of food  E a r l y work has demonstrated t h a t f o l a t e s a r e v e r y to destruction. cooking  o f f o o d s and c o n c l u d e d t h a t o f a l l v i t a m i n s s t u d i e d , f o l a t e s w e r e  losses of f o l i c investigated  how  a c i d content  thrice boiling  and f o u n d l o s s e s o f 18-48%. of foods destroyed  in  the milk.  (1967)  the losses being  directly  during  ultra-high-temperature  increases  ascorbate  folate retention.  (Stokstad  t o b e due t o t h e  e^t a l . , 1 9 4 7 ) .  a 20-80% l o s s o f f o l a t e d u r i n g  recently, slight  that  r e l a t e d t o t h e presence of oxygen  L o s s e s o f f o l a t e have a l s o been r e p o r t e d light  folates  f i n e l y d i v i d e d and cooked i n w a t e r  These a u t h o r s a l s o found t h a t t h e presence o f  action of u l t r a v i o l e t  described  p r o c e s s e s and t h a t t h e l o s s e s  G h i t i s ( 1 9 6 6 ) a n d F o r d j i t a l . ( 1 9 6 8 ) showed  i n m i l k was n e c e s s a r y t o s t a b i l i s e  reported  o f eggs on  t h a t a s much a s 9 5 % o f t h e i n i t i a l  v a r i a b l e losses of f o l a t e i n milk occurred processing,  Herbert  (1949)  the majority of f o l a t e a c t i v i t y .  f o o d s c o u l d be l o s t by o x i d a t i v e h e a t i n g  long periods.  reported  H a n n i n g and M i t t s  t h e e f f e c t o f f r y i n g , b o i l i n g and s c r a m b l i n g  w e r e a g g r a v a t e d when t h e f o o d was for  S c h w e i g e r t ejt a l . ( 1 9 4 6 )  a c i d i n meat u p o n c o o k i n g .  I n 1968b, he a l s o e s t i m a t e d in  susceptible  C h e l d i n jet a l . ( 1 9 4 3 ) c a r r i e d o u t a s u r v e y o n t h e  t h e most s u s c e p t i b l e t o breakdown.  folic  processing  i n the t o t a l  the m i l l i n g  Schroeder of f l o u r .  f o l a t e content  of  (1971) More  Brussels  - 12 -  s p r o u t s t r e a t e d w i t h v a r i o u s p r o c e s s i n g methods have been r e p o r t e d by M a l i n  (1977).  CHAPTER I I .  A COMPARISON OF MICROBIOLOGICAL ASSAY AND RADIOASSAY METHODS FOR MEASURING FOLATE  INTRODUCTION  The d i f f i c u l t i e s  and l i m i t a t i o n s o f a s s a y methods f o r  naturally  occurring  monoglutamate and p o l y g l u t a m a t e f o l a t e s a r e  reflected  i n the v a r i a b i l i t y  of reported l e v e l s f o r t h i s vitamin i n  foodstuffs. Until recently, frequently  microbiological  a s s a y s have been t h e most  u s e d method f o r q u a n t i t a t i v e l y m e a s u r i n g f o l a t e c o n t e n t o f  f o o d , serum and o t h e r m a t e r i a l s . binding fraction i n milk folate-binding  The r e p o r t e d p r e s e n c e o f a f o l a t e -  ( G h i t i s , 1967) and t h e l a t e r a s s o c i a t i o n  a c t i v i t y with the lactoglobulin  fraction of milk  ( F o r d , e t a l . , 1 9 6 9 ) h a s b e e n shown t o p r o v i d e t h e b a s i s sensitive  test  f o rfolates  (Waxman e t a l . , 1 9 7 1 ; A r c h i b a l d  Rothenberg e t a l . , 1972; T a j u d d i n and Gardyna, Because o f t h e i n c r e a s i n g quantitative  measurement  of this protein  f o r a r a p i d and e t a l . , 1972;  1973).  u s e o f t h e r a d i o a s s a y method f o r t h e  o f f o l a t e and t h e f r e q u e n t l y  contradictory  r e p o r t s on t h e c o r r e l a t i o n o f r e s u l t s o b t a i n e d b y t h i s method w i t h o b t a i n e d by m i c r o b i o l o g i c a l 1972;  assays  those  (Waxman e t a l . , 1 9 7 1 ; R o t h e n b e r g e t a l . ,  M i n c e y e t a l . , 1 9 7 3 ; Dunn & F o s t e r , 1 9 7 3 ; T a j u d d i n a n d G a r d y n a , 1 9 7 3 ;  Shaw e t a l . , 1 9 7 4 ; Kamen a n d C a s t o n , 1 9 7 4 ; W a d d e l l et a l . , 1 9 7 6 ; R u d z k i e t a l . , 1 9 7 6 ) i t was o f i n t e r e s t t o d e t e r m i n e b o t h t h e r e p r o d u c i b i l i t y of  s t a n d a r d c u r v e s and t h e c o r r e l a t i o n o f r e s u l t s o b t a i n e d by b o t h methods  using buffered  solutions  o f N^CH FH. a s t h e s t a n d a r d a n d a s t h e unknown.  - 14 -  Food m a t e r i a l s were t h e n a n a l y z e d b y b o t h methods o f a s s a y and  v a l u e s f o r f r e e and t o t a l  activities ( J  folate activities  b e f o r e and a f t e r treatment  ( i . e . the folate  of the food e x t r a c t with  conjugase  g l u t a m y l p e p t i d a s e ) ) o b t a i n e d b y each method were compared. In  an attempt  t o e x p l a i n the d i f f e r e n c e s i n food f o l a t e  levels  o b t a i n e d b y t h e two methods and t h e v a r i a t i o n i n p r e v i o u s l y r e p o r t e d results, (Fig.  standard  curves  obtained using pteroylglutamic acid  (PteGlu)  2) w e r e t h e n c o m p a r e d w i t h t h o s e o b t a i n e d u s i n g N^CH^FH^. M i c r o b i o l o g i c a l methods f o r t h e d e t e r m i n a t i o n o f f o l a t e  been w e l l documented ( T e p l y and E l v e h j e m , 1967;  Herbert  experiments  and B e r t i n o , 1967).  s i n c e t h i s organism  The r a d i o a s s a y t e c h n i q u e  1945; H e r b e r t ,  L_. c a s e i was u t i l i z e d  have  1966; Cooperman, i n these  e x h i b i t s a growth response  t o N^CH^FH^.  u s e d was a m o d i f i c a t i o n o f t h a t d e s c r i b e d b y  R o t h e n b e r g et a l . ( 1 9 7 2 ) a n d u t i l i z e d  a two-step  sequential addition  technique.  MATERIALS AND METHODS  Preparation of N -Methyltetrahydrofolic Acid  S a m p l e s o f N^CH^FH^ ( C l i n i c a l A s s a y s ,  Standards  Inc. Massachusetts)  were  d i l u t e d w i t h 0.1M p h o s p h a t e b u f f e r (pH 7.3) c o n t a i n i n g 0.3%  mercaptoethanol  to  stock  g i v e a n i n i t i a l N^CH^FH^ c o n c e n t r a t i o n o f 50 n g / m l .  s o l u t i o n was t h e n s e r i a l l y Table  diluted  I. Additional dilutions  This  t o g i v e t h e c o n c e n t r a t i o n s shown i n  t o g i v e c o n c e n t r a t i o n s o f 5.0, 7.5, 1 0 . 0 ,  Table  I.  N CH FH. c o n c e n t r a t i o n s u s e d i i i p r e p a r a t i o n o f s t a n d a r d  curves.  N CH FH / 5  3  Dilution  4  100 m i c r o l i t r e s (ng)  Sample E q u i v a l e n t (ng/ml)  1/1  5.000  50.00  1/3  1.667  16.67  1/9  0.556  5.56  1  hUi  1  1/27  0.185  1.85  1/81  0.062  0.62  i  - 16 -  15.0 a n d 20.0 n g / m l N CH^FH^ w e r e l a t e r p r e p a r e d i n o r d e r t o i n c r e a s e the p r e c i s i o n o f t h e m i c r o b i o l o g i c a l assay standard curve.  Because o f  t h e r e p o r t e d s e n s i t i v i t y o f N^CH^FH^ t o a i r o x i d a t i o n a n d t o t h e a c t i o n of u l t r a v i o l e t standard v i a l ,  light,  assay tubes were kept i n d i f f u s e l i g h t and a f r e s h  reconstituted  f o r t h e p r e p a r a t i o n o f each  i m m e d i a t e l y p r i o r t o u s e , was  utilized  s e t of standard curves.  P r e p a r a t i o n o f Unknown C o n c e n t r a t i o n s o f N^CH FH.  A s e r i e s o f unknown c o n c e n t r a t i o n s o f N CH^FH^ i n p h o s p h a t e (0.3% mercaptoethanol)  buffer  w a s made up s u c h t h a t a l l s a m p l e s w e r e w i t h i n t h e  c o n c e n t r a t i o n range o f t h e standard curves.  P r e p a r a t i o n o f Food  Three and  'total'  Extracts  samples o f s t r a i n e d baby foods were a n a l y z e d f o r ' f r e e '  folate.  The c o n t e n t s o f each j a r were mixed  thoroughly before  the preparation of the extracts. The t e s t e x t r a c t s w e r e p r e p a r e d a s f o l l o w s : 2g s t r a i n e d phosphate ly,  to approximately  f o o d s a m p l e i n a 15 m l c e n t r i f u g e t u b e was a d d e d 5 m l 0.1M  buffer  (pH 7.3, 1% a s c o r b a t e ) .  using a vortex mixer.  The e x t r a c t s w e r e m i x e d  thorough-  The p r e p a r e d t u b e s w e r e t h e n t r e a t e d i n  a b o i l i n g w a t e r b a t h f o r 10 m i n u t e s  and c o o l e d i n c o l d  water.  - 17 -  Desiccated water  chicken pancreas:  (5 mg/ml), t h o r o u g h l y  37°C f o r 45 m i n u t e s . No.  mixed w i t h a g l a s s r o d and i n c u b a t e d a t  After filtration  of the slurry  1 p a p e r , t h e c l e a r f i l t r a t e was u s e d For  the determination  Toluene  t h e s a m p l e t u b e s a n d enzyme b l a n k s  p r e p a r a t i o n ) were i n c u b a t e d the f l a s k were then  adjusted  After further mixing,  treatment to  10 m l , r e m i x e d ,  ( 5 m l b u f f e r p l u s 1 m l enzyme  below.  of 'free folate'  and t h e c o n t e n t s  activity,  the conjugase  o f t h e tubes were a d j u s t e d  c e n t r i f u g e d and a f t e r a p p r o p r i a t e d i l u t i o n ,  directly  assayed.  Standards  ( N u t r i t i o n a l Biochemicals  i n 0.1 M p h o s p h a t e b u f f e r  Corp., Ohio) were  (pH 7.3) c o n t a i n i n g 0.3%  mercaptoethanol  g i v e c o n c e n t r a t i o n s o f 5 0 . 0 0 , 1 6 . 6 7 , 5 . 5 6 , 1.85 a n d 0.62 n g / m l P t e G l u .  Maintenance of Stock  C u l t u r e and P r e p a r a t i o n o f Inoculum  L a c t o b a c i l l u s c a s e i ATCC 7469 was m a i n t a i n e d in  buffer.  c e n t r i f u g a t i o n and a p p r o p r i a t e d i l u t i o n , t h e  Samples o f P t e G l u  to  The c o n t e n t s o f  t o 10 m l w i t h p h o s p h a t e - a s c o r b a t e  Preparation of Pteroylglutamic Acid  diluted  1 ml of the  ( a s a p r e s e r v a t i v e ) was a d d e d  a t 37°C f o r 16 h o u r s .  the determination  was o m i t t e d  Whatman  immediately.  e x t r a c t s were assayed by t h e methods d e s c r i b e d For  through  of 'total folate a c t i v i t y '  f i l t r a t e was a d d e d t o e a c h t u b e . and  ( D i f c o ) was s u s p e n d e d i n  tubes o f prepared  B a c t o - L a c t o b a c i l l i Agar.  as a stab c u l t u r e  C u l t u r e s were  incubated  - 18 -  a t 35-37 C f o r 1 8 - 2 4 h o u r s a n d t h e n s t o r e d  i n a r e f r i g e r a t o r a t 4°C.  T r a n s f e r s w e r e made a t m o n t h l y i n t e r v a l s .  I n o c u l u m f o r t h e a s s a y was  made b y s u b c u l t u r i n g f r o m t h e s t o c k c u l t u r e i n t o a t u b e 10 m l p r e p a r e d B a c t o - L a c t o b a c i l l i B r o t h .  After  containing  i n c u b a t i o n f o r 16-18  h o u r s a t 37°C t h e t u b e s w e r e c e n t r i f u g e d , t h e s u p e r n a t a n t was d i s c a r d e d a n d c e l l s w e r e r e s u s p e n d e d i n 10 m l 0 . 8 5 % s t e r i l e r e c e n t r i f u g e d and suspended was d i l u t e d  saline.  T h e c u l t u r e was  i n f r e s h s a l i n e t w i c e more.  100-fold i n a s t e r i l e  container with s t e r i l e  An a l i q u o t s a l i n e and one  d r o p o f t h i s was u s e d t o i n o c u l a t e t h e a s s a y t u b e s .  M i c r o b i o l o g i c a l Assay Procedure  5 m l o f r e h y d r a t e d B a c t o - F o l i c A c i d C a s e i Medium were d i s p e n s e d i n t o  test tubes.  (Difco)  0.1 m l o f t h e N^CH^FH^ d i l u t i o n ,  ( o r P t e G l u d i l u t i o n ) was a d d e d t o e a c h t u b e a n d t h e v o l u m e made up t o 10 m l w i t h 0.1 M p h o s p h a t e b u f f e r c o n t a i n i n g 1% a s c o r b a t e . s t r a i n e d baby f o o d s were a s s a y e d , d i l u t i o n s assay) of untreated e x t r a c t ,  When t h e  ( w i t h i n t h e range o f t h e  conjugase-treated e x t r a c t o r conjugase  b l a n k s w e r e a d d e d t o d u p l i c a t e t u b e s c o n t a i n i n g 5 m l r e h y d r a t e d medium. V o l u m e was made up t o 10 m l w i t h 0.1 M p h o s p h a t e b u f f e r c o n t a i n i n g 1% ascorbate.  A l l t u b e s w e r e a u t o c l a v e d f o r 5 m i n u t e s a t 15 p . s . i . g .  ( 1 2 1 C) a n d t h e n c o o l e d t o r o o m t e m p e r a t u r e . L.casei c e l l  One d r o p o f t h e d i l u t e d  s u s p e n s i o n was a d d e d t o e a c h t u b e . T u b e s w e r e i n c u b a t e d f o r  18 h o u r s a t 37°C. A f t e r t h o r o u g h l y m i x i n g , t u r b i d i t y was m e a s u r e d . T h i s was e x p r e s s e d i n t e r m s o f a b s o r b a n c e w h i c h was r e a d a t a w a v e l e n g t h o f 660 nm.  - 19 -  Radioassay  Procedure  The  p r o c e d u r e f o r r a d i o m e t r i c a s s a y was t h a t o u t l i n e d  i n the  3 C l i n i c a l Assays  H F o l i c Acid Radioassay K i t ( C l i n i c a l  M a s s a c h u s e t t s , 1975). A l l s o l u t i o n s used  Assays,  i n t h e r a d i o a s s a y w e r e made  up a n d t h e a s s a y p r o c e d u r e f o l l o w e d e x a c t l y a s d e s c r i b e d i n t h e k i t w i t h t h e e x c e p t i o n t h a t 0.1 M p h o s p h a t e tris-NaCl buffer  (Appendix A ) .  b u f f e r pH 7.3 was u s e d  i n place of  D u p l i c a t e s a m p l e s o f N^CH^FH^, P t e G l u ,  or food e x t r a c t d i l u t i o n s were i n c u b a t e d w i t h m i l k f o l a t e b i n d e r f o r  3 20 m i n u t e s  a t room t e m p e r a t u r e .  a f u r t h e r 20 m i n u t e ,  A f t e r addition of  room t e m p e r a t u r e i n c u b a t i o n .  H P t e G l u t h e r e was The p r o t e i n - b o u n d  3 and  the free  H P t e G l u were t h e n s e p a r a t e d by a d s o r p t i o n o f t h e l a t t e r  on d e x t r a n c o a t e d c h a r c o a l .  Supernatant and c h a r c o a l were s e p a r a t e d  by c e n t r i f u g a t i o n i n a r e f r i g e r a t e d a t 4 C, 2,000 x g f o r 20 m i n u t e s . vials  a n d 12 m l s c i n t i l l a t i o n  C o c k t a i l ) were added. f o r 10 m i n u t e s  fluid  centrifuge  ( S o r v a l l M o d e l RC2-B)  The s u p e r n a t a n t was d e c a n t e d  ( S c i n t i v e r s e - F i s h e r U n i v e r s a l LSC  A f t e r c o o l i n g , s a m p l e r a d i o a c t i v i t y was c o u n t e d  i n a N u c l e a r C h i c a g o I s o c a p 300 l i q u i d  u s i n g t h e t r i t i u m program f o r low quench samples. efficiency  into  scintillation  counter  Correction f o r counting  ( q u e n c h c o r r e c t i o n ) , was c a r r i e d o u t a t e a c h a s s a y u s i n g t h e  channels r a t i o  technique (Appendix B ) .  A maximum b i n d i n g c o n t r o l was r u n i n d u p l i c a t e  with  every  a s s a y t o d e t e r m i n e t h e amount o f r a d i o a c t i v e m a t e r i a l w h i c h was a b l e t o c o m p e t e w i t h c o l d N^CH^FH^ f o r m i l k p r o t e i n b i n d i n g  sites.  In addition,  3 background  c o n t r o l samples c o n t a i n i n g b u f f e r and  H P t e G l u were r u n i n  -  d u p l i c a t e w i t h each assay  20 -  t o determine  levels of radioactivity not  removed b y d e x t r a n c o a t e d c h a r c o a l . When f o o d e x t r a c t s w e r e a s s a y e d , c o n t a i n i n g b u f f e r , e x t r a c t and  3 H  d u p l i c a t e background  P t e G l u were a n a l y z e d  samples  f o r each  e x t r a c t i n order t o c o r r e c t f o rthe food's n o n - s p e c i f i c p r o t e i n b i n d i n g capacity. who,  T h i s p r e c a u t i o n was recommended b y T a j u d d i n a n d G a r d y n a  when m e a s u r i n g  human s e r u m f o l a t e l e v e l s ,  correct for non-specific binding of count  r a t e s were found  3 H  (1973)  added a serum b l a n k t o  PteGlu.  When e x t r a c t  background  t o be g r e a t e r than 20% o f e x t r a c t samples t h e t o t a l  3  count  rate f o r  Experimental  H P t e G l u was a l s o m e a s u r e d .  Procedure  M i c r o b i o l o g i c a l and r a d i o a s s a y s were c a r r i e d o u t c o n c u r r e n t l y u s i n g d u p l i c a t e s a m p l e s o f t h e f r e s h l y p r e p a r e d N^CH^FH^.  This  t h a t any v a r i a t i o n i n t h e s t a n d a r d c u r v e s due t o d i f f e r i n g  concentrations  o f N CH^FH^ b e t w e e n b a t c h e s Twenty-five methods.  prepared  was t a k e n i n t o a c c o u n t  by b o t h methods.  u n k n o w n d i l u t i o n s o f N^CH^FH^ w e r e a s s a y e d  C o n c e n t r a t i o n s were determined  a t t h e same t i m e a n d u n d e r  -  ensured  f r o m N^CH^FH, s t a n d a r d 3 4  by both curves  c o n d i t i o n s i d e n t i c a l t o t h e unknown  samples. F i v e d i l u t i o n s o f each food e x t r a c t e x t r a c t s ) were prepared standard  (both t o t a l and f r e e  i n d u p l i c a t e f o r each assay method.  curves were prepared  s i m u l t a n e o u s l y by each assay  folate  N^CH^FH^ method.  -  21 -  F i v e P t e G l u s t a n d a r d c u r v e s were p r e p a r e d by each method using  d u p l i c a t e samples o f each d i l u t i o n . P t e G l u s t a n d a r d curves were  p r e p a r e d a t t h e same t i m e a s a n a d d i t i o n a l f i v e N^CH^FH^ s t a n d a r d curves i n order t o ensure that as p o s s i b l e  f o reach standard  l^CH^FH, R e c o v e r y  o f assay remained as s i m i l a r  used.  Study  A powered m i l k buffer  conditions  s a m p l e ( 3 ) was g  mixed w i t h  (pH 7.3, 1% a s c o r b a t e ) i n a 15 m l c e n t r i f u g e  5 m l 0.1 M p h o s p h a t e tube.-  T h e t u b e was  t h e n h e a t e d i n a b o i l i n g w a t e r b a t h f o r 10 m i n u t e s a n d c o o l e d water.  The c o n t e n t s o f t h e t u b e w e r e t h e n a d j u s t e d  i n cold  t o 10 m l ,  remixed, and c e n t r i f u g e d . A radioassay outlined  i n Table I I .  was t h e n c a r r i e d o u t u s i n g Samples o f t h e e x t r a c t  each o f f i v e tubes c o n t a i n i n g  the milk  e x t r a c t as  (100 u l ) were added t o  t h e s t a n d a r d N^CH^FH^ d i l u t i o n s .  In addition,  3 d u p l i c a t e background samples c o n t a i n i n g  b u f f e r , e x t r a c t and  were a n a l y z e d t o c o r r e c t  non-specific  capacity.  Duplicate  f o rthe milk's  samples c o n t a i n i n g  protein  H PteGlu binding  buffer, extract, binding  protein  and  3 H P t e G l u were assayed i n o r d e r t o determine t h e f o l a t e content o f  the  powdered m i l k .  greater for  When e x t r a c t b a c k g r o u n d c o u n t s w e r e f o u n d t o b e  t h a n 20% o f e x t r a c t and s t a n d a r d samples, t h e t o t a l  3 H P t e G l u was a l s o  measured.  count  rate  - 22 -  Table I I .  Volumes o f s o l u t i o n s used i n N CH FH recovery s t u d y . 3  Tube No. T T 12 1,2  Buffer  Milk Extracts  Standard Dilution  Binding Protein  3  H  PteGlu  1,100  —  50  500  —  50  3,4  500  50  50  5-14  400  100  50  50  5-14M  300  1 00  100  50  50  15,16  400  100  —  50  17,18  400  100  50  50  —  a A l l volumes i n m i c r o l i t e r s Tubes:T T 12 1,2  T o t a l count Binding  c o n t r o l background  3,4  Binding  control  5,6  Standard d i l u t i o n (5ng/100ul)  5,6M  Standard d i l u t i o n (5ng/100ul) + m i l k  7,8  Standard d i l u t i o n (1.667ng/100ul)  7,8M  Standard d i l u t i o n  9,10  Standard d i l u t i o n (0.556ng/100ul)  9,10M  Standard d i l u t i o n (0.556ng/100ul) + m i l k  11,12  Standard d i l u t i o n  (0.185 ng/lOOul)  11.12M  Standard d i l u t i o n  (0.185 ng/lOOul) + m i l k  13,14  Standard d i l u t i o n  (0.062 ng/lOOul)  13.14M  S t a n d a r d d i l u t i o n (0.062 ng/lOOul) + m i l k  15,16  M i l k e x t r a c t b i n d i n g c o n t r o l background  17,18  M i l k e x t r a c t assay  extract  (1.667ng/100;il) + m i l k  extract  extract extract extract  - 23 -  RESULTS  N CH.FH, S t a n d a r d  Curves  Microbiological  Regression standard  Assay  a n a l y s i s was c a r r i e d o u t o n t h e d a t a f r o m t h e t e n  curves obtained by t h e m i c r o b i o l o g i c a l  assay method.  This i s  2 shown i n F i g u r e 3. illustrates  that  The c o e f f i c i e n t o f d e t e r m i n a t i o n  t h e r e i s a s i g n i f i c a n t (p  c o n c e n t r a t i o n o f N^CH^FH^ a n d g r o w t h r e s p o n s e by  a b s o r b a n c e a t 6 6 0 nm.  ( r ) o f 0.61  0.01) r e l a t i o n s h i p b e t w e e n o f _L. c a s e i a s m e a s u r e d  T h e s t a n d a r d e r r o r o f e s t i m a t e was f o u n d  t o be  0.11.  Radioassay  Standard  curves were o b t a i n e d by p l o t t i n g t h e p e r c e n t  b o u n d f o r e a c h amount o f s t a n d a r d  on s e m i l o g paper.  b o u n d f o r e a c h l e v e l o f s t a n d a r d was c a l c u l a t e d corrected) counts  %  H  per minute,  The p e r c e n t  tracer of tracer  from t h e normalized  thus:  P t e G l u bound =  (CPM - CPM ) , * „ " x (CPM - CPM^)  x 100 2  B  where  CPM  n  = Average counts  CPM = Average counts B G  per minute f o r standard  samples  per minute f o r background  controls  CPMg = A v e r a g e c o u n t s p e r m i n u t e f o r b i n d i n g c o n t r o l s  (quench  - 24 -  - 25 -  Regression shown i n F i g u r e 4.  a n a l y s i s performed on t h e t e n s e t s o f data i s A c u b i c e q u a t i o n was f i t t e d t o t h e d a t a t o i m p r o v e 2  t h e goodness o f f i t . (The c o e f f i c i e n t o f d e t e r m i n a t i o n c u b i c e q u a t i o n was 0.98 c o m p a r e d t o a c o e f f i c i e n t 0.97 o b t a i n e d when a q u a d r a t i c e q u a t i o n was f i t t e d is a highly significant N^CH^FH^ a n d p e r c e n t was  found  (p ^  (r ) f o r the  o f determination of to the data).  There  0.01) r e l a t i o n s h i p b e t w e e n c o n c e n t r a t i o n o f  binding of tracer.  The s t a n d a r d  error of estimate  t o b e 4.01.  Comparison o f F o l a t e Concentrations Using  Both  Methods  F o l a t e c o n c e n t r a t i o n s f o r t h e 25 unknown N^CH^FH^ s a m p l e s w e r e c a l c u l a t e d by r e f e r e n c e t o s t a n d a r d  curves  obtained by both  methods.  R e s u l t s o b t a i n e d b y t h e r a d i o a s s a y a n d m i c r o b i o l o g i c a l m e t h o d s a r e shown i n F i g u r e 5.  Radioassay  v a l u e s r a n g e d f r o m 0.62 t o 35 n g / m l  m i c r o b i o l o g i c a l a s s a y v a l u e s r a n g e d f r o m 0.75 t o 38 n g / m l .  while The c o e f f i c i e n t  2 of determination and  ( r ) f o r v a l u e s o b t a i n e d b y t h e t w o m e t h o d s was 0.93  t h e r e g r e s s i o n l i n e was f o u n d  o f 3.10 n g / m l .  The s t a n d a r d  Comparison o f Free Both Methods  t o h a v e a s l o p e o f 0.76 a n d a n i n t e r c e p t  e r r o r o f e s t i m a t e was 3.65 n g / m l .  and T o t a l F o l a t e L e v e l s o f Baby Foods O b t a i n e d  Using  Free and t o t a l f o l a t e c o n c e n t r a t i o n s o f t h e food e x t r a c t s were measured by t h e r a d i o a s s a y and m i c r o b i o l o g i c a l assay  methods.  -0-5  0-5  0-0 LOG  F i g u r e 4.  cone  10  1-5  (ng^/ml  R e g r e s s i o n a n a l y s i s performed on N CH^FH^ s t a n d a r d c u r v e s o b t a i n e d by r a d i o a s s a y method.  20  - 27 -  MICROBIOLOGICAL  F i g u r e 5.  Regression  ASSAY  a n a l y s i s o f r a d i o a s s a y on L,. c a s e i m i c r o b i o l o g i c a l  assay method o f measuring N^CH^FH^ c o n c e n t r a t i o n , y = x i s the r e g r e s s i o n l i n e t h a t would be o b t a i n e d had m i c r o b i o l o g i c a l assay v a l u e s e q u a l l e d the r a d i o a s s a y  the  values.  - 28 -  F o r t h e r a d i o a s s a y method, t h e b i n d i n g o f each food was f i r s t  c o r r e c t e d . f o r i t s own n o n - s p e c i f i c b i n d i n g  (CPM H P t e G l u bound  where  =  Average  c  o  u  n  =  t  s  - CPM  thus:  )  ( PM^ - CPM^)  X  1  0  0  %  C  per minute f o r extract  sample  CPMgg = A v e r a g e c o u n t s p e r m i n u t e f o r e x t r a c t n CPM  extract  = Average counts p e r minute f o r b i n d i n g  backgrounds  controls  CPMgg = A v e r a g e c o u n t s p e r m i n u t e f o r b a c k g r o u n d  controls.  Where f o o d e x t r a c t b a c k g r o u n d c o u n t s w e r e f o u n d t o b e g r e a t e r t h a n 2 0 % o f t h e e x t r a c t s a m p l e c o u n t s a c o r r e c t i o n was made t o t h e c a l c u l a t i o n o f v a l u e s to take i n t o account the lower t r a c e r concentration a v a i l a b l e f o r binding during the extract  sample i n c u b a t i o n .  T h e f o l l o w i n g e q u a t i o n was u s e d :  (CPM %  H P t e G l u bound  - CPM n  (CPM ( C P ^ - CPM  where  ) x 100%  = B G n  ) x  (  C  P  M  t  - CPM _ C  P  M  b  ) g  )  CPM^, = T o t a l c o u n t s p e r m i n u t e  This equation reduces t o :  (CPM %  Normalized  H P t e G l u bound  =  - CPM  ) x 100%  (CPM^ - C P M ^ ) x % T o t a l Bound T 1 0 0 T BGn  (quench c o r r e c t e d ) c o u n t s p e r m i n u t e were used i n a l l  calculations.  - 29 -  Values  o f f r e e and t o t a l f o l a t e s o b t a i n e d b y t h e two methods o f a s s a y f o r  t h e t h r e e s t r a i n e d b a b y f o o d s a m p l e s a r e shown i n T a b l e I I I .  C o m p a r i s o n o f N^CH^FH^ a n d P t e G l u S t a n d a r d  R e g r e s s i o n a n a l y s e s were p e r f o r m e d curves produced  Curves  on t h e f i v e s e t s o f standard  b y b o t h methods o f a s s a y u s i n g t h e two f o r m s o f f o l a t e .  Results are presented  i n F i g u r e s 6 a n d 7.  were c a r r i e d o u t on t h e d a t a .  P a i r e d comparison  These r e s u l t s a r e p r e s e n t e d  Student's t - t e s t s  i n Table IV.  A s i g n i f i c a n t d i f f e r e n c e i n t h e two s t a n d a r d c u r v e s  i s evident.  T h i s i s p a r t i c u l a r l y t h e c a s e a t h i g h e r c o n c e n t r a t i o n s o f f o l a t e s when t h e r a d i o a s s a y method i s used.  A t c o n c e n t r a t i o n s l o w e r t h a n 6.0 n g / m l t h e  d i f f e r e n c e i n t h e two s t a n d a r d c u r v e s  i s l e s s e v i d e n t i n t h e r a d i o a s s a y method  where s i m i l a r p e r c e n t b i n d i n g v a l u e s a r e f r e q u e n t l y o b t a i n e d f o r b o t h standards.  The d i f f e r e n c e i n t h e g r o w t h r e s p o n s e  o f L. c a s e i w i t h P t e G l u  and N^CH^FH^ i s s i g n i f i c a n t a t a l l c o n c e n t r a t i o n s o f f o l a t e . the case a t lower  This i s particularly  c o n c e n t r a t i o n s o f f o l a t e a l t h o u g h t h e d i f f e r e n c e does n o t  a p p e a r t o be e v i d e n t from  the regression analysis.  I t would appear from  the l i m i t e d data presented  that the milk  p r o t e i n shows a g r e a t e r a f f i n i t y  f o r b i n d i n g t o P t e G l u t h a n t o N^CH^FH^  a t pH 7.3 a n d t h a t P t e G l u c a u s e s  a g r e a t e r growth response  than  o f L_. c a s e i  N CH FH.. 3 4 5  0  N CH FH^ Recovery 5  3  Study  Results of this N^CH^FH^is  s t u d y a r e shown i n T a b l e V.  The r e c o v e r y o f  shown t o b e b e t w e e n 7 5 - 8 0 % f o r t h e l o w e s t c o n c e n t r a t i o n s o f  Table I I I .  F r e e and T o t a l F o l a t e A c t i v i t y  o f Baby F o o d s .  Free Folate A c t i v i t y | ^ng/lOOg p r o d u c t j '  Range  Mean  Total Folate Activity \ug/100g p r o d u c t ]  Range  Mean  MIXED VEGETABLES Radioassay  2.0 - 4.3  3.2  13.0 - 18.0  17.5  L.  4.0 - 4.6  4.2  17.5 - 19.0  18.3  casei  u> o  HAM WITH VEGETABLES Radioassay  2.6 - 5.7  4.9  18.5 - 22.4  21.0  L.  6.5 - 9.5  7.8  10.0 - 13.7  12.6  Radioassay  4.7-7.0  6.5  20.0 - 24.5  22.5  L. c a s e i  8.0 - 1 3 . 5  12.1  26.0 - 29.0  27.0  casei  VEGETABLES AND L I V E R WITH BACON  - 31 -  00 -0-5  00 LOG  F i g u r e 6.  10  0-5 cone  (ngy/mlj  R e g r e s s i o n a n a l y s e s p e r f o r m e d on P t e G l u and N s t a n d a r d c u r v e s o b t a i n e d b y L. assay  method.  2 0  1-5  casei  CH^FH^  microbiological  - 32 -  Table  IV.  P a i r e d c o m p a r i s o n Student's t - t e s t v a l u e s o f P t e G l u a n d N CH„FH,  3 standard  curves  o b t a i n e d by m i c r o b i o l o g i c a l and r a d i o a s s a y  Concentration of Folate 50.00  16.70  4 techniques.  (ng/ml)  5.57  1.85  0.62  9.07  4.06  12.06  MICROBIOLOGICAL ASSAY t  value  degrees o f freedom •P  i  8.10 4 0.01  6.27 4  4  4  4  0.01  0.001  0.02  0.001  7.08  4.74  2.50  3.74  RADIOASSAY t  value  degrees o f freedom P  *  11.51 4 0.001  4 0.01  4 0.02  4 0.10  4 0.05  T a b l e V.  Recovery o f N^CH^FH^ from m i l k e x t r a c t as measured by t h e r a d i o a s s a y  method.  Concentration of N CH FH 5  3  4  added  (ng/lOOul)  Recovery %  5.000  35  1.667  63  0.556  76  0.185  80  0.062  76  - 35 -  a d d e d N^CH^FH^. T h e p e r c e n t  recovery  a d d e d N^CH^FH^ i n c r e a s e s h o w e v e r . high  counting  In a l l cases, 20%  r a t e recorded  drops r a p i d l y as t h e c o n c e n t r a t i o n o f  This  i s due, i n p a r t , t o t h e v e r y  f o rt h e m i l k e x t r a c t background  sample.  t h e m i l k e x t r a c t b a c k g r o u n d was f o u n d t o be g r e a t e r  of the milk extract plus  the c a l c u l a t i o n of values  standard  t o take  sample.  A c o r r e c t i o n was made t o  i n t o account t h e lower  tracer  concentration available for binding during the extract plus sample i n c u b a t i o n .  standard  (See "Comparison o f Free and T o t a l F o l a t e  o f Baby Foods O b t a i n e d U s i n g  than  Levels  Both Methods".)  DISCUSSION  M i c r o b i o l o g i c a l a s s a y s have b e e n t h e most f r e q u e n t l y u s e d methods f o r measuring f o l a t e l e v e l s .  However, such a s s a y s a r e e x t r e m e l y  time  consuming r e q u i r i n g b o t h maintenance o f t h e s t o c k c u l t u r e and an o v e r n i g h t incubation  period. _L. c a s e i h a s b e e n t h e m o s t f r e q u e n t l y u s e d m i c r o o r g a n i s m f o r  folate  assay b u t i t has been found t o be a f f e c t e d by s e v e r a l u n i d e n t i f i e d growth factors  (Stokstad  various other  and H u t c h i n g s ,  1947).  I t i s capable  growth f a c t o r s t o f o l a t e during periods  et: a l . , 1 9 7 1 ) , a n d i t c a n a l s o b e s u b j e c t t o m e t a b o l i c  of converting  of stress  (Baker,  changes such  that  f o l a t e i s no l o n g e r r e q u i r e d .  These f a c t s ,  together with the various  e f f e c t s on growth o f d i f f e r e n t  c o m m e r c i a l m e d i a , t e m p e r a t u r e and t i m e  o f a u t o c l a v i n g , t e m p e r a t u r e a n d t i m e o f i n c u b a t i o n , make t h e u s e o f t h i s method u n s u i t a b l e f o r a c c u r a t e  determination  of folate  levels.  - 36  The  -  r a d i o a s s a y method, by  comparison,  i s rapid  c a r r i e d out w i t h s i m p l e l a b o r a t o r y equipment over Although and  t i m e o f i n c u b a t i o n , pH  1975; not  m i l k f o l a t e b i n d i n g i s i n f l u e n c e d by  Z e t t n e r and D u l y ,  and  1974,  curve  T h u s , a s shown b y  can be  can  such  f a c t o r s as  1975), the e f f e c t s of these factors affecting  these r e s u l t s ,  be  a period of 3 to 4  hours.  temperature  c h a r c o a l p r e p a r a t i o n ( G i v a s and  as g r e a t as t h e a f o r e m e n t i o n e d  method.  and  Gutcho,  factors  are  the m i c r o b i o l o g i c a l  a much m o r e r e p r o d u c i b l e  standard  o b t a i n e d u s i n g t h e r a d i o a s s a y method t h a n by  the m i c r o b i o l o g i c a l  It  s h o u l d be n o t e d  prepared  be  a d i f f e r e n c e i n the f o l a t e b i n d i n g a c t i v i t y of the m i l k p r o t e i n  assay.  t h e r e may f r o m one  k i t t o another  t h a t when u s i n g c o m m e r c i a l l y  causing d i f f e r e n c e s i n standard curves  to  Previous p u b l i c a t i o n s p e r t a i n i n g to the comparison of l e v e l s o b t a i n e d u s i n g t h e r a d i o a s s a y and m i c r o b i o l o g i c a l a s s a y vary considerably i n their i n every and et  case.  conclusions.  o b t a i n e d by  methods,  Serum f o l a t e l e v e l s w e r e c o m p a r e d  G a r d y n a ( 1 9 7 3 ) showed g o o d a g r e e m e n t b e t w e e n t h e two  as d e t e r m i n e d  by  W a d d e l l e t a l . (1976) found  t h e m i c r o b i o l o g i c a l method but  methods.  still  lower  Rudzki  half  than  those  that  o b t a i n e d by  e t a l . (1976) c o n c l u d e d  the  m i c r o b i o l o g i c a l assay  gave t h e more a c c u r a t e i n d i c a t i o n o f f o l a t e  i n s e r u m . I n a l l c a s e s , N^CH^FH^ was  used as s t a n d a r d  method b u t P t e G l u o r an u n i d e n t i f i e d  s t a n d a r d was  that  radioassay  and  curve.  levels  those  m i c r o b i o l o g i c a l assay  of the m i c r o b i o l o g i c a l standard  Mincey  o f f e r e d t h e same d e g r e e  R o t h e n b e r g et_ a l . ( 1 9 7 2 ) f o u n d  f o l a t e c o n c e n t r a t i o n s were a p p r o x i m a t e l y  Tajuddin  t h a t mean s e r u m f o l a t e  t h e r a d i o a s s a y method were s l i g h t l y  of d i a g n o s t i c i n f o r m a t i o n .  occur.  folate  A r c h i b a l d e t a l . ( 1 9 7 2 ) , Waxman e t a l . ( 1 9 7 1 ) a n d  a l . (1973) and  kits,  f o r the  the levels  radioassay  used f o r p r e p a r a t i o n  - 37  It  c a n be  seen from the r e s u l t s o b t a i n e d i n t h i s  t h a t t h e g r o w t h o f L_. c a s e i and dependent, to  -  experiment  the milk p r o t e i n binding a b i l i t y  are  i n p a r t , on t h e form o f f o l a t e p r e s e n t i n t h e s y s t e m .  It is  b e e x p e c t e d , t h e r e f o r e , t h a t c o m p a r i s o n s o f t h e two m e t h o d s o f a s s a y  u s i n g P t e G l u a s s t a n d a r d f o r t h e m i c r o b i o l o g i c a l a s s a y a n d N^CH^FH^ a s standard f o r the radioassay w i l l  yield  different  results.  S i n c e P t e G l u a n d N^CH^FH^ r e a c t d i f f e r e n t l y a s s a y , i t may  i n each system  a l s o be e x p e c t e d t h a t r e s u l t s r e a d f r o m a s t a n d a r d c u r v e  prepared from a s i n g u l a r pure form of f o l a t e w i l l  n o t g i v e an a c c u r a t e  e s t i m a t e of f o l a t e c o n t a i n e d i n a m u l t i p l e system. d o c u m e n t e d t h a t N^CH^FH^ i s t h e p r e d o m i n a n t (Herbert et a l . ,  1962).  I t has been  H o w e v e r , f o r m s o f f o l a t e s o t h e r t h a n N^CH„FH, 3 4  a l s o b e p r e s e n t w h i c h a r e a b l e t o show a d i f f e r e n t b i n d i n g  for  t h e m i l k p r o t e i n o r c a u s e a g r e a t e r g r o w t h r e s p o n s e o f L. partially  coefficient  e x p l a i n why  Shaw et^ a l .  (1974) o b t a i n e d a p o o r  a s s a y e d f o r f o l a t e by b o t h methods.  f o l a t e s h a v e b e e n a s s a y e d u s i n g b o t h _L. c a s e i  the  correlation though  a l s o b e one r e a s o n f o r t h e g r e a t v a r i a t i o n i n t h e  o b t a i n e d when b a b y f o o d was  1972;  S_. f a e c a l i s  casei.  used as s t a n d a r d f o r b o t h a s s a y s .  T h i s may  al.,  affinity  f o r serum f o l a t e l e v e l s measured by b o t h methods even  N^CH„FH. was 3 4  et  well  f o r m o f f o l a t e f o u n d i n serum  may  T h i s may  of  1973;  Butterfield  (Lin et a l . ,  1967;  C a l l o w a y , 1972;  1 9 7 5 ; D o n g a n d O a c e , 1973  response to the d i f f e r e n t  ( S t o k s t a d and K o c h ,  and  (Hoppner,  1971;  Food  Hoppner  and o t h e r ) and and o t h e r s ) .  f o r m s o f f o l a t e v a r y among t h e s e  Dong a n d O a c e , 1 9 7 3 ) .  results  However, organisms  - 38 -  Other f a c t o r s and m a t e r i a l s p r e s e n t  w i t h i n t h e complex food  may a l s o a f f e c t a s s a y r e s u l t s p a r t i c u l a r l y when t h e r a d i o a s s a y  system  method i s  used. Many p r o t e i n s h a v e b e e n shown t o e x h i b i t f o l a t e b i n d i n g Binders  o f f o l a t e and/or bound forms o f f o l a t e have been i d e n t i f i e d  ( G h i t i s , 1 9 6 7 ; M e t z e t a l . , 1 9 6 8 ) a n d many o t h e r b i o l o g i c a l (Rothenberg e t a l . , 1977). basis of the radioassay method. in for  Indeed, t h e a b i l i t y  to bind  Thus t h e p r e s e n c e o f o t h e r  their  specific  i n milk  materials  f o l a t e forms t h e  method and p o s s i b l y a l s o , t h e m i c r o b i o l o g i c a l a s s a y proteins with  folate binding  t h e s y s t e m t o b e a s s a y e d , may c a u s e e r r o r s u n l e s s  Gardyna  properties.  folate binding a b i l i t y .  ability  c o r r e c t i o n s a r e made  For t h i s reason,  (1973) s u g g e s t e d t h e a d d i t i o n o f a serum b l a n k  Tajuddin  and  to correct f o r  3 non-specific binding a b i l i t y (1969) s t a t e d t h a t  H  heating milk  bound f o l a t e and d e s t r o y e d (1973) e x t r a c t e d  of  their  released  the protein binding a b i l i t y .  serum samples b y b o i l i n g  t h e bound f o l a t e .  a g e n t was a d d e d t o t h e i r  extraction process involved heating  t h e serum  binder  reducing  heating).  food m a t e r i a l s described  t h e food  the protein  i n a w a t e r b a t h f o r 15 m i n u t e s  ( I t i s i n t e r e s t i n g t o n o t e t h a t no  serum e x t r a c t s d u r i n g  eta l . ,  T h u s , Dunn a n d F o s t e r  This procedure denatured  I n t h e two e x p e r i m e n t s u s i n g  w a t e r b a t h and a s s a y i n g  t o the assay material.Ford  f o r 10 m i n u t e s a t 100°C r e l e a s e d  i n 0.05 M l y s i n e b u f f e r , pH 1 0 . 5 . and  PteGlu  here, the  s a m p l e s f o r 10 m i n u t e s i n a b o i l i n g  an e x t r a c t background sample.  Thus, t h e f o l a t e  b i n d i n g p r o t e i n s n o t denatured by h e a t i n g were compensated f o r i n t h e background sample. vegetables,  I n a l l foods tested w i t h  the exception  o f t h e mixed  t h e e x t r a c t background counts were found t o be g r e a t e r  20%  o f t h e e x t r a c t sample counts;  not  sufficient  to destroy  i n d i c a t i n g that the heating  a l l the protein binding  activity.  than  p r o c e s s was Therefore,  a  - 39 -  c o r r e c t i o n was made t o t h e c a l c u l a t i o n o f p e r c e n t food  sample.  the v a l i d i t y  High e x t r a c t background counts  H  PGA b o u n d t o e a c h  however, tend  t o decrease  o f t h e assay.  O t h e r f a c t o r s s u c h a s pH, m e t a l pro- o r a n t i - oxidants w i t h i n t h e food  c a t a l y s t s and t h e p r e s e n c e o f  s y s t e m may a l s o a f f e c t  the v a l i d i t y  of t h e assay. The r e c o v e r y o f N^CH^FH^ f r o m a however, t h a t p a r t i c u l a r l y procedure i sv a l i d . folate,  a t lower  food  system does i n d i c a t e  concentrations of f o l a t e the experimental  A s shown i n t h i s e x p e r i m e n t ^ a t  high concentrations of  t h e f o l a t e b i n d i n g p r o t e i n becomes s a t u r a t e d and i s t h u s u n a b l e t o  bind a l l the f o l a t e present  i n t h e sample thus  decreasing  the v a l i d i t y of  t h e method.  CONCLUSIONS  These e x p e r i m e n t s d e m o n s t r a t e t h a t t h e r a d i o a s s a y method described i sa faster, o f N^CH^FH^ t h a n curves  herein  s i m p l e r a n d more a c c u r a t e method f o r m e a s u r i n g  theJL,. c a s e i m i c r o b i o l o g i c a l m e t h o d .  When N^CH^FH^  levels standard  a r e u s e d , c o n c e n t r a t i o n s o f b u f f e r e d N^CH^FH^ a s d e t e r m i n e d b y t h e  r a d i o a s s a y method compare w e l l w i t h c o n c e n t r a t i o n s o b t a i n e d b y t h e L. c a s e i m i c r o b i o l o g i c a l assay.  When f r e e a n d t o t a l f o l a t e l e v e l s i n s t r a i n e d b a b y  f o o d s w e r e a s s a y e d r e s u l t s o b t a i n e d b y t h e two methods d i d n o t compare When t h e r a d i o a s s a y m e t h o d o f f o l a t e a s s a y t h e r e i s a marked d i f f e r e n c e i n t h e a b i l i t y P t e G l u a n d N^CH^FH^.  well.  i s c a r r i e d o u t a t pH  7.3  of t h e milk p r o t e i n t o bind  S i m i l a r l y L. c a s e i a l s o e x h i b i t s a d i f f e r e n c e i n i t s  g r o w t h r e s p o n s e t o t h e two forms o f f o l a t e .  T h e two a s s a y  systems t h e r e f o r e ,  when c a r r i e d o u t b y t h e m e t h o d s h e r e i n d e s c r i b e d , a r e s u i t a b l e o n l y f o r  - 40 -  measuring s i n g u l a r forms o f f o l a t e u s i n g data  obtained  using  t h e same  standard  curves  constructed  folate.  N^CH^FH^ a n d P t e G l u  are just  two o f t h e many f o r m s o f f o l a t e  h a v e b e e n i d e n t i f i e d . O t h e r f o r m s may show g r e a t e r o r l e s s e r b i n d i n g and  from  that  affinities  may e x h i b i t f u r t h e r d i f f e r e n c e s i n t h e g r o w t h r e s p o n s e o f m i c r o o r g a n i s m s .  I n a d d i t i o n , t h e p r e s e n c e o f two o r more f o l a t e t y p e s m a t e r i a l may a l s o c a u s e v a r i a t i o n s .  together  i n a n y one  I t becomes o b v i o u s t h e r e f o r e , t h a t t h e  f o l a t e l e v e l s o f f o o d s t u f f s w h i c h a r e k n o w n t o c o n t a i n many f o r m s o f f o l a t e cannot be e s t i m a t e d  a c c u r a t e l y by e i t h e r o f t h e two m e t h o d s d e s c r i b e d  until  more i n f o r m a t i o n p e r t a i n i n g t o t h e number, f o r m s and d i s t r i b u t i o n o f f o l a t e i n f o o d s t u f f s and t h e i n t e r a c t i o n o f t h e v a r i o u s forms w i t h each o t h e r and with b i o l o g i c a l materials i s obtained. At best, p r e s e n t l y a v a i l a b l e data be  compared t o o t h e r v a l u e s  be  recognized  obtained  on f o o d  folate levels  by an i d e n t i c a l method.  t h a t any f o l a t e c o n c e n t r a t i o n v a l u e s  can only  I t should  also  a v a i l a b l e a t t h i s t i m e may  not n e c e s s a r i l y be a r e f l e c t i o n o f t h e i r n u t r i t i o n a l  s i g n i f i c a n c e t o man.  - 41 -  CHAPTER I I I .  K I N E T I C STUDIES  INTRODUCTION  Q u a l i t y d e t e r i o r a t i o n i n the form of n u t r i t i o n a l can occur  i n any  food during heat processing.  The  degradation  r a t e s of the r e a c t i o n s  r e s p o n s i b l e f o r n u t r i t i o n a l d e g r a d a t i o n a r e dependent upon such as t e m p e r a t u r e and p r e s e n c e oxidants. of  and  l e n g t h o f t i m e o f h e a t i n g , amount o f o x y g e n  or absence of other chemicals  The  factors  presence  such as p r o - o r  present  anti-  o r a b s e n c e o f o t h e r c h e m i c a l s makes t h e  prediction  n u t r i t i o n a l d e g r a d a t i o n p a r t i c u l a r l y d i f f i c u l t s i n c e w i t h i n most  systems >  t h e r e a r e many s u b s t a n c e s  present which  food  i n f l u e n c e the r a t e of  degradation of the v a r i o u s n u t r i e n t s . Very  little  r e s e a r c h has  been conducted  p r o c e s s i n g on f o l a t e c o n t e n t o f f o o d s . et  a l . (1946),  (1966), Herbert Malin  have found by  C h e l d i n et a l . (1943),  Schweigert  S t o k s t a d ^ t a l . ( 1 9 4 7 ) , H a n n i n g and M i t t s ( 1 9 4 9 ) , (1967,  1968b), Ford et a l . (1968),  stability  Schroeder  of f o l a t e s w i t h i n d i f f e r e n t  Ghitis,  (1971)  food systems  t h e m t o b e v e r y s u s c e p t i b l e to ^ d e g r a d a t i o n b y  the a c t i o n of u l t r a v i o l e t  light.Herbert  t h a t a s much a s 9 5 % o f t h e i n i t i a l  f o l a t e s i n food c o u l d be  e f f e c t s o f UHT  s u b s e q u e n t s t o r a g e o f m i l k on  content, concluded  lost  a l . (1968), i n v e s t i g a t i n g  that v a r i a t i o n i n the s t a b i l i t y  r e l a t e d d i r e c t l y to the presence  on and  (1968b) e s t i m a t e d  Ford  p r o c e s s i n g and  and  oxidative heating  oxidative heating processes.  of  of  (1977) have s t u d i e d t h e e f f e c t s o f v a r i o u s p r o c e s s i n g methods  the n u t r i t i o n a l  and  on t h e e f f e c t  by the  the v i t a m i n  of f o l a t e s  was  of oxygen i n the m i l k , the d e a e r a t i o n  m i l k b e f o r e p r o c e s s i n g e f f e c t i v e l y r e d u c i n g the v i t a m i n l o s s .  - 42 -  N CH^FH^ i s a f o r m o f f o l a t e r e p o r t e d t o b e v e r y s u s c e p t i b l e t o o x i d a t i o n b y v a r i o u s means ( W a t e r s found  a heat  l a b i l e f r a c t i o n present  f r a c t i o n c o u l d be p r o t e c t e d from acid although the author  found  and M o l l i n ,  o x i d a t i o n by t h e presence  The  N^CH^FH^  complexity o f foods  m e a s u r e d , makes i t  difficult  The  of ascorbic  folate fraction  present during  m i c r o b i o l o g i c a l assay  method  levels.  i n which  to predict  of f o l a t e w i t h i n a food system  (1966)  that levels of ascorbate n a t u r a l l y  G h i t i s used t h e L a c t o b a c i l l u s c a s e i  f o r q u a n t i t a t i v e l y measuring  Ghitis  i n m i l k r e s e m b l i n g N^CH^FH^.  i n m i l k were n o t s u f f i c i e n t t o p r o t e c t t h e l a b i l e boiling.  1961).  f o l a t e l e v e l s have been  the thermal degradation  o t h e r t h a n t h e one t e s t e d .  characteristics  Several recent  s t u d i e s have i n v o l v e d measurement o f o t h e r n u t r i e n t d e g r a d a t i o n r a t e s u s i n g model systems. carried  Examples o f such  s t u d i e s a r e those o f thiamine  degradation  o u t b y M u l l e y e t a l . (1975a, b ) . I n t h i s s e c t i o n , a s t u d y o f t h e r e a c t i o n k i n e t i c s o f N^CH^FH^  d e g r a d a t i o n b y h e a t i n g i n o x y g e n was c a r r i e d o u t u s i n g a s i m p l e m o d e l system  o f N^CH FH i n p h o s p h a t e b u f f e r . j 4  i n Chapter  I I was u s e d f o r m e a s u r i n g  The r a d i o a s s a y method a s d e s c r i b e d  N^CH^FH^ c o n c e n t r a t i o n s .  d e g r a d a t i o n o f N^CH^FH^ b y h e a t i n g i n o x y g e n was t h o u g h t r e a c t i o n , a n attempt the above experiment assayed,  was made t o i d e n t i f y  t o be an o x i d a t i o n  t h e r e a c t i o n mechanism b y r e p e a t i n g  i n t h e a b s e n c e o f a n y r e d u c i n g a g e n t . N ^ C H ^ F H ^ was  t h e r e f o r e , i n t h e absence o f mercaptoethanol.  Singh e t a l .  ( 1 9 7 6 ) h a v e shown t h a t t h e o v e r a l l r e a c t i o n o f a s c o r b i c a c i d and  oxygen uptake  f o l l o w s second  o f N^CH^FH^ i n t h e p r e s e n c e also studied.  Since the  order reaction ,kinetics.  of a limited  degradation  The d e g r a d a t i o n  o x y g e n s u p p l y was t h e r e f o r e  - 43  In  -  order to study the e f f e c t s of a heat treatment, i t i s  d e s i r a b l e t o o b t a i n n e a r l y i n s t a n t a n e o u s and u n i f o r m h e a t i n g t o t h e r e q u i r e d temperature and, a f t e r  a h o l d i n g time at the r e q u i r e d  a n e a r l y i n s t a n t a n e o u s c o o l i n g t o room t e m p e r a t u r e . ( 1 9 7 6 ) d e v e l o p e d a new  Beumer of  They found t h a t  the  t i m e o f t h e pouch compared f a v o r a b l y w i t h t h e c o n v e n t i o n a l  thermal death time tube. in  E r d t s i c k and  method f o r d e t e r m i n i n g t h e h e a t r e s i s t a n c e  m i c r o - o r g a n i s m s u s i n g a t h e r m a l d e a t h t i m e pouch. come-up  temperature,  the i n i t i a l  kinetic  T h u s , t h e t h e r m a l d e a t h t i m e p o u c h was  studies  used  reported here.  METHODS  P r e p a r a t i o n o f N C H FH  Solutions  5  S o l u t i o n s o f N CH^FH^ w e r e made up pH  7.3.  kinetic  The  studies.  standard  Kinetic  approximate  i n 0.1  s t a r t i n g c o n c e n t r a t i o n was  M phosphate  buffer  35 u g / m l f o r a l l  No m e r c a p t o e t h a n o l o r a s c o r b i c a c i d was  added t o t h e  solutions.  S t u d i e s U s i n g Thermal  Death  Time Pouches  P o u c h e s w e r e p r e p a r e d f r o m 40 yna. R i l s a n ( A m e r i c a n Company, A t l a n t a , USA) sausages.  a m a t e r i a l n o r m a l l y used  From t h i s m a t e r i a l , r e c t a n g u l a r f l a t  b e t w e e n s e a l s ) w e r e made. solution.  After f i l l i n g ,  Each  Lecithin  f o r m a k i n g meat a n d pouches  e n v e l o p e c o n t a i n e d 1.5  (4 cm x 6  agar  cm  m l o f N^CH^FH^  t h e e n v e l o p e s were s e a l e d and used  f o r the  - 44 -  d e t e r m i n a t i o n o f N CH^FH^ d e g r a d a t i o n c u r v e s . stretched f l a t seal  The pouches were  i n a v e r t i c a l p o s i t i o n by weights a t t a c h e d t o t h e lower  ( F i g . 8 ) . By a p p l y i n g t h i s p r o c e d u r e  t o b e l e s s t h a n 1 mm. weredetermined  prior  t h e p o u c h t h i c k n e s s was f o u n d  H e a t i n g and c o o l i n g l a g times i n t h e pouches to nutrient destruction studies  (Appendix C ) .  P o u c h e s c o n t a i n i n g N^CH^FH^ s o l u t i o n w e r e h e a t e d i n a water bath.  t o 100°C  C o n c e n t r a t i o n s o f N^CH^FH^ a f t e r v a r i o u s t i m e s o f h e a t i n g  were^assayed u s i n g t h e r a d i o a s s a y method as p r e v i o u s l y d e s c r i b e d .  Kinetic  S t u d i e s U s i n g U n l i m i t e d Oxygen  Supply  A g l a s s f l a s k was p l a c e d i n a n a g i t a t e d w a t e r b a t h . was  condenser  p l a c e d on t h e t o p o f t h e f l a s k and a tube c a r r y i n g oxygen t o t h e base  o f t h e f l a s k was i n t r o d u c e d t h r o u g h a n a r m ( F i g . 9 ) . i n 0.1 M p h o s p h a t e was  A  bubbled  buffer  S a m p l e s o f N^CH^FH^  (pH 7.3) w e r e p l a c e d i n t h e f l a s k a n d o x y g e n  c o n s t a n t l y t h r o u g h t h e s a m p l e w h i l e i t was h e a t e d  temperatures.  A l l apparatus were k e p t i n d i f f u s e l i g h t  N^CH^FH^ d e g r a d a t i o n d u e t o u l t r a v i o l e t samples were removed f r o m t h e f l a s k ,  radiation.  to various  t o minimize  D u p l i c a t e 100 ;ul  a t r e g u l a r i n t e r v a l s and assayed  by t h e r a d i o a s s a y method. N CH FH 5  3  40°C.  4  samples were heated  t o 1 0 0 ° , 9 0 ° , 7 5 ° , 6 0 ° , 55° a n d  P r e l i m i n a r y studies s i m i l a r t o those o u t l i n e d  i n Appendix  C  i n d i c a t e d t h a t h e a t i n g and c o o l i n g l a g t i m e s were n e g l i g i b l e i n t h i s experiment.  - 45 -  Figure  8.  Thermal death t i m e pouch w i t h w e i g h t  attached.  - 46 -  condenser  oxygen  i  water bath  sample  F i g u r e 9.  Apparatus used f o r k i n e t i c nutritional  s t u d i e s of the  d e g r a d a t i o n o f N~*CH„FH. .  3  4  - 47 -  K i n e t i c S t u d i e s U s i n g U n l i m i t e d Oxygen S u p p l y and a M e r c a p t o e t h a n o l - F r e e Assay System  S a m p l e s o f N^CH^FH^ w e r e t r e a t e d  i n a n i d e n t i c a l manner t o  that described i n the previous experiment. h o w e v e r , no m e r c a p t o e t h a n o l was u s e d protein or the t r i t i a t e d PteGlu. in diffuse light  I n the assay  system,  i n the buffer, the milk binding  A l l r e a g e n t s were k e p t on i c e and  throughout t h e experiment  to minimize degradation.  N^CH^FH^ s a m p l e s w e r e h e a t e d t o 9 0 , 7 5 , a n d 40°C i n t h i s  Kinetic  S t u d i e s U s i n g L i m i t e d Oxygen  Supply  Since a dissolved-oxygen meter I n s t r u m e n t C o . , O h i o ) was u s e d  ( M o d e l 57 Y e l l o w S p r i n g s  f o r measuring  N^CH^FH^ s a m p l e s w e r e d i s s o l v e d  experiment.  changes i n oxygen  i n tris/NaCl buffer  concentration,  (pH 7.3) f o r t h i s  experiment.  Samples o f b u f f e r were b u b b l e d w i t h oxygen o r d e a e r a t e d t o  give i n i t i a l  o x y g e n c o n c e n t r a t i o n s o f 6.3, 9.6 a n d 14.4 ppm b y v o l u m e .  N^CH^FH^ was t h e n a d d e d t o e a c h b u f f e r a n d t h e o x y g e n l e v e l s r e c o r d e d . The  samples were t h e n drawn up i n t o h y p o d e r m i c  syringes which  were  s u b s e q u e n t l y s e a l e d and p l a c e d i n a c o n s t a n t l y s t i r r e d water b a t h a t 20°C.  N^CH^FH^ a n d o x y g e n c o n c e n t r a t i o n s w e r e m e a s u r e d a t i n t e r v a l s . The  r a d i o a s s a y s y s t e m c o n t a i n i n g m e r c a p t o e t h a n o l was u s e d f o r  e v a l u a t i n g N^CH~FH, c o n c e n t r a t i o n s . 3 4 b u f f e r a t 20° C w i t h a n u n l i m i t e d t h e same t i m e  period.  A s a m p l e o f N^CH„FH. i n t r i s / N a C l J 4  s u p p l y o f o x y g e n was a l s o a s s a y e d  over  - 48 -  THEORETICAL  CONSIDERATIONS  F i r s t Order  Reactions  Experimental r e s u l t s indicated that the degradation of N^CH^FH^ ( i n t h e p r e s e n c e first  o r d e r mechanism.  o f an u n l i m i t e d oxygen s u p p l y )  T h e r e a c t i o n scheme a t a c o n s t a n t  follows a temperature  and p r e s s u r e may t h u s b e w r i t t e n :  ->  (degradation  (N C H F H ) 3  4  (1)  Product  product)  where k i s t h e r a t e c o n s t a n t . T h u s t h e r a t e o f r e a c t i o n may b e e x p r e s s e d a s : - d =  .k'  ng  £ A J  ml  min  (2)  dt  w h i c h may b e i n t e g r a t e d a n d r e a r r a n g e d t o  In  where  | A  o  is the i n i t i a l  L A J  o  . -1 mm  c o n c e n t r a t i o n o f N CH„FH. a n d 3 4  equals the concentration a f t e r  t minutes  (3)  Al  - 49 -  However,  should oxygen be i n v o l v e d i n t h e d e g r a d a t i o n o f  N^CH„FH, then t h e r e a c t i o n must be c o n s i d e r e d  3  4  t o beJ pseudo f i r s t  order.  T h i s i s the case s i n c e t h e c o n c e n t r a t i o n o f oxygen i s v e r y much g r e a t e r than t h a t o f N^CH^FH^ and t h e r e f o r e t h e change i n i t s c o n c e n t r a t i o n i s negligible  compared w i t h t h e change i n c o n c e n t r a t i o n o f t h e o t h e r  reactant present.  Thus, the o v e r a l l o r d e r i s reduced.  T h i s r e a c t i o n scheme i s w r i t t e n : k A  +  (N CH FH ) 5  B  »  (0 )  (4)  Product  (degradation  product)  where k i s the r a t e c o n s t a n t . The r a t e o f r e a c t i o n may be expressed as  d  [product] L Product, dt  ng  ml  mxn  (5)  If t h e oxygen c o n c e n t r a t i o n i s e s s e n t i a l l y unchanged throughout the r e a c t i o n compared t o the c o n c e n t r a t i o n o f N^CH^FH^, then i t i s regarded constant  as a  and t h e r a t e e x p r e s s i o n i s w r i t t e n  d  I ProductJ dt  =  k'  -.-1 ng  ml  . -1 mm  (6)  where k'  = k  mm  -1  (7)  - 50 -  Thus t h e r e a c t i o n e x h i b i t s f i r s t  order k i n e t i c s although i t involves  more t h a n one r e a c t a n t and i s r e f e r r e d first  The  order  reaction.  Arrhenius  Equation  Arrhenius of  t o a c c o r d i n g l y as a pseudo  (1889) found  the rate constant  k = Ae"  that t h e e x p e r i m e n t a l l y observed  (k) w i t h a b s o l u t e temperature  (T) c a n be  expressed:  (8)  E / R T  where A ( t h e f r e q u e n c y  f a c t o r ) and E ( t h e a c t i v a t i o n energy) a r e c o n s t a n t s  for  t h e p a r t i c u l a r r e a c t i o n and R i s t h e u n i v e r s a l gas c o n s t a n t .  and  E a r e determined  and  E a r e known f o r a r e a c t i o n ,  temperature  Second Order  i t s r a t e may b e c a l c u l a t e d  Once A  f o r any  Reactions  of a limited  t h a t t h e d e g r a d a t i o n o f N^CH^FH^  oxygen s u p p l y ) c o u l d f o l l o w a second  order  I n t h i s c a s e , t h e r e a c t i o n scheme i s a s f o l l o w s : ,.k A  A  from t h e above e q u a t i o n .  the presence  mechanism.  Both  by t h e p r o p e r t i e s o f t h e r e a c t i n g m o l e c u l e s .  Experimental results indicated (in  variation  +  B  (N^CH^FH^) ( o x y g e n )  where k i s t h e r a t e c o n s t a n t .  »  Product  (degradation  (9) product)  - 51 -  If  t h e d i s s o l v e d oxygen, B, undergoes a second  reaction of f i n i t e  s p e e d w i t h t h e d i s s o l v e d N^CH^FH^, A , t h e r a t e  of r e a c t i o n can be expressed  -d dt  Let £AJ  t  |VJ  A  =  and  and^BJ  ^  order  as:  k  £BJ ^  BJ  ng  ml ^  be t h e i n i t i a l  min ^  (10)  c o n c e n t r a t i o n o f r e a c t a n t s and  be t h e c o n c e n t r a t i o n o f r e a c t a n t s a t t i m e ' t ' . L e t  X b e t h e amount o f A o r B t h a t h a s r e a c t e d  ' t ' . Then a t any t i m e  [X  A  t  at time  . J o  '] t - H o -  (ID  M  (12)  Therefore,  -44  Substituting into the rate equation  -d  [A]  (13)  dt  dt  T  d  dt  I n t e g r a t i o n from t = 0 gives  gives  [x] dt  k  (W o - M)(H  O  - H]  <14)  t h e c o n c e n t r a t i o n o f A o r B o r X a t any t i m e  - 52 -  RESULTS  I t s h o u l d be n o t e d t h a t throughout t h e s e e x p e r i m e n t s , an 3 increase i n the binding of  H  PteGlu to the milk protein i n the  r a d i o a s s a y system i s r e f e r r e d t o as a d e c r e a s e i n t h e c o n c e n t r a t i o n o f N^CH^FH^ as determined from t h e r a d i o a s s a y s t a n d a r d c u r v e .  Thus, t h e  assumption i s made t h a t t h e d e g r a d a t i o n p r o d u c t o f N^CH^FH^ does n o t bind to the m i l k p r o t e i n . K i n e t i c S t u d i e s U s i n g Thermal Death Time Pouches  5  o  The d e g r a d a t i o n c u r v e f o r N CH^FH^ a t 100 C i n phosphate  buffer  (pH 7.3) i s shown i n F i g . 10 as a s e m i l o g p l o t o f N^CH^FH^ r e t a i n e d v e r s u s h e a t i n g time a t c o n s t a n t temperature.  Because i t was d i f f i c u l t  t o e l i m i n a t e a l l a i r from t h e pouches d u r i n g s e a l i n g , and because oxygen i s a b l e t o d i f f u s e s l o w l y t h r o u g h n y l o n i t was found t o be i m p o s s i b l e t o a c c u r a t e l y m o n i t o r t h e amount o f oxygen p r e s e n t i n t h e system  throughout  - 53 -  - 54  the heating period.  -  S i n c e o x y g e n i s t h o u g h t t o be  important i n the  n u t r i t i o n a l d e g r a d a t i o n o f N^CH^FH^, t h e t h e r m a l d e a t h t i m e p o u c h e s w e r e o n l y u s e d i n t h i s p r e l i m i n a r y s t u d y t o i n d i c a t e t h e f a c t t h a t N^CH^FH^ i n d e e d d e g r a d e s when h e a t e d t o 100°C.  K i n e t i c S t u d i e s U s i n g U n l i m i t e d Oxygen  The 55 and  Supply  r a t e of degradation curves f o r N CH FH 5  3  40°C i n p h o s p h a t e b u f f e r  (pH 7.3)  4  a t 100,  Two  stages to the r e a c t i o n are apparent.  The  be  first  part  The  second p a r t of t h e r e a c t i o n  along the s l o p e p o r t i o n of the curve  (shown a s Y-Z  of  (shown a s  f o r 40°C) w h e r e no a p p r e c i a b l e d i f f e r e n c e i n p r o t e i n b i n d i n g  concentration) i s occurring.  60,  seen i n  the r e a c t i o n occurs along the h o r i z o n t a l p o r t i o n of the curve X-Y  75,  ( a s a s e m i l o g p l o t o f N^CH^FH^  r e t a i n e d v e r s u s h e a t i n g t i m e a t c o n s t a n t t e m p e r a t u r e ) may F i g u r e 11.  90,  (N^CH^FH  occurs  f o r 40°C) w h e r e  an  i n c r e a s e i n p r o t e i n b i n d i n g t a k e s p l a c e a s shown b y a d e c r e a s e i n f o l a t e -concentration.  The H o r i z o n t a l  The  X-Y  l e n g t h o f t i m e f r o m X t o Y was  temperature of r e a c t i o n decreased.  found t o i n c r e a s e as  A p l o t of temperature of  reaction  v e r s u s l o g t i m e o f r e a c t i o n i s shown i n F i g u r e 12 a n d i n d i c a t e s these i n t e r v a l s are temperature  dependent.  the  that  -  F i g u r e 11.  55  -  D e g r a d a t i o n o f N CH^FH^ heated t o v a r i o u s i n an u n l i m i t e d oxygen s u p p l y .  temperatures  - 56 -  - 57 -  The S l o p e s  Y-Z  S i n c e oxygen has been c i t e d as necessary  f o r the degradation  o f N^CH^FH^, t h e d a t a u s e d t o c o n s t r u c t t h e s l o p i n g p o r t i o n o f t h e c u r v e s were p l o t t e d a c c o r d i n g t o a pseudo f i r s t temperature.  order k i n e t i c equation f o r each  The r e g r e s s i o n a n a l y s e s f o r t h e d a t a a r e i l l u s t r a t e d  The h i g h c o e f f i c i e n t s o f d e t e r m i n a t i o n o b t a i n e d i n d i c a t e t h a t t h e r e a c t i o n f o l l o w s pseudo f i r s t  i n Fig.13.  (Table VI)  order r e a c t i o n k i n e t i c s .  A s d i s c u s s e d e a r l i e r , t h e r e a c t i o n i s r e f e r r e d t o a s 'pseudo f i r s t s i n c e o x y g e n was p r e s e n t and in  i n an abundant s u p p l y throughout  order'  thereaction  t h u s i t s c h a n g e i n c o n c e n t r a t i o n was n e g l i g i b l e c o m p a r e d t o t h e c h a n g e c o n c e n t r a t i o n o f N^CH^FH^ m o l e c u l e s  first  present.  order r a t e constants f o rt h i s experiment A plot of the rate constants  r e a c t i o n i s shown i n F i g u r e 14. i n c r e a s e as t h e temperature  a r e reported i n Table V I .  (k')against temperature  f a c t o r ) was f o u n d  a c t i v a t i o n e n e r g y ) was f o u n d  The s t r a i g h t l i n e o b t a i n e d  t o b e 1440 m i n  t o b e 7.10 k c a l m o l e \  RT 1  T h i s r a t e e x p r e s s i o n i s f o r a pseudo f i r s t  order  From t h e s e  data,  Thus t h e c o m p l e t e  -7100  min"  demonstrates  while E (the  r a t e e x p r e s s i o n may b e w r i t t e n .  k' = 1440 e  of  o f t h e r e a c t i o n was r a i s e d , a n A r r h e n i u s - t y p e  of the Arrhenius equation i n t h i s reaction.  A (the frequency  pseudo  S i n c e t h e r a t e c o n s t a n t was f o u n d t o  p l o t was c o n s t r u c t e d ( F i g u r e 1 5 ) . the v a l i d i t y  The c a l c u l a t e d  reaction.  - 58 -  3-5  0  25  50  75 TIME  F i g u r e 13.  100  125  (minutes)  R e g r e s s i o n a n a l y s e s performed on d a t a p r e s e n t e d i n F i g u r e 11 a c c o r d i n g t o pseudo f i r s t o r d e r r e a c t i o n equation (6).  150  Table VI.  Pseudo f i r s t o r d e r r a t e c o n s t a n t s i n an abundant.supply o f oxygen.  Rate  Temperature of  Reaction  ( k ' j , o f N CH^FH^ d e g r a d a t i o n  Intercept  Coefficient  Constant (min  1  ]  of determination  fr ] 2  0.012  0.108  0.96  90  0.009  0.076  0.96  75  0.011  0.053  0.96  60  0.032  0.028  0.94  55  0.087  0.026  0.96  40  0.056  0.018  0.96  100  - 60 -  - 61 -  F i g u r e 15.  P l o t o f d a t a i n F i g u r e 14 a c c o r d i n g t o A r r h e n i u s equation (8).  - 62 -  K i n e t i c S t u d i e s U s i n g U n l i m i t e d Oxygen Supply and a M e r c a p t o e t h a n o l - F r e e Assay System  The r a t e o f d e g r a d a t i o n c u r v e s f o r N~*CH„FH, a t AO, 75 and 90°C 3 4 i n phosphate b u f f e r (pH 7.3) measured i n a m e r c a p t o e t h a n o l  - f r e e assay  a r e shown a s a s e m i l o g p l o t o f N^CH^FH^ r e t a i n e d v e r s u s h e a t i n g time a t c o n s t a n t temperature  i n F i g u r e 16.  The d e g r a d a t i o n o f N^CH^FH^ i s observed t o o c c u r  immediately  when samples a r e heated t o 90° and 75°C i n t h e p r e s e n c e o f an u n l i m i t e d oxygen s u p p l y .  D e g r a d a t i o n o c c u r s a f t e r 90 minutes i n t h e sample heated t o  40°C. A pseudo f i r s t o r d e r . k i n e t i c e q u a t i o n was a p p l i e d t o a l l t h e d a t a o f t h e samples heated t o 75° and 90°C and t o d a t a r e c o r d e d a f t e r 90 minutes i n t h e sample heated t o 40°C. d a t a a r e shown i n F i g u r e 17.  L i n e a r r e g r e s s i o n analyses of the  The h i g h c o e f f i c i e n t s o f d e t e r m i n a t i o n o b t a i n e d  (Table V I I ) i n d i c a t e t h a t t h e r e a c t i o n f o l l o w s pseudo f i r s t o r d e r r e a c t i o n .kinetics.  The c a l c u l a t e d pseudo f i r s t o r d e r r a t e c o n s t a n t s ( k ' ) a r e  r e p o r t e d i n T a b l e V I I and a r e observed t o be v e r y s i m i l a r t o t h e v a l u e s o b t a i n e d when t h e N^CH^FH^ was assayed i n a system c o n t a i n i n g m e r c a p t o e t h a n o l .  K i n e t i c S t u d i e s U s i n g a L i m i t e d Oxygen Supply  The r a t e o f d e g r a d a t i o n c u r v e s , f o r N^CH^FH^ a t 20°C i n phosphate b u f f e r (pH 7.3) and i n t h e presence o f a l i m i t e d oxygen s u p p l y a r e shown i n F i g u r e 18.  The f i g u r e a l s o i n c l u d e s r e s u l t s f o r samples i n w h i c h  d i s s o l v e d oxygen was m a i n t a i n e d a t l e v e l s above s a t u r a t i o n .  - 63 -  0-00  •H5r-  2-30h  -3-45 0  180  90 TIME F i g u r e 16.  (minutes)  D e g r a d a t i o n o f N CH^FH^ h e a t e d t o v a r i o u s t e m p e r a t u r e s i n an u n l i m i t e d oxygen s u p p l y and assayed u s i n g a mercaptoethanol-free  system.  270  - 64 -  0  25  75  50 TIME  F i g u r e 17.  100  v  40°  •  75°  o  90°  125  (minutes)  R e g r e s s i o n a n a l y s e s performed on d a t a p r e s e n t e d i n F i g u r e 16 a c c o r d i n g t o pseudo f i r s t o r d e r r e a c t i o n e q u a t i o n (6) . s  150  Table V I I .  P s e u d o f i r s t o r d e r r a t e c o n s t a n t s ( k ' ) o f N CH^FH^ d e g r a d a t i o n i n an abundant s u p p l y o f oxygen measured by a m e r c a p t o e t h a n o l - f r e e assay system.  Rate  Temperature of  Reaction  Intercept  Constant  . -1  lo.  Coefficient of  Determination 21  mm  40  0.060  0.018  0.96  75  0.062  0.057  0.94  90  -0.012  0.077  0.96  S i  - 66 -  o-oo  -115  -2-30  Concentration • •  Unlimited 14-4 p p m  0  9-6ppm  A  6 -3 p p m  -3-45 0  6  12 TIME  F i g u r e 18.  18  (hours)  I n f l u e n c e o f d i s s o l v e d oxygen c o n t e n t on N^CH^FH^ d e g r a d a t i o n a t 20°C.  24  -  As is  in  p r e v i o u s l y o b s e r v e d , t h e c o n c e n t r a t i o n o f N^CH^FH^  essentially  first  67 -  constant  and does n o t change a p p r e c i a b l y d u r i n g t h e  s i x to e i g h t hours o f t h e experiment  the radioassay  (mercaptoethanol  was  present  system).  A p l o t o f change i n c o n c e n t r a t i o n o f oxygen t h r o u g h o u t t h e d u r a t i o n o f t h e experiment, however, i n d i c a t e s t h a t oxygen was  d e c r e a s i n g more r a p i d l y d u r i n g t h e f i r s t  concentration  6 t o 8 hours than  during  the remainder o f the experiment  (Figure 19).  oxidation reaction taking place  t h r o u g h o u t t h e 23 h o u r s o f t h e e x p e r i m e n t  but  T h i s i s i n d i c a t i v e o f an  the rate of o x i d a t i o n decreases a f t e r the f i r s t The  degradation  of d i s s o l v e d oxygen. of degradation  o f N^CH^FH^ a p p e a r s t o d e p e n d o n t h e c o n c e n t r a t i o n  This  i s o b s e r v e d f r o m F i g u r e 18 w h e r e t h e r a t e  o f N^CH^FH^ i s f a s t e r f o r h i g h e r  concentration.  6 t o 8 hours.  initial  d i s s o l v e d oxygen  I t i s a l s o apparent that i n t h e presence of a l i m i t e d  oxygen supply  t h e d a t a do n o t f i t a s t r a i g h t l i n e p l o t o n  coordinates.  To t e s t t h e a s s u m p t i o n t h a t t h e r e a c t i o n f o l l o w s s e c o n d  order  kinetics,  the data  plotted according  from t h e s l o p i n g p o r t i o n o f each curve  t o a second order  of v i t a m i n c o n c e n t r a t i o n  the i n t e g r a t e d second order  (Figure 20).  respectively.  S i n c e o x y g e n was  t o avoid t h e use o f negative rate equation  d i s s o l v e d oxygen c o n c e n t r a t i o n s shown i n F i g u r e 2 0 .  as  equation  were The u n i t s  (B) were present  a g r e a t e r c o n c e n t r a t i o n than t h e v i t a m i n , t h e i n t e g r a t e d second  r a t e e q u a t i o n was r e v e r s e d  two  kinetic  (A) and d i s s o l v e d oxygen c o n c e n t r a t i o n  m g / l i t e r and p a r t s p e r m i l l i o n , in  semilog  initial  values.  Plots of  f o r samples c o n t a i n i n g  initial  o f 14.4 a n d 9.6 p a r t s p e r m i l l i o n a r e  The c a l c u l a t e d second o r d e r  rate constants  f o r the  d i s s o l v e d oxygen c o n c e n t r a t i o n s were found t o be v e r y  illustrated  i n Table  VIII.  order  similar  - 68 -  0  6  12  TIME F i g u r e 19.  18  (hours)  C h a n g e i n o x y g e n c o n c e n t r a t i o n a t 20°C i n t h e p r e s e n c e o f N^CH-FH .  24  - 69 -  0  4  8 TIME  F i g u r e 20.  12  16  (hours)  R e g r e s s i o n a n a l y s e s performed on p a r t o f t h e d a t a p r e s e n t e d i n F i g u r e 18 a c c o r d i n g t o second o r d e r r e a c t i o n e q u a t i o n ( 1 6 ) .  Table V I I I .  Initial  S e c o n d o r d e r r a t e c o n s t a n t s (k) f o r N CH^FH^ d e g r a d a t i o n i n a l i m i t e d s u p p l y o f o x y g e n a t 20°C.  cone. ppm  Coefficient  Rate  dissolved  Constant  Intercept  |mg 1 _  1  hr  of  determination 2\  9.6  0.028  0.036  0.92  14.4  -0.011  0.038  0.94  The r a t e c o n s t a n t s a r e b a s e d o n d u p l i c a t e  trials.  - 71 -  DISCUSSION  Folic  a c i d h a s b e e n shown b y many w o r k e r s  s u s c e p t i b l e t o degradation by o x i d a t i v e heating. S c h w e i g e r t eit a l . , Ghitis,  S t o k s t a d e t a l . , 1947; Hanning  and M i t t s , 1949;  Methods o f measurement o f f o l i c  acid  degradation i n  t h e s e experiments have been m i c r o b i o l o g i c a l i n n a t u r e and have  been based the  ( C h e l d i n e t a l . , 1943;  1966; H e r b e r t , 1 9 6 7 , 1968b; F o r d e t a l . , 1 9 6 8 ; S c h r o e d e r , 1 9 7 1 ;  M a l i n , 1977). all  1946;  t o be v e r y  o n t h e a s s u m p t i o n t h a t t h e d e g r a d a t i o n p r o d u c t s do n o t s u p p o r t  growth of t h e t e s t Throughout  microorganisms. t h e e x p e r i m e n t s r e p o r t e d h e r e t h e a s s u m p t i o n was made  t h a t t h e p r o d u c t o f d e g r a d a t i o n o f N^CH^FH^ d i d n o t b i n d t o t h e f o l a t e b i n d i n g p r o t e i n o f t h e r a d i o a s s a y method. Initial  experiments u t i l i s i n g  t h e t h e r m a l death time pouches  showed t h a t , when N^CH^FH^ i n a p h o s p h a t e  b u f f e r was h e a t e d  i na boiling  w a t e r b a t h , a c h a n g e i n t h e s t r u c t u r e o f N^CH^FH^ t o o k p l a c e s u c h it  no l o n g e r h a d t h e a b i l i t y  to bind to the folate binding  S i n c e o x y g e n was t h o u g h t  that  protein.  t o be i n v o l v e d i n t h e d e g r a d a t i o n  r e a c t i o n , N^CH^FH^ was t h e n h e a t e d t o v a r i o u s t e m p e r a t u r e s w h i l e o x y g e n was  c o n s t a n t l y bubbled through t h e sample.  Degradation curves  indicated  an i n i t i a l p e r i o d o f t i m e , (which i n c r e a s e d as t h e temperature o f h e a t i n g d e c r e a s e d ) , w h e n t h e N^CH^FH^ c o n c e n t r a t i o n , a s m e a s u r e d b y t h e r a d i o a s s a y method, d i d n o t appear  t o change.  i n N^CH^FH^ c o n c e n t r a t i o n . order  T h i s was f o l l o w e d b y a r a p i d  T h i s r e a c t i o n appeared  k i n e t i c s and t h e pseudo f i r s t  decrease  t o f o l l o w pseudo  first  o r d e r r a t e c o n s t a n t s w e r e shown t o  decrease as t h e temperature o f r e a c t i o n  decreased.  -  72 -  Conformity with the Arrhenius  equation  made i t p o s s i b l e t o  c a l c u l a t e t h e a c t i v a t i o n e n e r g y f o r t h e t h e r m a l b r e a k d o w n o f N^CH^FH^ in  a phosphate b u f f e r system and i n t h e p r e s e n c e o f an u n l i m i t e d  oxygen  supply. Gupta and Huennekens N^CH^FH^ y i e l d s a d i h y d r o This  ( 1 9 6 7 ) h a v e shown t h a t o x i d a t i o n o f  compound, p r e s u m a b l y 5 m e t h y l - 5 , 6 d i h y d r o f o l a t e .  compound was shown b y t h e a u t h o r s t o b e r e d u c e d r e a d i l y b a c k t o  N^CH^FH^ u p o n t h e a d d i t i o n o f m e r c a p t o e t h a n o l . S i n c e m e r c a p t o e t h a n o l was p r e s e n t i n t h e b u f f e r , t h e m i l k 3 p r o t e i n and t h e H P t e G l u  of the radioassay  t h a t t h i s may b e i n f l u e n c i n g t h e f i r s t where no a p p r e c i a b l e  was o b s e r v e d .  e x h i b i t e d an i n i t i a l time p e r i o d  changes were d e t e c t e d . rate constants  oxygen supply,  f o rthe degradation  from t h e assay system and t h e  The r a t e c o n s t a n t s  i nrelatively  concentrations. order  experiment.  change i n  H o w e v e r , s a m p l e s h e a t e d t o 40° C before  N^CH^FH^  concentration  reaction.  o f N^CH^FH^ was o b s e r v e d i n a l i m i t e d  t h e r e a c t i o n appeared t o f o l l o w second order  element o f c a u t i o n , however. were s t i l l  place.  The a b s e n c e o f m e r c a p t o e t h a n o l d i d n o t a f f e c t  When t h e d e g r a d a t i o n  first  was t a k i n g  o f t i m e i n t h e r e a c t i o n when l i t t l e  N^CH^FH^ c o n c e n t r a t i o n  kinetics.  reaction  e f f e c t o f t h e e l i m i n a t i o n o f m e r c a p t o e t h a n o l was t o  decrease the period  the  eliminated  speculated  of the degradation  o f N^CH^FH^ was e x a m i n e d a s i n t h e p r e v i o u s The  still  part  c h a n g e i n N^CH^FH^ c o n c e n t r a t i o n  M e r c a p t o e t h a n o l was t h e r e f o r e degradation  m e t h o d , i t was  c a l c u l a t e d must be r e g a r d e d w i t h a c e r t a i n Oxygen c o n c e n t r a t i o n s ,  although  limited,  l a r g e a m o u n t s c o m p a r e d t o t h e N^CH^FH^  Thus, t h e r e a c t i o n c o u l d  although  reaction  coefficients  still  be r e g a r d e d as pseudo  of determination, f o r the regression  lines  - 73 -  o b t a i n e d when a p s e u d o f i r s t  order k i n e t i c  t h e d a t a w e r e n o t a s h i g h a s when a s e c o n d was  applied.  Greater i n i t i a l  e q u a t i o n was a p p l i e d t o order ^kinetic  equation  c o n c e n t r a t i o n s o f N^CH^FH^ a r e n e c e s s a r y  in  order t o accurately ascertain the rate constants f o r t h i s  It  does appear,  however, t h a t t h e presence  reaction.  o f oxygen i s n e c e s s a r y f o r  t h e d e g r a d a t i o n o f t^CH^FH^. The  decrease  i n oxygen c o n c e n t r a t i o n i n t h e i n i t i a l  stages  of  the reaction indicates that o x i d a t i o n i s taking place although  is  n o t d e t e c t e d b y a c h a n g e i n N^CH^FH^ c o n c e n t r a t i o n s .  that the mercaptoethanol  present  i n the assay  system  this  Thus, i t appears  i s reducing the  d e g r a d a t i o n p r o d u c t b a c k t o N^CH^FH^ d u r i n g t h e i n i t i a l  stages of the  reaction.  CONCLUSIONS  N^CH„FH, d e g r a d a t i o n i n t h e p r e s e n c e  3  4  s u p p l y c a n be d e s c r i b e d by a pseudo f i r s t  o f an u n l i m i t e d oxygen  order reaction.  o r d e r r a t e c o n s t a n t s i n c r e a s e as t h e temperature and  of reaction  t h i s r e l a t i o n s h i p can be d e s c r i b e d by t h e A r r h e n i u s In  t h e presence  of a limited  r e a c t i o n appears t o be second decrease the assay  throughout system,  increases  oxygen s u p p l y t h e o v e r a l l  order i n nature.  Oxygen c o n c e n t r a t i o n s i s present i n  i n N^CH^FH^ c o n c e n t r a t i o n i s n o t o b s e r v e d  immediately.When mercaptoethanol c o n c e n t r a t i o n s were observed  first  equation.  t h e r e a c t i o n b u t , when m e r c a p t o e t h a n o l  a decrease  Pseudo  i s e l i m i n a t e d from  t o decrease  earlier.  d e g r a d a t i o n r a t e constant f o r each temperature  the assay,  N^CH^FH^  No c h a n g e i n t h e  was d e t e c t e d .  -  74 -  The d e g r a d a t i o n o f J^CH^FH^ i s t h e r e f o r e thought t o be an o x i d a t i o n r e a c t i o n p r o b a b l y t o t h e N"* m e t h y l d i h y d r o f o l a t e .  This  compound does n o t b i n d t o t h e f o l a t e b i n d i n g p r o t e i n i n t h e r a d i o a s s a y method.  The o x i d a t i o n p r o d u c t i s r e a d i l y reduced back t o N^CH^FH^  i n t h e presence o f m e r c a p t o e t h a n o l . Care must be t a k e n , t h e r e f o r e , t o e x c l u d e t h e presence o f mercaptoethanol  i n t h e assay system when a t t e m p t i n g t o measure t h e r a t e  of breakdown o f N^CH^FH^ s i n c e , i n t h e i n i t i a l s t a g e s o f t h e r e a c t i o n , no d e g r a d a t i o n w i l l be observed i f i t i s used.  The presence o f o t h e r  r e d u c i n g agents i n a f o o d system may have a s i m i l a r e f f e c t on t h e r a t e of d e g r a d a t i o n o f N CH^FH . 5  A  -  CHAPTER I V .  75 -  I D E N T I F I C A T I O N OF THE DEGRADATION PRODUCT AND THE EFFECT OF ASCORBIC A C I D ON RATE OF DEGRADATION OF N  5  METHYLTETRAHYDROFOLIC A C I D  INTRODUCTION  N^CH^FH^ h a s b e e n shown t o b e s u s c e p t i b l e t o by  heat i n t h e presence o f oxygen.  There a r e s e v e r a l  degradation publications  p e r t a i n i n g t o t h e i d e n t i f i c a t i o n o f t h e N^CH^FH^ d e g r a d a t i o n O ' B r d i n e t a l . (1975) r e p o r t e d unstable" that was  product.  t h a t N^CH^FH^ was " n u t r i t i o n a l l y  p a r t i c u l a r l y a t a c i d pH v a l u e s .  the r e l a t i v e l y rapid n u t r i t i o n a l  The a u t h o r s s t a t e d  i n a c t i v a t i o n of this  very  however,  compound  u n l i k e l y t o b e d u e t o c l e a v a g e o f t h e Cg - N ^ Q b o n d , s i n c e  form o f t h e v i t a m i n being  this  s u b s t i t u t e d i n t h e 5 p o s i t i o n would be  expected t o be r e s i s t a n t t o such  reactions.  Donaldson and Keresztesy  ( 1 9 6 2 ) h a v e shown t h a t  N^CH^FH^  i s o x i d i z e d t o t h e d i h y d r o f o l a t e f o r m b y a number o f o x i d i z i n g a g e n t s . The  o x i d i z e d d e r i v a t i v e was c h a r a c t e r i z e d  acid.  Gupta and Hennekens (1967) c o n f i r m e d t h e s e r e s u l t s . In t h i s part  the  a s 5 m e t h y l 5, 6, d i h y d r o f o l i c  degradation  of the research  a n a t t e m p t was made t o i d e n t i f y  p r o d u c t o f N^CH^FH^ u s i n g u l t r a v i o l e t  Donaldson and K e r e s z t e s y agents a r e required  spectroscopy.  ( 1 9 6 2 ) showed t h a t v a r i o u s  f o rt h e conversion  reducing  o f N^CH^FH^ t o 5, 1 0 m e t h y l e n e -  t e t r a h y d r o f o l i c a c i d b y t h e enzyme 5, 1 0 - m e t h y l e n e t e t r a h y d r o f o l i c reductase. was  The a u t h o r s s p e c u l a t e d  t o prevent the degradation  that the r o l e of the reducing  acid  agent  o f N^CH^FH^ t o t h e d e h y d r o - c o m p o u n d .  Homocysteine, mercaptoethanol and a s c o r b i c  a c i d were a l l found t o be  - 76 -  e f f e c t i v e reducing agents. a c i d i n phosphate  a t lower concentrations of f o l a t e .  previous chapter indicated  able t o reduce  ascorbic  buffer afforded better protection against oxidation  than mercaptoethanol The  0 ' B r a i n e t a l . (1975) s t a t e d t h a t  that, mercaptoethanol  was  t h e d e g r a d a t i o n p r o d u c t o f h e a t i n g N^CH^FH^ i n o x y g e n  b a c k t o t h e N^CH^FH^ compound.  I t was s u g g e s t e d  that the presence of:  o t h e r r e d u c i n g a g e n t s w i t h i n a f o o d s y s t e m may h a v e a n e f f e c t o n t h e r a t e o f o x i d a t i o n o f N^CH^FH^. commonly f o u n d i n f o o d s .  Ascorbic acid  i s a reducing  agent  To o b s e r v e i t s e f f e c t o n t h e r a t e o f  d e g r a d a t i o n o f N^CH^FH^ a known amount o f a s c o r b a t e was a d d e d t o a f o o d , t h e f o o d was h e a t e d a n d t h e d e g r a d a t i o n o f N^CH^FH^ was m e a s u r e d u s i n g t h e r a d i o a s s a y method.  METHODS  Ultraviolet  Spectroscopy  A s a m p l e o f N^CH^FH^ i n p h o s p h a t e h e a t e d t o 70°C i n a w a t e r b a t h .  buffer  O x y g e n was b u b b l e d  (pH 7.3 0.1M) was through t h e sample  w h i l e i t was h e a t e d . Samples were removed f o r a n a l y s e s b y b o t h  ultraviolet  s p e c t r o s c o p y a n d t h e r a d i o a s s a y m e t h o d e v e r y 15 m i n u t e s . was  used  No  mercaptoethanol  i n t h e r a d i o a s s a y method. S a m p l e s o f N^CH^FH^ a t g r e a t e r c o n c e n t r a t i o n s t h a n  used were then heated  t o 100°C f o r 2 h o u r s .  the sample throughout  the heating period.  then analyzed by u l t r a v i o l e t  spectroscopy.  previously  Oxygen was b u b b l e d  through  T h e r e s u l t i n g s a m p l e was  - 77 -  The E f f e c t of A s c o r b i c A c i d on The D e g r a d a t i o n o f N CH FH^ i n a Food  A baby f o o d was  chosen f o r t h i s experiment s i n c e t h i s  a b l e n d i n g p r o c e s s p r i o r t o the a s s a y .  negated  Care was a l s o t a k e n t o ensure  t h a t t h e f o o d chosen c o n t a i n e d l i t t l e o r no a s c o r b i c a c i d ( a c c o r d i n g to m a n u f a c t u r e r ' s n u t r i e n t c o n t e n t t a b l e s ) . Two  lOOg samples o f "bacon and egg b r e a k f a s t " baby f o o d were  placed i n f l a s k s .  15 mg o f a s c o r b i c a c i d were added t o one  sample.  The f l a s k s were then p l a c e d i n a b o i l i n g water b a t h and oxygen was bubbled t h r o u g h the samples.  Both samples were c o n s t a n t l y  throughout a two-hour h e a t i n g p e r i o d .  Care was  stirred  t a k e n t o ensure t h a t  v e r y l i t t l e e v a p o r a t i o n of t h e sample took p l a c e . Samples were removed e v e r y 15 minutes f o r t h e f i r s t hour of h e a t i n g and e v e r y 30 minutes t h e r e a f t e r .  A f t e r c o o l i n g i n an i c e water  b a t h samples were assayed f o r f r e e and t o t a l f o l a t e by the r a d i o a s s a y method d e s c r i b e d i n Chapter  2.  RESULTS  U l t r a v i o l e t Spectroscopy  A s i n g l e a b s o r p t i o n peak a t 265 nm was observed when a sample of  unheated N^CH^FH^ i n phosphate b u f f e r was a n a l y z e d .  No o t h e r  a b s o r p t i o n peaks were d e t e c t e d a t t h e c o n c e n t r a t i o n s t e s t e d .  The  d e c r e a s e i n a b s o r p t i o n peak h e i g h t w i t h l e n g t h of t i m e of h e a t i n g t h e N^CH^FH, sample i n phosphate b u f f e r i s shown i n F i g u r e 21.  After  -  78 -  30 m i n u t e s o f h e a t i n g a t 70°C, t h e a b s o r p t i o n p e a k was n o detectable  ( c a r e was t a k e n  t o ensure t h a t samples were n o t exposed  to u l t r a v i o l e t l i g h t f o rtoo long a period p r i o r ultraviolet very  t o o b t a i n i n g an  s c a n a s i t was f o u n d t h a t t h e a b s o r p t i o n p e a k  rapidly  upon exposure t o l i g h t a t t h i s  Corresponding  radioassay  results  heating  shown i n T a b l e I X i n d i c a t e  t h e s a m p l e f o r 4 5 m i n u t e s , a 54 p e r c e n t  c o n c e n t r a t i o n was o b s e r v e d . ascorbate  present  stages  decrease i n  t h e N^CH^FH^ f r o m o x i d a t i o n  of the reaction.  S a m p l e s o f N^CH^FH^ a t g r e a t e r c o n c e n t r a t i o n s in 290  the previous  a n d 249 nm a f t e r h e a t i n g  Effect  A standard  The  T h i s i s shown i n F i g u r e 2 2 .  curve  food  folate  ascorbate  curves  ( a s measured by t h e  system, heated i n a b o i l i n g water bath  o f t h e two f o o d  i s shown t o d e c r e a s e d u r i n g  c o n t a i n i n g 15 mg a s c o r b i c a c i d 30 m i n u t e s o f h e a t i n g .  with  F r e e and t o t a l  s a m p l e s a r e shown.  f r e e f o l a t e concentration o f t h e food  A s i m i l a r decrease i n the f r e e f o l a t e  after  for folate  o f o x y g e n a r e shown i n F i g u r e 2 3 .  concentrations The  concentrations.  rate of degradation  an u n l i m i t e d s u p p l y  o f N^CH^FH^ i n a F o o d  c o n s t r u c t e d u s i n g N^CH^FH^ s a m p l e s was u s e d  r a d i o a s s a y method) i n a f o o d  folate  used  t o 100°C f o r 2 h o u r s i n t h e p r e s e n c e o f  o f A s c o r b i c A c i d on The D e g r a d a t i o n  for calculating  than those  e x p e r i m e n t y i e l d e d two u l t r a v i o l e t a b s o r p t i o n p e a k s a t  an u n l i m i t e d oxygen s u p p l y .  The  immediately.  I t i s assumed t h e r e f o r e t h a t t h e r e s i d u a l  i n t h e sample p r o t e c t e d  during the i n i t i a l  disappeared  wavelength).  t h a t a d e c r e a s e i n N^CH^FH^ c o n c e n t r a t i o n i s n o t d e t e c t e d After  longer  the f i r s t  sample c o n t a i n i n g no  15 m i n u t e s o f h e a t i n g .  c o n c e n t r a t i o n o f t h e sample  p e r 100 m l o f f o o d I t i s of interest  does n o t o c c u r  t o note that very  until little  -  F i g u r e 21.  79  -  Decrease i n u l t r a v i o l e t time  absorption w i t h length of  ( i n m i n u t e s ) o f h e a t i n g N^CH^FH^ s a m p l e s i n  p h o s p h a t e b u f f e r t o 70°C.  Table  IX.  Time  E f f e c t o f h e a t i n g o n N CH FR, s t a b i l i t y .  Concentration of  Change  Concentration  of Heating  N CH FH  (mins)  (ng/ml)  0  27.31  0  15  26.70  2  30  27.54  0  45  12.64  54  60  5.23  81  5  3  4  i n  (%)  - 82 -  F i g u r e 22.  U l t r a v i o l e t a b s o r p t i o n spectrum of N  methyldihydrofolic  acid.  ^-  max  = 290 nm  ( € = 31.0  x 10  3  M  _1  cm" ) 1  - 84 -  120  TIME  Figure  23.  (minutes)  The i n f l u e n c e of a s c o r b i c a c i d on t h e d e g r a d a t i o n of f r e e and t o t a l f o l a t e i n a food heated t o 100°C i n the presence o f an u n l i m i t e d oxygen  supply.  - 85 -  d i f f e r e n c e i s observed i n t h e t o t a l  f o l a t e content  o f t h e two s a m p l e s .  W i t h i n t h e two-hour h e a t i n g no a p p r e c i a b l e d e c r e a s e i n t o t a l  folate  c o n c e n t r a t i o n o f e i t h e r sample i s o b s e r v e d .  DISCUSSION  Consultation w i t h C l i n i c a l Assays  (Massachusetts) confirmed  t h a t t h e p e a k a t 2 6 5 nm o b s e r v e d when N^CH^FH^ s a m p l e s w e r e s u b j e c t e d to u l t r a v i o l e t  s p e c t r o s c o p y was d u e t o t h e p r e s e n c e o f r e s i d u a l  a s c o r b i c a c i d a d d e d t o t h e N^CH^FH^ p r i o r  to lyophilization.  The  p r e s e n c e o f t h i s p e a k made i t i m p o s s i b l e t o o b s e r v e t h e c h a r a c t e r i s t i c ultraviolet  a b s o r p t i o n s p e c t r a o f t h e p u r e N^CH^FH^ s a m p l e .  and K e r e s z t e s y (1962) and G u p t a  and Huennekens  Donaldson  (1967) have i n d i c a t e d  that  t h e maximum a b s o r p t i o n f o r p u r e N^CH^FH^ o c c u r s a t 290 nm (E = 30.8 x 10  3  M  _ 1  cm" ). 1  The  presence of ascorbic acid  t h e r a t e o f d e g r a d a t i o n o f N^CH^FH^.  i n t h e s a m p l e i s shown t o a f f e c t  I t would  appear  that ascorbic  r e d u c e s t h e d e g r a d a t i o n p r o d u c t b a c k t o N^CH^FH^, i t s e l f b e i n g t o t h e d e h y d r o a s c o r b i c a c i d w h i c h does n o t absorb u l t r a v i o l e t  acid  oxidized light.  H e n c e , t h e a b s o r p t i o n p e a k a t 2 6 5 nm i s o b s e r v e d t o d e c r e a s e w h e n t h e s a m p l e c o n t a i n i n g N^CH^FH^ i s h e a t e d . The why  presence of ascorbic acid  i n t h e N^CH^FH^ s a m p l e s  explains  t h e s a m p l e h e a t e d t o 40°C a n d a s s a y e d i n a m e r c a p t o e t h a n o l - f r e e  system  ( d e s c r i b e d i n C h a p t e r I I I ) e x h i b i t e d a d e c r e a s e i n N^CH^FH^  c o n c e n t r a t i o n o n l y a f t e r 1% h o u r s o f h e a t i n g . chromatography  o f N^CH^FH^ s a m p l e s  T h i n l a y e r and column  heated f o r short p e r i o d s of time  y i e l d e d o n l y t h e o r i g i n a l s t a r t i n g p r o d u c t f o r t h e same r e a s o n  thus  - 86 -  making t h e i d e n t i f i c a t i o n o f t h e degradation The when h i g h e r  ultraviolet  product  a b s o r p t i o n p e a k s a t 290  concentrations  impossible.  a n d 249 nm o b s e r v e d  o f sample were used c o u l d o n l y be  detected  when t h e a b s o r p t i o n p e a k f o r a s c o r b i c a c i d h a d d i s a p p e a r e d . a b s o r p t i o n peaks c o n f i r m the  that the product  o f N^CH^FH^ o x i d a t i o n i s  dihydro-compound. Since  ascorbic acid  i s an e f f e c t i v e reducing  r e a c t i o n , i t s e f f e c t on t h e f o l a t e d e g r a d a t i o n interest.  Ghitis  (1966) a n d F o r d et: a l .  the presence o f ascorbate  the a s c o r b i c a c i d  content  i n milk i s necessary  normally  p r o t e c t N^CH^FH^ f r o m d e g r a d a t i o n that i fascorbic acid  r a t e s i n foods i s of  to s t a b i l i z e  folate  (1966) s t a t e d , h o w e v e r , t h a t  f o u n d i n m i l k was n o t s u f f i c i e n t t o during boiling.  i s used t o e n r i c h processed  The a u t h o r  suggested  m i l k s then i t should  added i n s u f f i c i e n t amounts t o p r o t e c t t h e f o l a t e s f r o m o x i d a t i o n  during  heating. Many f o o d s r i c h i n f o l a t e a r e a l s o r i c h  and  agent i n t h i s  (1968) h a v e o b s e r v e d t h a t  retention during heating processes.Ghitis  be  These  green vegetables  a r e examples o f such foods.  such as eggs, which a r e r e l a t i v e l y r i c h ascorbic acid.  i n ascorbic acid. However, o t h e r  Liver  foods  i n f o l a t e s , do n o t c o n t a i n  From t h e e x p e r i m e n t d e s c r i b e d h e r e i n , i t i s o b s e r v e d  t h a t a s c o r b i c a c i d can p r o t e c t f r e e f o l a t e from degradation The  t o t a l f o l a t e content  of t h e food  examined d i d n o t change  two  hours o f heating i n a b o i l i n g water  bath.  during during  heating.  - 87 -  CONCLUSIONS  When N^CH^FH^ i s heated i n t h e presence  o f oxygen,  d e g r a d a t i o n p r o d u c t i s t h e d i h y d r o f o l a t e compound. t h e work o f Donaldson and K e r e s z t e s y (1966).  the  This confirms  (1962) and Gupta and Hennekens  The d e g r a d a t i o n o f N^CH^FH^ by h e a t i n g i n oxygen i s d e l a y e d  by t h e presence  of ascorbic acid.  The a s c o r b i c a c i d a c t s as a r e d u c i n g  agent, c o n v e r t i n g t h e d i h y d r o - compound back t o t h e t e t r a h y d r o - compound. The presence  o f a s c o r b i c a c i d i n food r i c h i n f o l a t e i s  t h e r e f o r e o f g r e a t importance  i f t h e food i s t o be s u b j e c t e d t o h e a t i n g .  Foods w h i c h a r e r i c h i n a s c o r b i c a c i d may be h e a t e d  f o r longer periods  t h a n foods w h i c h c o n t a i n l i t t l e o r no a s c o r b i c a c i d b e f o r e e x h i b i t i n g a decrease  i n f o l a t e c o n t e n t J If foods a r e t o be e n r i c h e d w i t h a s c o r b a t e ,  t h e n l e v e l s s h o u l d be s u f f i c i e n t t o p r o t e c t t h e f o l a t e from o x i d a t i o n . F u t u r e s t u d i e s s h o u l d be d i r e c t e d towards t h e d e t e r m i n a t i o n o f t h e r e s i s t a n c e o f o t h e r f o l a t e compounds t o f o o d p r o c e s s i n g o p e r a t i o n s . The e f f e c t s o f o t h e r food c o n s t i t u e n t s on t h e r a t e o f f o l a t e  degradation  and t h e d e t e r m i n a t i o n o f r e d u c i n g agent l e v e l s n e c e s s a r y t o p r o t e c t f o l a t e s d u r i n g heat p r o c e s s i n g s h o u l d a l s o be s t u d i e d . Only a s a r e s u l t  o f a b e t t e r u n d e r s t a n d i n g o f t h i s complex  v i t a m i n c a n a t t e m p t s be made t o ensure maximum r e t e n t i o n o f t h e n u t r i e n t i n food d u r i n g p r o c e s s i n g and c o o k i n g o p e r a t i o n s .  - 88 -  REFERENCES  A r c h i b a l d , E . L . , E.K. M i n c e y a n d R.T. M o r r i s o n . 1972. E s t i m a t i o n o f serum f o l a t e l e v e l s by c o m p e t i t i v e p r o t e i n b i n d i n g a s s a y . C l i n . Biochem. 5:232. Asenjo,  F.  1948. 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Use and s i g n i f i c a n c e of f o l a t e b i n d e r s . I n : " F o l i c A c i d - B i o c h e m i s t r y and P h y s i o l o g y i n R e l a t i o n t o Human N u t r i t i o n Requirements". N a t . Acad, o f S c i e n c e s . Washington, D.C. R u d z k i , Z., M. Nazaruk and R.J. Kimber. 1976. The c l i n i c a l v a l u e o f t h e r a d i o a s s a y o f serum f o l a t e . J . L a b . C l i n . Med. 87:859. Schroeder,  H.A. 1971. Losses o f v i t a m i n s and t r a c e m i n e r a l s r e s u l t i n g from p r o c e s s i n g and p r e s e r v a t i o n o f f o o d s . Am. J . C l i n . N u t r . 24:562.  - 92 -  S c h w e i g e r t , B. S., A.E. P o l l a r d and C A . Elvehjem. 1946. The f o l i c a c i d c o n t e n t o f meats and t h e r e t e n t i o n o f t h i s v i t a m i n i n c o o k i n g . A r c h . Biochem. B i o p h y s . 10:107. Shaw, W., B.A. S l a d e , J.W. H a r r i s o n and H.V. Nino. 1974. 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H e r b e r t . 1971. R a d i o i s o t o p i c assay f o r measurement o f serum f o l a t e l e v e l s . B l o o d 38:219. Waxman, S. and C. S c h r e i b e r . 1973. Measurement o f serum f o l a t e l e v e l s andserum f o l i c a c i d b i n d i n g p r o t e i n by HPGA r a d i o a s s a y . Blood 42:281. J  - 94 -  Z e t t n e r , A. and P.E. D u l y . 1974. P r i n c i p l e s o f c o m p e t i t i v e b i n d i n g assays ( s a t u r a t i o n analyses) I I S e q u e n t i a l s a t u r a t i o n . C l i n . Chem. 20:5. Z e t t n e r , A. and P.E. D u l y . 1975. R e l a t i v e e f f i c a c y o f s e p a r a t i o n o f ' f r e e ' and 'bound' ( 3 ' 5' - -%) p t e r o y l g l u t a m a t e by c h a r c o a l coated w i t h v a r i o u s m a t e r i a l s . C l i n . Chem. 21:1927.  - 95  - 96 -  Replacement o f T r i s - N a C l B u f f e r w i t h Phosphate B u f f e r i n t h e R a d i o a s s a y Method  T r i s - N a C l b u f f e r a s s u p p l i e d b y C l i n i c a l A s s a y s I n c . was r e p l a c e d by  0.1M p h o s p h a t e b u f f e r ( N a ^ P C y N a H ^ O ^ O )  r a d i o a s s a y method.  Standard  curves  pH 7.3 i n t h e  o b t a i n e d u s i n g t h i s method d i d 3  not  differ significantly  a s shown b y t h e v a l u e s o f p e r c e n t  H  PteGlu  bound f o r each s t a n d a r d used i n T a b l e A - l .  3 Table A - l .  H P t e G l u bound i n r a d i o a s s a y method u s i n g two b u f f e r systems.  Concentration of (ng/100 u l )  *  Standard  3  H P t e G l u bound (Tris/NaCl)  (%)  3  H  P t e G l u bound (Phosphate)*  5.0  11.3  11.5  1.67  24.1  23.7  0.555  32.0  31.6  0.185  62.1  63.0  0.062  72.6  74.1  R e s u l t s f o r d u p l i c a t e samples.  (%)  - 97 -  APPENDIX B  - 98 -  Method o f Quench  Liquid r a d i a t i o n and  Correction  scintillation  c o u n t i n g i s a method o f  detecting  i s used m a i n l y f o r low energy b e t a e m i t t e r s .  method i s b a s e d  on t h e e n e r g y  t r a n s f e r from the i o n i z i n g  t o t h e s o l v e n t m o l e c u l e s and hence t o t h e p r i m a r y s o l u t e w h i c h e m i t s some o f t h e e n e r g y the  as l i g h t .  light  particle (fluor)  i s d e t e c t e d by  p h o t o m u l t i p l i e r tube w h i c h responds by p r o d u c i n g a charge  w h i c h when a m p l i f i e d , i s c o u n t e d .  scintillation of energy detected.  However, i n l i q u i d  c o u n t i n g s e v e r a l p r o c e s s e s may  reduce the  efficiency  t r a n s f e r a n d t h u s t h e number o f e m i t t e d p a r t i c l e s T h i s i s known a s  to correct  pulse  I t i s d e s i r a b l e t h a t t h e maximum  number o f e m i t t e d p a r t i c l e s b e d e t e c t e d .  used  The  The  'quenching'.  that  are  A number o f m e t h o d s h a v e b e e n  f o r t h e l o s s o f d e t e c t a b l e a c t i v i t y by c o u n t i n g .  t h i s r e s e a r c h , t h e c h a n n e l s r a t i o method o f quench c o r r e c t i o n  In  was  utilized. A standard curve r e l a t i n g counting e f f i c i e n c y to the o f t h e n e t c o u n t r a t e s o f a s a m p l e i n two constructed.  d i f f e r e n t channels i s  T h i s i s done by c o u n t i n g v a r i o u s l y quenched s a m p l e s  t a i n i n g known a m o u n t s o f r a d i o a c t i v i t y i n two  counted  c h a n n e l s and, by r e f e r e n c e t o t h e s t a n d a r d c u r v e , t h e  corresponding to the observed  channels r a t i o  con-  appropriate channels.  Unknown s a m p l e s o f t h e same r a d i o n u c l e i d e c a n t h e n b e same two  ratio  i s obtained.  i n these efficiency  Such i n f o r m a t i o n  i s necessary i n cross-comparing the c o u n t i n g r e s u l t s of samples.  A  s e r i e s o f q u e n c h e d s a m p l e s w e r e p r e p a r e d w i t h c o m p o s i t i o n s shown i n Table  B-l.  - 99 -  Table B - l .  C o m p o s i t i o n of quenched  S a m p l e No.  Buffer  Scintiverse  (ml)  H PteG3u( l) u  Chloroform (ill) 0  50  12  500  1 2  100  3  200  4  300  5  400  6  500  All Liquid ratio  samples were counted  scintillation f o r each  efficiency plot  i n the Nuclear Chicago  c o u n t e r u s i n g t h e t r i t i u m program^  s a m p l e was  counting efficiency  The  (ul)  samples.  a u t o m a t i c a l l y c a l c u l a t e d by  f o r e a c h o f t h e s a m p l e s was  v e r s u s c h a n n e l s r a t i o was  performed efficiency  on t h i s p l o t f o r any  counts per minute  From a l i n e a r  of  .50 -  The Counting  samples. regression  then p o s s i b l e to c a l c u l a t e the counting  c o u l d t h e n be c a l c u l a t e d  t h e samples counted  channels  the counter.  f o r a l l the  sample g i v e n i t s channels r a t i o .  of  .75.  i t was  The'A/B  then c a l c u l a t e d .  then p l o t t e d  s o c o n s t r u c t e d i s shown i n F i g u r e , B l .  I s o c a p , 300  throughout  f o r each  t h i s r e s e a r c h had  The  quench  sample.  corrected  The m a j o r i t y  channels r a t i o  values  - 100 -  Figure Bl. 0  Quench c o r r e c t i o n c u r v e .  - 101 -  APPENDIX C  - 102  -  D e t e r m i n a t i o n o f H e a t i n g and C o o l i n g L a g f o r T h e r m a l D e a t h Time Pouches  were used  Times  Thermocouples  ( c o p p e r / c o n s t a h t a n , w i r e d i a m e t e r .005mm)  f o r measuring  t h e h e a t i n g and c o o l i n g l a g t i m e s i n t h e  t h e r m a l death time pouches. i n t o t h e end s e a l .  A Digitec  The 128  thermocouples were d a t a l o g g e r was  incorporated  used f o r temperature  recording. Pouches  (with thermocouples incorporated i n t o  w e r e f i l l e d w i t h 1.5  m l 0.1M  phosphate b u f f e r and, w h i l e  a s much a i r a s p o s s i b l e , t h e e n d s w e r e h e a t s e a l e d .  r e c o r d e d a t one  the  recorder.  of  recorded u n t i l of  channel of  immediately placed temperature  i t reached the temperature of the water bath.  After conversion of m i l l v o l t the  removed and  Once a g a i n , t h e i n t e r n a l p o u c h  v a l u e s were o b t a i n e d f o r h e a t i n g and  r e a d i n g s t o °C i t was  h e a t i n g and c o o l i n g l a g t i m e s f o r pouches  c o n t a i n i n g 1.5  t h i s o r d e r were r e g a r d e d as n e g l i g i b l e i n t h e r e a c t i o n  investigation.  was  Ten  series  found  that  cooling.  p h o s p h a t e b u f f e r w e r e o f t h e o r d e r o f 11 s e c o n d s + 2 s e c o n d s . of  was  temperature reached the temperature  t h e b o i l i n g w a t e r b a t h , t h e p o u c h was  i n a cold waterbath.  eliminating  temperature  second i n t e r v a l s u s i n g the m i l l i v o l t  When t h e i n t e r n a l p o u c h  seal)  Each pouch  t h e n p l a c e d i n a b o i l i n g w a t e r b a t h and i n t e r n a l pouch was  t h e end  ml  0.1M Lag  under  times  

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