UBC Theses and Dissertations

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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 FOLIC ACID by JANE ELIZABETH RUDDICK B . S c , H o n s . U n i v e r s i t y o f London, 1972 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 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 c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1978 "cv) Jane E l i z a b e t h R u d d i c k , 1978 i n In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his 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 Brit ish Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 MAY 31st 1978. - i i -ABSTRACT F o l i c a c i d i s present i n foods i n many d i f f e r e n t forms. Concentrations of 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 of f o l a t e ) were determined by a radioassay and the L a c t o b a c i l l u s c a s e i micro-b i o l o g i c a l assay methods. The c o e f f i c i e n t of determination f o r samples analyzed by both methods was 0.86 but the radioassay method was found to be f a s t e r , simpler and more accurate than the 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 binder and L. c a s e i were found to react d i f f e r e n t l y to p t e r o y l g l u t a m i c a c i d (another form of f o l a t e ) compared to 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 . The two methods of assay were found to be s u i t a b l e only f o r measuring the form of f o l a t e used to construct the standard curve f o r the assay. I t was found t h a t n e i t h e r method could be used to quantify- 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, at best, p r e s e n t l y a v a i l a b l e data on food f o l a t e l e v e l s can only be compared to other values obtained by an i d e n t i c a l method. Al s o f o l a t e c o n c e n t r a t i o n values p r e s e n t l y a v a i l a b l 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 of 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 to man. The radioassay method was used f o r the measurement of 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 under v a r i o u s c o n d i t i o n s . I t was found that 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 i n the presence of an u n l i m i t e d oxygen supply could be described as a pseudo f i r s t order r e a c t i o n . Rate constants f o r the r e a c t i o n were found to increase as the temperature of the r e a c t i o n increased and were described i n terms of the Arrhenius equation. - i i i -The o v e r a l l r e a c t i o n appeared t o be second o r d e r i n t h e p r e s e n c e o f a l i m i t e d oxygen s u p p l y . The p r e s e n c e o f m e r c a p t o e t h a n o l i n t h e a s s a y s y s t e m d e l a y e d t h e b e g i n n i n g o f t h e 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 r e a c t i o n . The p r e s e n c e o f a s c o r b i c a c i d i n t h e 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 d e l a y e d t h e s t a r t o f t h e r e a c t i o n . The r e a c t i o n r a t e c o n s t a n t s were n o t a l t e r e d by t h e p r e s e n c e o f t h e two r e d u c i n g a g e n t s , however. The 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 h e r e f o r e c o n c l u d e d t o be an o x i d a t i o n r e a c t i o n . The d e g r a d a t i o n p r o d u c t was 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 s p e c t r o s c o p y . The r e s u l t s o f t h i s r e s e a r c h i m p l i c a t e t h e i m p o r t a n c e o f r e d u c i n g a g e n t s i n f o o d s c o n t a i n i n g f o l a t e w h i c h a r e s u b j e c t e d t o h e a t p r o c e s s i n g . E x p e r i m e n t s w i t h f o o d m a t e r i a l s c o n t a i n i n g a s c o r b i c a c i d and exposed t o h i g h t e m p e r a t u r e s i n d i c a t e d t h a t t h e d e g r a d a t i o n of f r e e f o l a t e was d e l a y e d by t h e p r e s e n c e o f t h e r e d u c i n g a g e n t . - iv -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES v i i i LIST OF FIGURES ix ACKNOWLEDGEMENTS x i i INTRODUCTION 1 CHAPTER I. REVIEW OF LITERATURE _ . . 4 DEFINITION 4 BIOCHEMICALLY ACTIVE FOLATES - THEIR  DISTRIBUTION AND AVAILABILITY IN FOODS 6 METHODS OF FOLATE MEASUREMENT. 7 Biological Methoda 8 Microbiological Methods 8 Biochemical, Chemical and Instrumental Methods 9 LOSSES OF FOLIC ACID IN FOOD PROCESSING . . . . 11 CHAPTER II. A COMPARISON OF MICROBIOLOGICAL ASSAY AND RADIOASSAY METHODS FOR MEASURING FOLATE . . . . 13 INTRODUCTION 13 MATERIALS AND METHODS 14 Preparation of N~*- Methyltetrahydrof o l i c Acid Standards 14 Preparation of Unknown Concentrations of N5CH0FH. 16 3 4 Preparation of Food Extracts 16 P r e p a r a t i o n o f P t e r o y l g l u t a m i c A c i d S t a n d a r d s . .17 M a i n t e n a n c e 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 o f Inoculum . . . 17 M i c r o b i o l o g i c a l A s s a y P r o c e d u r e 18 R a d i o a s s a y P r o c e d u r e 19 E x p e r i m e n t a l P r o c e d u r e 20 N 5CH 3FH^ R e c o v e r y Study 21 RESULTS 23 N 5CH„FH, S t a n d a r d C u r v e s 23 j 4 - M i c r o b i o l o g i c a l A s s a y 23 -Rad i o a s s a y 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 B o t h Methods 25 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 o f Baby Foods O b t a i n e d U s i n g B o t h Methods. . . 25 Comparison o f N^CH^FH^ and P t e G l u S t a n d a r d Curves 29 N 5 C H 3 F H 4 R e c o v e r y Study ?9 DISCUSSION 3 5 CONCLUSIONS 39 CHAPTER I I I . KINETIC STUDIES Ci1 INTRODUCTION 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 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 4 3 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. . 4 3 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 . . . .47 K i n e t i c S t u d i e s U s i n g L i m i t e d Oxygen S u p p l y . . 47 - v i -THEORETICAL CONSIDERATIONS. 48 F i r s t O rder R e a c t i o n s 48 The A r r h e n i u s E q u a t i o n • -50 Second Order R e a c t i o n s 50 RESULTS 52 K i n e t i c S t u d i e s U s i n g T h ermal D e a t h Time Pouches 52 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 . .54 The H o r i z o n t a l X-Y 54 The S l o p e s Y-Z . .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 . . . . 62 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 S u p p l y . .62 DISCUSSION 7 1 CONCLUSIONS 7 3 CHAPTER IV. IDENTIFICATION OF THE DEGRADATION PRODUCT AND THE EFFECT OF ASCORBIC ACID ON RATE OF DEGRADATION OF N 5-METHYLTETRAHYDROFOLIC ACID 75 INTRODUCTION 7 5 METHODS. I6 U l t r a v i o l e t S p e c t r o s c o p y 7 ^ 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 o f N 5CH.FH. i n a Food 7 7 3 4 RESULTS 7 7 U l t r a v i o l e t S p e c t r o s c o p y 7 7 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 o f N 5CH 0FH. i n a Food 7 8 DISCUSSION 8 5 CONCLUSIONS- 8 7 - 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 P h osphate B u f f e r i n t h e R a d i o a s s a y Method . . . .95 B. Method o f Quench C o r r e c t i o n 97 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 Lag Times f o r Thermal Death Time Pouches 101 - v i i i -T a b l e LIST OF TABLES Page I N^CH^FH^ c o n c e n t r a t i o n s used i n p r e p a r a t i o n o f s t a n d a r d c u r v e s 15 I I Volumes o f s o l u t i o n s used i n N^CH^FH^ r e c o v e r y s t u d y . .22 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 . 30 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 and 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 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 Re c o v e r y o f N 5 C H 3 F H 4 from m i l k e x t r a c t 34 V I Pseudo f i r s t o r d e r c o n s t a n t s ( k ' ) of N 5CH„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 59 V I I 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') of 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 a s s a y s y s t e m 65 V I I I Second 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 oxygen a t 20°C. . . .70 IX E f f e c t o f h e a t i n g on N^CH^FH^ s t a b i l i t y 81 3 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 system 96 B - l C o m p o s i t i o n o f quenched samples 99 - i x -LIST OF FIGURES F i g u r e Page 1 S t r u c t u r e 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 3 2 B a s i c s t r u c t u r e o f a l l f o l a t e compounds-p t e r o y l g l u t a m i c a c i d 5 3 R e g r e s s i o n a n a l y s i s p e r f o r m e d 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 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 24 4 R e g r e s s i o n a n a l y s i s p e r f o r m e d 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 26 5 R e g r e s s i o n 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 a s s a y method o f m e a s u r i n g N 5CH„FH, c o n c e n t r a t i o n 2 7 3 4 6 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^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 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 31 7 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^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 32 8 Thermal d e a t h t i m e pouch w i t h w e i g h t a t t a c h e d 45 9 A p p a r a t u s used f o r k i n e t i c s t u d i e s . 46 10 D e g r a d a t i o n o f N 5CH FH^ h e a t e d t o 100°C i n t h e r m a l d e a t h t i m e pouches 53 - x -11 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 55 12 I n f l u e n c e o f t e m p e r a t u r e 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 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 56 13 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 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 e q u a t i o n (6) 58 14 V a r i a t i o n o f r a t e c o n s t a n t (k') w i t h t e m p e r a t u r e of r e a c t i o n 60 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 e q u a t i o n (8) 61 16 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 a s s a y e d u s i n g a m e r c a p t o e t h a n o l - f r e e s y s t e m 63 17 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 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) 64 18 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 66 19 Change i n oxygen 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 5CH„FH. 68 3 4 20 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 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 (16) 69 - x i -21 Decrease i n u l t r a v i o l e t absorption w i t h l e n g t h of time ( i n minutes) of heating N^CH^FH^ samples i n phosphate b u f f e r to 70°C 79 22 U l t r a v i o l e t spectrum of 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 of a s c o r b i c a c i d on the degradation of f r e e and t o t a l f o l a t e i n a food heated to 100°C i n the presence of an u n l i m i t e d oxygen supply 84 B l Quench c o r r e c t i o n curve . . . . 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 , Dr. J . F . R i c h a r d s and Dr. J . V a n d e r s t o e p f o r t h e i r g u i d a n c e , s u p p o r t and encouragement t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y ; - t h e members o f my committee, Dr. M.A. Tung, P r o f e s s o r B. March, Dr. S. N a k a i and Dr. J . L e i c h t e r f o r t h e i r h e l p f u l 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 t h e s i s ; - John 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 and t h e courage t o c a r r y out t h i s r e s e a r c h . - 1 -INTRODUCTION The f o l a t e s a r e a group o f w a t e r s o l u b l e compounds w i t h i n t h e v i t a m i n B complex. They a r e p h y s i o l o g i c a l l y and b i o c h e m i c a l l y i m p o r t a n t e s p e c i a l l y t o h i g h e r a n i m a l s and t o man, p l a y i n g an i m p o r t a n t r o l e i n amino a c i d m e t a b o l i s m and i n t h e s y n t h e s i s o f p u r i n e and p y r i m i d i n e n u c l e o t i d e s . 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 t o d i e t a r y i n a d e q u a c y , f a i l u r e o f a l i m e n t a r y a b s o r p t i o n o r e l e v a t e d m e t a b o l i c r e q u i r e m e n t p r o d u c e s a c h a r a c t e r i s t i c change i n t h e b l o o d known as m e g a l o b l a s t i c anemia. F o l a t e d e f i c i e n c y i s p r o b a b l y t h e most p r e v a l e n t v i t a m i n d e f i c i e n c y i n man and i s common t h e w o r l d o v e r . I t has been s u g g e s t e d t h a t one o f t h e m a j o r causes of f o l a t e d e f i c i e n c y i s t h e 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 t h e p r o c e s s i n g and c o o k i n g o f f o o d ( H e r b e r t , 1968b). Because o f 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 i t becomes i m p o r t a n t 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 of f o l a t e s d u r i n g p r o c e s s i n g . Such measures can o n l y be a c h i e v e d as 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 e r e s p e c t i v e r e s i s t a n c e t o d e s t r u c t i v e p r o c e s s e s o f t h e v a r i o u s f o l a t e compounds. A s e a r c h o f t h e l i t e r a t u r e i n d i c a t e s t h a t w h i l e some s t u d i e s have been c o n d u c t e d on l o s s e s of f o l a t e w i t h i n f o o d m a t e r i a l s ( C h e l d i n e t a l . , 1943; S c h w e i g e r t e t a l . , 1946; Hanning and M i t t s , 1949; G h i t i s , 1966; H e r b e r t , 1967; 1968b; F o r d e t a l . , 1968; S c h r o e d e r , 1971), t h e r e i s a c o m p l e t e 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 t h e k i n e t i c s o f f o l a t e breakdown w i t h i n a s i m p l e system. ( T h i s may be due t o b o t h t h e - 2 -c o m p l e x i t y 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 s i m p l e a s s a y method). K i n e t i c i n f o r m a t i o n s u c h as t h i s i s t h e f i r s t s t e p t o c o r r e l a t i n g l o s s e s o f t h e v i t a m i n t o f o o d c o m p o s i t i o n and t o v a r i o u s p r o c e s s e s and s t o r a g e t e c h n i q u e s . S i n c e 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 t h e k i n e t i c 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 (N^CH^FH^). The s t r u c t u r e o f t h i s f o r m o f f o l a t e i s i l l u s t r a t e d i n F i g u r e 1. The e f f e c t o f h e a t i n g N^CH^FH^ i n pho 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 and a t v a r i o u s oxygen c o n c e n t r a t i o n s was e v a l u a t e d . The e f f e c t o f v a r i o u s r e d u c i n g 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 o f N^CH^FH, was a l s o s t u d i e d . 3 4 F o l l o w i n g i n i t i a l c o m p a r a t i v e s t u d i e s w i t h t h e 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 a s s a y method, a r a d i o a s s a y s y s t e m was chosen f o r m e a s u r i n g N 5CH„FH, l e v e l s . Figure 1. S t r u c t u r e of 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 . - 4 -CHAPTER 1. REVIEW OF LITERATURE DEFINITION F o l i c a c i d has been t h e s u b j e c t o f many i n v e s t i g a t i o n s i n v o l v i n g s e v e r a l s p e c i e s o f a n i m a l s and m i c r o o r g a n i s m s . Hence, t h e v i t a m i n had a c o n f u s i n g r o s t e r o f names b e f o r e i t was l e a r n e d t h a t t h e same o r s i m i l a r s u b s t a n c e s were b e i n g s t u d i e d . As e a r l y as 1935, a n u t r i t i o n a l d e f i c i e n c y i n monkeys,which e v e n t u a l l y l e d t o anemia,now known t o r e s p o n d t o f o l i c a c i d , was d e s c r i b e d . T h i s f a c t o r was c a l l e d " V i t a m i n M" (Day e t a l . , 1935). I n 1939, Hogan and P a r r o t t d e s c r i b e d an anemia i n c h i c k s due t o a d e f i c i e n c y o f an unknown f a c t o r w h i c h t h e y termed " V i t a m i n B". I n 1943, S t o k s t a d . r e p o r t e d a f a c t o r n e c e s s a r y i n t h e n u t r i t i o n o f L a c t o b a c i l l u s c a s e i , w h i c h he named " t h e L_. c a s e i f a c t o r " and M i t c h e l l et_ a l . (1941) i s o l a t e d a c r y s t a l l i n e compound fr o m s p i n a c h w h i c h t h e y c a l l e d " f o l i c a c i d " , from t h e L a t i n t e r m f o r l e a f ( f o l i u m ) . These and s e v e r a l o t h e r f a c t o r s were shown t o b e l o n g t o the same n u t r i t i o n a l l y and c h e m i c a l l y r e l a t e d f a m i l y o f compounds, f o l i c a c i d . The t e r m " f o l i c a c i d " i s p r e s e n t l y a c c e p t e d t o have two meanings ( M a l i n , 1975). G e n e r i c a l l y , i t c o v e r s a b r o a d range o f c l o s e l y r e l a t e d p t e r o i c a c i d compounds w h i c h i n c l u d e t h e monoglutamate, t h e p o l y g l u t a m a t e forms and t h e i r r e d u c e d o r s u b s t i t u t e d a n a l o g u e s . S p e c i f i c a l l y , i t i s u sed t o d e s c r i b e t h e compound, p t e r o y l g l u t a m i c a c i d . I t has been pr o p o s e d by t h e IUPAC-IUB commission ( S t o k s t a d and Thenen, 1972) t h a t t h e terms " f o l i c a c i d " and " f o l a t e " be used i n t h e g e n e r a l sense f o r any member of t h e f a m i l y o f compounds, o r a m i x t u r e o f t h e s e , w i t h t h e b a s i c s t r u c t u r e shown i n F i g u r e 2, e i t h e r f r e e o r c o n j u g a t e d w i t h two o r more g l u t a m i c GLUTAMIC ACID 2 - A M I N O - 4 -H YDROX Y-6-METHYLPTERIN AMINOBENZOIC ACID o 1 r OH CH, 9 CH, -NH CH COOH i i OH Figure 2. Basic structure of a l l f o l a t e compounds-pteroylglutamic a c i d . - 6 -a c i d r e s i d u e s . BIOCHEMICALLY ACTIVE FOLATES - THEIR DISTRIBUTION AND AVAILABILITY IN FOODS The forms o f f o l i c a c i d t h a t a r e b i o c h e m i c a l l y a c t i v e d i f f e r f r o m t h e 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 ways. F i r s t l y , t h e b i o -c h e m i c a l l y a c t i v e forms a r e r e d u c e d - i . e . t h e y have a d d i t i o n a l hydrogens 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 . 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 h o l d 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 p-amino 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 o r as a b r i d g e between t h e s e two p o s i t i o n s as m e t h y l e n e o r m e t h e n y l . The t h i r d i m p o r t a n t b i o c h e m i c a l change 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 forms a r e c o n j u g a t e d i n p e p t i d e -l i k e 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 groups t o one o r more g l u t a m i c a c i d r e s i d u e s . M a l i n (1975) has 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 t i v e forms o f f o l i c a c i d t h a n o f any known v i t a m i n . A t t e m p t s have been made t o s e p a r a t e t h e many n a t u r a l f o l i c 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 ( S i l v e r m a n e t a l . , 1961; S h i n e t a l . , 1972a, 1972b; Kas and C e r n a , 1976). By e m p l o y i n g s u c h p r o c e d u r e s , t h e d i s t r i b u t i o n of f o l i c a c i d has been d e t e r m i n e d f o r c e r t a i n r e p r e s e n t a t i v e f o o d s and t i s s u e s . 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 f o l a t e d e r i v a t i v e s . Rat k i d n e y and l i v e r 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 m e t h y l d e r i v a t i v e s - m a i n l y p e n t a g l u t a m a t e s - whereas r e d c e l l s c o n t a i n 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 p e n t a - a n d h e x a g l u t a m a t e ( S h i n e t a l . , 1972a, 1974). M i l k a l s o c o n t a i n s m a i n l y m e t h y l d e r i v a t i v e s , 60 p e r c e n t - 7 -o f w h i c h a r e p r e s e n t as t h e monoglutamate ( S h i n , et^ _ a l . , 1975). P l a n t 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 o f f o l a t e d e r i v a t i v e s . L e t t u c e cabbage and orange 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 . , 1973; Chan e t a l . , 1973, Tamura e t a l . , 1976) b u t soybeans c o n t a i n o n l y 15 p e r c e n t o f t h i s f o r m , t h e r e s t b e i n g e i t h e r t h e 5- o r 10- f o r m y l d e r i v a t i v e ( S h i n e t a l . , 1975). Much work has been p u b l i s h e d on t h e a v a i l a b i l i t y and a b s o r p t i o n o f t h e f o l i c a c i d d e r i v a t i v e s i n f o o d (Swendseid e t a l . , 1947; S p r a y , 1952; J a n d l and L e a r , 1956; S t r e i f f and Rosenberg, 1967; H e r b e r t , 1968a; B u t t e r w o r t h et_ a l . , 1969; Rosenberg and Godwin, 1971; Tamura and - S t o k s t a d , 1973). I t 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 ^ g l u t a m y l p e p t i d e c o n j u g a t e s a r e a c t e d upon by i n t e s t i n a l enzymes ( ^ g l u t a m y l c a r b o x y p e p t i d a s e - " c o n j u g a s e " ) r e l e a s i n g t h e monoglutamic f o l a t e . I n t h e absence 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 p r e s e n t i n v a r i o u s f o o d m a t e r i a l s ( S t o k s t a d et_ a l . , 1977)» t h e r e i s l i t t l e d i f f e r e n c e i n t h e a v a i l a b i l i t y o f t h e c o n j u g a t e and t h e monoglutamate f o r m o f f o l a t e . An e s t i m a t e based on t h e i r s t u d i e s w i t h t r i t i u m l a b e l l e d f o l a t e mono-g l u t a m a t e s u g g e s t s t h a t 80% may be a b s o r b e d . METHODS OF FOLATE MEASUREMENT Methods f o r d e t e r m i n i n g f o l i c a c i d l e v e l s may be grouped i n t o t h r e e c a t e g o r i e s " . 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) 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' - 8 -B i o l o g i c a l Methods O ' D e l l and Hogan (1943) and C a m p b e l l e t a l . (1944) d e v e l o p e d t h e c h i c k a s s a y method o f f o l a t e measurement. R e c o v e r y f r o m d e f i c i e n c y symptoms when f e d c r y s t a l i n e f o l i c a c i d and foods: o f unknown p o t e n c y form t h e b a s i s o f t h i s method. R a t s have a l s o been used f o r b i o l o g i c a l a s s a y s (Ransome and Elvehjem>, 1943, A s e n j o , 1948). The main d i s -a d vantages o f a n i m a l a s s a y s a r e t h e i r expense and t h e t i m e f a c t o r i n v o l v e d . F r e e d (1966) however, a d v o c a t e d t h e use o f a n i m a l a s s a y s t o supplement m i c r o b i o l o g i c a l d a t a . M i c r o b i o l o g i c a l 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 a n a l y s i s 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 . T h i s o r g a n i s m has s i n c e become t h e most 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 a r e , o r have been, employed:- B a c i l l u s c o a g u l a n s (Baker et_ a l . , 1955) P e d i c o c c u s c e r e v i s i a e (Kavanagh, 1963) S t r e p t o c o c c i s p e c i e s (Luckey el: a l . , 1944). The c i l i a t e d p r o t o z o a n , Tetrahymena g e l e i i ( J u k e s , 1955) and t h e f l a g e l l a t e , C r i t h i d i a f a s c i c u l a t a (Nathan e t a l • , 1 958). Cooperman (1967) has emphasised t h a t t h e c h o i c e o f m i c r o -o r g a n i s m f o r any 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 of f o l a t e s l i k e l y t o be p r e s e n t i n t h e sample. F o r example, 1/. c a s e i i s t h e o n l y o r g a n i s m r e s p o n d i n g w e l l t o m e t h y l f o l a t e s - (as p r e v i o u s l y s t a t e d , 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 . f a e c a l i s (AOAC recommended a s s a y method) does 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, - 9 -i t does grow w e l l i n t h e p r e s e n c e o f p t e r o i c a c i d w h i c h i s m e t a b o l i c a l l y i n a c t i v e f o r man. F o l a t e s a r e f r e q u e n t l y f ound i n n a t u r e t o be p r e s e n t i n t h e p o l y g l u t a m a t e form. Thus, i t was f o u n d n e c e s s a r y by many w o r k e r s t o l i b e r a t e f r e e f o l a t e s p r i o r t o m i c r o b i o l o g i c a l a s s a y . Conjugase has been employed f o r t h i s p u r p o s e ( B i r d e t a l . , 1945, Dabrowska e t a l . , 1 9 4 9 ) . Because r e d u c e d forms o f f o l i c a c i d have been shown t o be h e a t l a b i l e , T o e n n i e s e t a l . (1956) employed a s c o r b i c a c i d i n t h e e x t r a c t i o n s o l v e n t t o p r e v e n t f o l i c a c i d d e s t r u c t i o n . The a s c o r b a t e was l a t e r shown by H e r b e r t (1961) t o a c t as a g r o w t h s t i m u l a n t t o L a c t o b a c i l l u s c a s e i . Thus he deemed i t n e c e s s a r y t o i n c l u d e a s c o r b a t e i n t h e s t a n d a r d s t o o b t a i n a t r u e assessment o f growth r e s p o n s e . 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 t h a n a n i m a l a s s a y s and a r e p a r t i c u l a r l y w e l l a d a p t e d t o s i m u l t a n e o u s a s s a y s o f s e v e r a l samples. However, 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 growth 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. I n a d d i t i o n , t h e i r growth may be a f f e c t e d by t h e p r e s e n c e o r absence o f s e v e r a l g r o w t h f a c t o r s . 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 complex f o o d m a t e r i a l s . 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 Most 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 a r e s u i t a b l e 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 o f f o l a t e s s i n c e t h e i r s e n s i t i v i t y i s i n a d e q u a t e t o d e t e c t t h e l o w c o n c e n t r a t i o n s o f f o l a t e s o c c u r r i n g i n f o o d s t u f f s . D e t a i l s o f much o f t h i s methodology w i l l t h e r e f o r e n o t be d i s c u s s e d h e r e . - 10 -A r e l a t i v e l y new method, however, d e v e l o p e d f o r t h e q u a n t i t a t i v e 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 , has been shown t o be s e n s i t i v e t o l ow l e v e l s o f f o l a t e , s i m i l a r t o t h o s e commonly found i n f o o d s . 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 b i n d i n g f r a c t i o n i n m i l k ( 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 w i t h t h e l a c t o g l o b u l i n f r a c t i o n o f m i l k p r o t e i n ( F o r d e t a l . , 1969) has been 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 and s e n s i t i v e a s s a y method. A r a d i o l i g a n d 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 r e p o r t e d by Metz et^ al. (1968). Waxman e t a l . (1971) d e s c r i b e d a s e n s i t i v e , d i r e c t c o m p e t i t i v e a s s a y u s i n g t r i t i a t e d 5-methyl t e t r a h y d r o f o l i c a c i d 3 5 ( H N CH„FH,). However, t h e a u t h o r s recommended t h a t t r i t i a t e d i 4 3 3 5 p t e r o y l g l u t a m i c a c i d ( H P t e G l u ) be s u b s t i t u t e d f o r t h e H N CH^FH^ i n a s e q u e n t i a l a s s a y system. A r c h i b a l d e t a l . ( 1 9 7 2 ) , R o t h e n b e r g e t a l . (1972) and T a j u d d i n and Gardyna (1973) employed t h e recommended p r o c e d u r e w i t h s l i g h t m o d i f i c a t i o n s . Waxman and S c h r e i b e r (1973) employed a c r y s t a l l i n e b o v i n e b e t a l a c t o g l o b u l i n as a s o u r c e 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 have been u t i l i z e d by Kamen and C a s t o n ( 1 9 7 4 ) . 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 has n o t been used f o r t h e a s s a y o f f o l a t e l e v e l s i n f o o d m a t e r i a l s a l t h o u g h t h e s e n s i t i v i t y o f t h e method - 0.62 t o 50 ng/ml o f sample ( f o r N^CH^FH^) - ap p e a r s t o be adequate f o r f o o d f o l a t e a s s a y . - 11 -LOSSES OF FOLIC ACID IN 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 a c i d r e s e a r c h i n v o l v e s t u d i e s o f i t s b i o c h e m i c a l and c l i n i c a l a s p e c t s . V e r y l i t t l e d a t a a r e a v a i l a b l e c o n c e r n i n g t h e e f f e c t o f f o o d p r o c e s s i n g on v i t a m i n c o n t e n t . E a r l y work has d e m o n s t r a t e d t h a t f o l a t e s a r e v e r y s u s c e p t i b l e t o d e s t r u c t i o n . C h e l d i n jet a l . (1943) c a r r i e d out a s u r v e y on t h e c o o k i n g 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 were t h e most s u s c e p t i b l e t o breakdown. S c h w e i g e r t ejt a l . (1946) r e p o r t e d l o s s e s o f f o l i c a c i d i n meat upon c o o k i n g . Hanning and M i t t s (1949) i n v e s t i g a t e d 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 o f eggs on f o l i c a c i d c o n t e n t and f o u n d l o s s e s o f 18-48%. H e r b e r t (1967) d e s c r i b e d how t h r i c e b o i l i n g o f f o o d s d e s t r o y e d t h e m a j o r i t y o f f o l a t e a c t i v i t y . I n 1968b, he a l s o e s t i m a t e d t h a t as much as 95% o f t h e i n i t i a l f o l a t e s i n 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 p r o c e s s e s and t h a t t h e l o s s e s were a g g r a v a t e d when t h e f o o d was f i n e l y d i v i d e d and cooked i n w a t e r f o r l o n g p e r i o d s . G h i t i s (1966) and F o r d j i t a l . (1968) showed t h a t v a r i a b l e l o s s e s o f f o l a t e i n m i l k o c c u r r e d d u r i n g u l t r a - h i g h - t e m p e r a t u r e p r o c e s s i n g , t h e l o s s e s b e i n g d i r e c t l y r e l a t e d t o t h e p r e s e n c e o f oxygen i n t h e m i l k . These a u t h o r s a l s o f ound t h a t t h e p r e s e n c e o f a s c o r b a t e 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 f o l a t e r e t e n t i o n . 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 t o be due t o t h e a c t i o n o f u l t r a v i o l e t l i g h t ( S t o k s t a d e^t a l . , 1947). S c h r o e d e r (1971) r e p o r t e d a 20-80% l o s s o f f o l a t e d u r i n g t h e m i l l i n g o f f l o u r . More r e c e n t l y , s l i g h t i n c r e a s e s i n t h e t o t a l f o l a t e c o n t e n t o f B r u s s e l s - 12 -sprouts t r e a t e d w i t h v a r i o u s processing methods have been reported 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 n a t u r a l l y o c c u r r i n g 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 r e f l e c t e d i n t h e v a r i a b i l i t y o f r e p o r t e d l e v e l s f o r t h i s v i t a m i n i n f o o d s t u f f s . U n t i l r e c e n t l y , m i c r o b i o l o g i c a l a s s a y s have been t h e most f r e q u e n t l y 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 . 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 -b i n d i n g f r a c t i o n i n m i l k ( 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 a c t i v i t y w i t h t h e l a c t o g l o b u l i n f r a c t i o n o f m i l k p r o t e i n ( F o r d , e t a l . , 1969) has been shown t o p r o v i d e t h e b a s i s f o r a r a p i d and s e n s i t i v e t e s t f o r f o l a t e s (Waxman e t a l . , 1971; A r c h i b a l d e t a l . , 1972; Rot h e n b e r g e t a l . , 1972; T a j u d d i n and Gardyna, 1973). Because o f t h e i n c r e a s i n g use o f t h e r a d i o a s s a y method f o r t h e q u a n t i t a t i v e measurement o f f o l a t e and t h e f r e q u e n t l y c o n t r a d i c t o r y 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 by t h i s method w i t h t h o s e o b t a i n e d by m i c r o b i o l o g i c a l a s s a y s (Waxman e t a l . , 1971; Ro t h e n b e r g e t a l . , 1972; M i n c e y e t a l . , 1973; Dunn & F o s t e r , 1973; T a j u d d i n and Gardyna, 1973; Shaw e t a l . , 1974; Kamen and C a s t o n , 1974; W a d d e l l et a l . , 1976; R u d z k i e t a l . , 1976) 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 o f 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 u s i n g b u f f e r e d s o l u t i o n s o f N^CH FH. as t h e s t a n d a r d and as 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 by 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 f o l a t e a c t i v i t i e s ( i . e . t h e f o l a t e a c t i v i t i e s b e f o r e and a f t e r t r e a t m e n t o f t h e f o o d e x t r a c t w i t h c o n j u g a s e ( J 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 by each method were compared. I n an attempt t o e x p l a i n t h e d i f f e r e n c e s i n f o o d f o l a t e l e v e l s o b t a i n e d by 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 r e s u l t s , s t a n d a r d c u r v e s o b t a i n e d u s i n g p t e r o y l g l u t a m i c a c i d ( P t e G l u ) ( F i g . 2) were t h e n compared 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 have been w e l l documented ( T e p l y and E l v e h j e m , 1945; H e r b e r t , 1966; Cooperman, 1967; H e r b e r t and B e r t i n o , 1967). L_. c a s e i was u t i l i z e d i n t h e s e e x p e r i m e n t s s i n c e t h i s o r g a n i s m e x h i b i t s a growth r e s p o n s e t o N^CH^FH^. The r a d i o a s s a y t e c h n i q u e used 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 by Rot h e n b e r g et a l . (1972) and u t i l i z e d a t w o - s t e p s e q u e n t i a l a d d i t i o n t e c h n i q u e . MATERIALS AND METHODS P r e p a r 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 S t a n d a r d s Samples o f N^CH^FH^ ( C l i n i c a l A s s a y s , I n c . M a s s a c h u s e t t s ) were d i l u t e d w i t h 0.1M phosphate b u f f e r (pH 7.3) c o n t a i n i n g 0.3% m e r c a p t o e t h a n o l t o g i v e an 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 ng/ml. T h i s s t o c k s o l u t i o n was t h e n s e r i a l l y d i l u t e d 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 a b l e I . A d d i t i o n a l d i l u t i o n s 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, 10.0, T a b l e I . N CH FH. c o n c e n t r a t i o n s used i i i p r e p a r a t i o n o f s t a n d a r d c u r v e s . 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 h-1 Ui 1/27 0.185 1.85 i 1/81 0.062 0.62 N 5 C H 3 F H 4 / D i l u t i o n 100 m i c r o l i t r e s (ng) - 16 -15.0 and 20.0 ng/ml N CH^FH^ were 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 t h e 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 a s s a y s t a n d a r d c u r v e . 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 and t o t h e a c t i o n o f u l t r a v i o l e t l i g h t , a s s a y t u b e s were k e p t i n d i f f u s e l i g h t and a f r e s h s t a n d a r d v i a l , r e c o n s t i t u t e d i m m e d i a t e l y p r i o r t o u s e , was u t i l i z e d f o r t h e p r e p a r a t i o n o f each s e t o f s t a n d a r d c u r v e s . 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 phosphate b u f f e r ( 0 . 3 % m e r c a p t o e t h a n o l ) was made up s u c h t h a t a l l samples were 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 s t a n d a r d c u r v e s . P r e p a r a t i o n o f Food E x t r a c t s Three samples o f s t r a i n e d baby f o o d s were a n a l y z e d f o r ' f r e e ' and ' t o t a l ' f o l a t e . The c o n t e n t s o f each j a r were mixed t h o r o u g h l y b e f o r e t h e p r e p a r a t i o n o f t h e e x t r a c t s . The t e s t e x t r a c t s were p r e p a r e d as f o l l o w s : t o a p p r o x i m a t e l y 2g s t r a i n e d f o o d sample i n a 15 m l c e n t r i f u g e t u b e was added 5 ml 0.1M phosphate b u f f e r (pH 7.3, 1% a s c o r b a t e ) . The e x t r a c t s were mixed t h o r o u g h -l y , u s i n g a v o r t e x m i x e r . The p r e p a r e d t u b e s were 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 w a t e r . - 17 -D e s i c c a t e d c h i c k e n p a n c r e a s : ( D i f c o ) was suspended i n w a t e r (5 mg/ml), t h o r o u g h l y 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 37°C f o r 45 m i n u t e s . A f t e r f i l t r a t i o n o f t h e s l u r r y t h r o u g h Whatman No. 1 p a p e r , t h e c l e a r f i l t r a t e was used i m m e d i a t e l y . F o r t h e d e t e r m i n a t i o n o f ' t o t a l f o l a t e a c t i v i t y ' 1 ml o f t h e f i l t r a t e was added t o each t u b e . T o l u e n e (as a p r e s e r v a t i v e ) was added and t h e sample t u b e s and enzyme b l a n k s (5 m l b u f f e r p l u s 1 m l enzyme p r e p a r a t i o n ) were i n c u b a t e d a t 37°C f o r 16 h o u r s . The c o n t e n t s o f t h e f l a s k were t h e n a d j u s t e d t o 10 m l w i t h p hosphate - a s c o r b a t e b u f f e r . A f t e r f u r t h e r m i x i n g , 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 e x t r a c t s were a s s a y e d by t h e methods d e s c r i b e d below. F o r t h e d e t e r m i n a t i o n o f ' f r e e f o l a t e ' a c t i v i t y , t h e c o n j u g a s e t r e a t m e n t was o m i t t e d and t h e c o n t e n t s o f t h e t u b e s were a d j u s t e d d i r e c t l y t o 10 m l , r e m i x 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 , a s s a y e d . P r e p a r a t i o n o f P t e r o y l g l u t a m i c A c i d S t a n d a r d s Samples o f P t e G l u ( N u t r i t i o n a l B i o c h e m i c a l s Corp., Ohio) were d i l u t e d i n 0.1 M phosphate b u f f e r (pH 7.3) c o n t a i n i n g 0.3% m e r c a p t o e t h a n o l t o g i v e c o n c e n t r a t i o n s o f 50.00, 16.67, 5.56, 1.85 and 0.62 ng/ml P t e G l u . M a i n t e n a n c e 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 o f Inoc u l u m 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 as a s t a b c u l t u r e i n t u b e s o f 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 A g a r . C u l t u r e s were i n c u b a t e d - 18 -a t 35-37 C f o r 18-24 ho u r s and 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 were made a t m o n t h l y i n t e r v a l s . I noculum f o r t h e a s s a y was made by 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 c o n t a i n i n g 10 ml 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 . A f t e r 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 tube s were 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 and c e l l s were r e s u s p e n d e d i n 10 m l 0.85% s t e r i l e s a l i n e . The c u l t u r e was r e c e n t r i f u g e d and suspended i n f r e s h s a l i n e t w i c e more. An a l i q u o t was d i l u t e d 1 0 0 - f o l d i n a s t e r i l e c o n t a i n e r w i t h s t e r i l e s a l i n e and one drop o f t h i s was used 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 A s s a y P r o c e d u r e 5 ml 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 ( D i f c o ) were d i s p e n s e d i n t o t e s t t u b e s . 0.1 ml 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 added t o each t u b e and t h e volume made up t o 10 ml w i t h 0.1 M phosphate 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 . When t h 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 ( w i t h i n t h e ra n g e o f t h e a s s a y ) o f u n t r e a t e d e x t r a c t , c o n j u g a s e - t r e a t e d e x t r a c t o r c o n j u g a s e b l a n k s were added 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 ml r e h y d r a t e d medium. Volume was made up t o 10 ml w i t h 0.1 M phosphate 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 . A l l t u b e s were 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 . (121 C) and t h e n c o o l e d t o room t e m p e r a t u r e . One drop o f t h e d i l u t e d L . c a s e i c e l l s u s p e n s i o n was added t o each t u b e . Tubes were 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 measured. T h i s was e x p r e s s e d i n terms o f ab 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 -R a d i o a s s a y P r o c e d u r e 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 t h e 3 C l i n i c a l A s s a y s H F o l i c A c i d R a d i o a s s a y K i t ( C l i n i c a l A s s a y s , 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 i n t h e r a d i o a s s a y were made up and 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 as 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 phosphate b u f f e r pH 7.3 was used i n p l a c e o f t r i s - N a C l b u f f e r (Appendix A ) . D u p l i c a t e samples o f N^CH^FH^, P t e G l u , o r f o o d 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 t e r a d d i t i o n o f H P t e G l u t h e r e was a f u r t h e r 20 m i n u t e , room t e m p e r a t u r e i n c u b a t i o n . The p r o t e i n - b o u n d 3 and t h e f r e e 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 . S u p e r n a t a n t 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 c e n t r i f u g e ( S o r v a l l M odel RC2-B) a t 4 C, 2,000 x g f o r 20 m i n u t e s . The s u p e r n a t a n t was d e c a n t e d i n t o v i a l s and 12 ml s c i n t i l l a t i o n f l u i 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 C o c k t a i l ) were added. A f t e r c o o l i n g , sample r a d i o a c t i v i t y was c o u n t e d f o r 10 m i n u t e s 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 s c i n t i l l a t i o n c o u n t e r u s i n g t h e t r i t i u m program f o r low quench samples. C o r r e c t i o n f o r c o u n t i n g e f f i c i e n c y (quench c o r r e c t i o n ) , was c a r r i e d o ut a t each a s s a y u s i n g t h e c h a n n e l s r a t i o t e c h n i q u e (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 w i t h e v e r y 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 compete 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 s i t e s . I n a d d i t i o n , 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 - 20 -d u p l i c a t e w i t h each a s s a y t o d e t e r m i n e l e v e l s o f r a d i o a c t i v i t y n o t removed by 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 were a s s a y e d , d u p l i c a t e b a c k g r o u n d samples 3 c o n t a i n i n g b u f f e r , e x t r a c t and H P t e G l u were a n a l y z e d f o r each e x t r a c t i n o r d e r t o c o r r e c t f o r t h e f o o d ' 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 c a p a c i t y . T h i s p r e c a u t i o n was recommended by T a j u d d i n and Gardyna (1973) who, when m e a s u r i n g human serum f o l a t e l e v e l s , added a serum b l a n k t o 3 c o r r e c t f o r n o n - s p e c i f i c b i n d i n g o f H P t e G l u . When e x t r a c t b a c k g r o u n d count r a t e s were f o u n d t o be g r e a t e r t h a n 20% o f e x t r a c t samples t h e t o t a l 3 count r a t e f o r H P t e G l u was a l s o measured. E x p e r i m e n t a l P r o c e d u r e 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 out 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 samples o f t h e f r e s h l y p r e p a r e d N^CH^FH^. T h i s e n s u r e d 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 c o n c e n t r a t i o n s o f N CH^FH^ between b a t c h e s was t a k e n i n t o a c c o u n t by b o t h methods. T w e n t y - f i v e unknown d i l u t i o n s o f N^CH^FH^ were a s s a y e d by b o t h methods. C o n c e n t r a t i o n s were d e t e r m i n e d from N^CH^FH, s t a n d a r d c u r v e s 3 4 p r e p a r e d a t t h e same t i m e and und e r - 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 f o o d e x t r a c t ( b o t h t o t a l and f r e e f o l a t e e x t r a c t s ) were p r e p a r e d i n d u p l i c a t e f o r each a s s a y method. N^CH^FH^ s t a n d a r d c u r v e s were p r e p a r e d s i m u l t a n e o u s l y by each a s s a y 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 u s i n g 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 c u r v e s were p r e p a r e d a t t h e same t i m e as an 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 c u r v e s i n o r d e r t o e n s u r e t h a t c o n d i t i o n s o f a s s a y remained as s i m i l a r as p o s s i b l e f o r each s t a n d a r d used. l^CH^FH, R e c o v e r y Study A powered m i l k sample ( 3 g ) was mixed w i t h 5 ml 0.1 M phosphate b u f f e r (pH 7.3, 1% a s c o r b a t e ) i n a 15 ml c e n t r i f u g e tube.- The tube was th 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 and c o o l e d i n c o l d w a t e r . The c o n t e n t s o f t h e t u b e were t h e n a d j u s t e d t o 10 m l , r e m i x e d , and c e n t r i f u g e d . A r a d i o a s s a y was t h e n c a r r i e d o ut u s i n g t h e m i l k e x t r a c t as o u t l i n e d i n T a b l e I I . Samples o f t h e e x t r a c t (100 u l ) were added t o each o f f i v e t u b e s c o n t a i n i n g t h e s t a n d a r d N^CH^FH^ d i l u t i o n s . I n a d d i t i o n , 3 d u p l i c a t e b a c k g r o u n d 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 H P t e G l u were a n a l y z e d t o c o r r e c t f o r t h e m i l k ' 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 c a p a c i t y . D u p l i c a t e samples c o n t a i n i n g b u f f e r , e x t r a c t , b i n d i n g p r o t e i n 3 and H P t e G l u were a s s a y e d i n o r d e r t o d e t e r m i n e t h e f o l a t e c o n t e n t o f t h e powdered m i l k . When e x t r a c t b ackground c o u n t s were found t o be g r e a t e r 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 count r a t e 3 f o r H P t e G l u was a l s o measured. - 22 -Table I I . Volumes of s o l u t i o n s used i n N CH FH recovery s t u d y . 3 Tube No. Buf f e r M i l k E x t r a c t s Standard D i l u t i o n Binding P r o t e i n 3H PteGlu T T 1 2 1,100 — 50 1,2 500 — 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 1 2 T o t a l count 1,2 Binding c o n t r o l background 3,4 Binding c o n t r o l 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 e x t r a c t 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 (1.667ng/100;il) + m i l k e x t r a c t 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 e x t r a c t 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 e x t r a c t 13,14 Standard d i l u t i o n (0.062 ng/lOOul) 13.14M Standard d i l u t i o n (0.062 ng/lOOul) + m i l k e x t r a c t 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 - 23 -RESULTS N CH.FH, S t a n d a r d Curves M i c r o b i o l o g i c a l A s s a y R e g r e s s i o n a n a l y s i s was c a r r i e d out on t h e d a t a f r o m t h e t e n s t a n d a r d c u r v e s o b t a i n e d by t h e m i c r o b i o l o g i c a l a s s a y method. T h i s i s 2 shown i n F i g u r e 3. 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 ( r ) o f 0.61 i l l u s t r a t e s t h a t t h e r e i s a s i g n i f i c a n t (p 0.01) r e l a t i o n s h i p between c o n c e n t r a t i o n o f N^CH^FH^ and growth r e s p o n s e o f _L. c a s e i as measured by a b s o r b a n c e a t 660 nm. The s t a n d a r d e r r o r o f e s t i m a t e was fo u n d t o be 0.11. R a d i o a s s a y S t a n d a r d c u r v e s 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 t r a c e r bound f o r each amount o f s t a n d a r d on s e m i l o g p a p e r . The p e r c e n t o f t r a c e r bound f o r each 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 from t h e n o r m a l i z e d (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 , t h u s : (CPM - CPM ) % H P t e G l u bound = , * „ " x x 100 2 (CPM B - CPM^) where CPM = Average c o u n t s p e r m i n u t e f o r s t a n d a r d samples n CPM B G= Average c o u n t s p e r m i n u t e f o r bac k g r o u n d c o n t r o l s CP Mg = Average 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 - 24 -- 25 -R e g r e s s i o n a n a l y s i s p e r f o r m e d on t h e t e n s e t s o f d a t a i s shown i n F i g u r e 4. 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 improve 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 ( r ) f o r t h e c u b i c e q u a t i o n was 0.98 compared t o a 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 of 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 t o t h e d a t a ) . There i s a h i g h l y s i g n i f i c a n t (p ^  0.01) r e l a t i o n s h i p between c o n c e n t r a t i o n o f N^CH^FH^ and p e r c e n t b i n d i n g o f t r a c e r . The 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 4.01. 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 B o t h 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^ samples were 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 c u r v e s o b t a i n e d by b o t h methods. R e s u l t s o b t a i n e d 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 methods a r e shown i n F i g u r e 5. R a d i o a s s a y v a l u e s r a n g e d from 0.62 t o 35 ng/ml w h i l e 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 from 0.75 t o 38 ng/ml. The c o e f f i c i e n t 2 o f d e t e r m i n a t i o n ( r ) f o r v a l u e s o b t a i n e d by t h e two methods was 0.93 and t h e r e g r e s s i o n l i n e was f o u n d t o have a s l o p e o f 0.76 and an i n t e r c e p t o f 3.10 ng/ml. The s t a n d a r d e r r o r o f e s t i m a t e was 3.65 ng/ml. 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 o f Baby Foods O b t a i n e d U s i n g B o t h Methods F r e e 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 f o o d 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 a s s a y methods. -0-5 0-0 L O G 0-5 c o n e (ng^/ml 1 0 1-5 2 0 Figure 4. Regression a n a l y s i s performed on N CH^FH^ standard curves obtained by radioassay method. - 27 -M I C R O B I O L O G I C A L A S S A Y Figure 5. Regression a n a l y s i s of radioassay on L,. c a s e i m i c r o b i o l o g i c a l assay method of measuring N^CH^FH^ con 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 that would be obtained had the m i c r o b i o l o g i c a l assay values equalled the radioassay 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 f o o d e x t r a c t was f i r s t c o r r e c t e d .for i t s own n o n - s p e c i f i c b i n d i n g t h u s : (CPM - CPM ) H P t e G l u bound = ( CPM^ - CPM^) X 1 0 0 % where = 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 sample CPMgg = Average 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 b a ckgrounds n CPM = Average 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 CPMgg = Average c o u n t s p e r m i n u t e f o r bac k g r o u n d c o n t r o l s . Where f o o d e x t r a c t b ackground c o u n t s were f o u n d t o be g r e a t e r t h a n 20% o f t h e e x t r a c t sample 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 t o t a k e i n t o a c c o u n t t h e l o w e r t r a c e r c o n c e n t r a t i o n a v a i l a b l e f o r b i n d i n g d u r i n g t h e e x t r a c t sample i n c u b a t i o n . The f o l l o w i n g e q u a t i o n was use d : (CPM - CPM ) x 100% % H P t e G l u bound = n (CPM - CPM ) ( C P ^ - C P M B G n ) x ( C P M t _ C P M b g ) where CPM^, = T o t a l c o u n t s p e r m i n u t e T h i s e q u a t i o n r e d u c e s t o : (CPM - CPM ) x 100% % H P t e G l u bound = (CPM^ - C P M ^ ) x % T o t a l Bound T 100 T BGn N o r m a l i z e d (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 c a l c u l a t i o n s . - 29 -V a l u e s 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 by 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 baby f o o d samples a r e shown i n T a b l e I I I . Comparison o f N^CH^FH^ and P t e G l u S t a n d a r d Curves 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 on t h e f i v e s e t s o f s t a n d a r d c u r v e s produced by b o t h methods o f a s s a y u s i n g t h e two forms o f f o l a t e . R e s u l t s a r e p r e s e n t e d i n F i g u r e s 6 and 7. P a i r e d c o m p a r i s o n S t u d e n t ' s t - t e s t s were c a r r i e d o ut on t h e d a t a . These r e s u l t s a r e p r e s e n t e d i n T a b l e I V. 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 e v i d e n t . T h i s i s p a r t i c u l a r l y t h e case 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 u s e d . 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 ng/ml 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 s t a n d a r d s . The d i f f e r e n c e i n t h e growth 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 . 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 l o w e r 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 appear t o be e v i d e n t from t h e r e g r e s s i o n a n a l y s i s . I t w o u l d appear f r o m t h e l i m i t e d d a t a p r e s e n t e d t h a t t h e m i l k 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 and t h a t P t e G l u causes a g r e a t e r growth r e s p o n s e o f L_. c a s e i t h a n N 5CH 0FH.. 3 4 N 5CH 3FH^ R e c o v e r y Study R e s u l t s o f t h i s 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 N^CH^FH^is shown t o be between 75-80% 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 T a b l e 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 Foods. F r e e F o l a t e A c t i v i t y | ^ ng/lOOg p r o d u c t j ' T o t a l F o l a t e A c t i v i t y \ug/100g p r o d u c t ] Range Mean Range Mean MIXED VEGETABLES R a d i o a s s a y L. c a s e i 2.0 - 4.3 4.0 - 4.6 3.2 4.2 13.0 - 18.0 17.5 - 19.0 17.5 18.3 HAM WITH VEGETABLES R a d i o a s s a y L. c a s e i 2.6 - 5.7 6.5 - 9.5 4.9 7.8 18.5 - 22.4 10.0 - 13.7 21.0 12.6 u> o VEGETABLES AND LIVER WITH BACON R a d i o a s s a y 4 . 7 - 7 . 0 6.5 L. c a s e i 8.0 -13.5 12.1 20.0 - 24.5 26.0 - 29.0 22.5 27.0 - 31 -0 0 -0-5 0 0 0-5 1 0 1-5 2 0 L O G c o n e (ngy/mlj F i g u r e 6. 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 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 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. - 32 -T a b l e 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 and N CH„FH, 3 4 s t a n d a r d c u r v e s 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 t e c h n i q u e s . 50.00 C o n c e n t r a t i o n o f F o l a t e (ng/ml) 16.70 5.57 1.85 0.62 MICROBIOLOGICAL ASSAY t v a l u e d e g r e e s o f freedom •P i 8.10 4 0.01 6.27 4 0.01 9.07 4 0.001 4.06 4 0.02 12.06 4 0.001 RADIOASSAY t v a l u e d e g r e e s o f freedom P * 11.51 4 0.001 7.08 4 0.01 4.74 4 0.02 2.50 4 0.10 3.74 4 0.05 Table V. Recovery of N^CH^FH^ from mi l k e x t r a c t as measured by the radioassay method. Concentration of N 5CH 3FH 4 added Recovery (ng/lOOul) % 5.000 1.667 0.556 0.185 0.062 35 63 76 80 76 - 35 -added N^CH^FH^. The p e r c e n t r e c o v e r y d r o p s 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 added N^CH^FH^ i n c r e a s e s however. T h i s i s due, i n p a r t , t o t h e v e r y h i g h c o u n t i n g r a t e r e c o r d e d f o r t h e m i l k e x t r a c t b a c k g r o u n d sample. I n a l l c a s e s , t h e m i l k e x t r a c t b ackground was found t o be g r e a t e r t h a n 20% o f t h e m i l k e x t r a c t p l u s s t a n d a r d sample. 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 t o t a k e i n t o a c c o u n t t h e l o w e r t r a c e r c o n c e n t r a t i o n a v a i l a b l e f o r b i n d i n g d u r i n g t h e e x t r a c t p l u s s t a n d a r d sample i n c u b a t i o n . (See "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 o f Baby Foods O b t a i n e d U s i n g B o t h Methods".) DISCUSSION M i c r o b i o l o g i c a l a s s a y s have been t h e most f r e q u e n t l y used methods f o r m e a s u r i n g 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 t i m e consuming r e q u i r i n g b o t h m a i n t e n a n c e 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 i n c u b a t i o n p e r i o d . _L. c a s e i has been t h e most f r e q u e n t l y used m i c r o o r g a n i s m f o r f o l a t e a s s a y 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 f a c t o r s ( S t o k s t a d and H u t c h i n g s , 1947). I t i s c a p a b l e o f c o n v e r t i n g v a r i o u s o t h e r g r o w t h f a c t o r s t o f o l a t e d u r i n g p e r i o d s o f s t r e s s ( B a k e r , et: a l . , 1 9 71), and i t can a l s o be s u b j e c t t o m e t a b o l i c changes such t h a t 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 , t o g e t h e r w i t h t h e v a r i o u s 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 and t i m e o f i n c u b a t i o n , make t h e use 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 d e t e r m i n a t i o n o f f o l a t e l e v e l s . - 36 -The r a d i o a s s a y method, by c o m p a r i s o n , i s r a p i d and can be 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 o v e r a p e r i o d o f 3 t o 4 h o u r s . A l t h o u g h 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 s u c h f a c t o r s as t e m p e r a t u r e and t i m e o f i n c u b a t i o n , pH and 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 Gutcho, 1975; Z e t t n e r and D u l y , 1974, 1 975), t h e e f f e c t s o f t h e s e f a c t o r s a r e n o t as g r e a t as t h e a f o r e m e n t i o n e d f a c t o r s a f f e c t i n g t h e m i c r o b i o l o g i c a l method. Thus, as shown by t h e s e r e s u l t s , a much more r e p r o d u c i b l e s t a n d a r d c u r v e can be 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 t h e m i c r o b i o l o g i c a l a s s a y . I t s h o u l d be n o t e d t h a t when u s i n g c o m m e r c i a l l y p r e p a r e d k i t s , t h e r e may be a d i f f e r e n c e i n t h e f o l a t e b i n d i n g a c t i v i t y o f t h e m i l k p r o t e i n f r o m one k i t t o a n o t h e r c a u s i n g d i f f e r e n c e s i n s t a n d a r d c u r v e s t o o c c u r . P r e v i o u s p u b l i c a t i o n s p e r t a i n i n g t o t h e c o m p a r i s o n o f f o l a t e 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 methods, v a r y c o n s i d e r a b l y i n t h e i r c o n c l u s i o n s . Serum f o l a t e l e v e l s were compared i n e v e r y c a s e . A r c h i b a l d e t a l . ( 1 9 7 2 ) , Waxman e t a l . (1971) and T a j u d d i n and Gardyna (1973) showed good agreement between t h e two methods. M i n c e y e t a l . (1973) and W a d d e l l e t a l . (1976) f o u n d t h a t mean serum f o l a t e l e v e l s as d e t e r m i n e d by t h e r a d i o a s s a y method were s l i g h t l y l o w e r t h a n t h o s e o b t a i n e d by t h e m i c r o b i o l o g i c a l method b u t s t i l l o f f e r e d t h e same degree o f d i a g n o s t i c i n f o r m a t i o n . R o t h e n b e r g et_ a l . (1972) found t h a t r a d i o a s s a y 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 h a l f t h o s e o b t a i n e d by t h e m i c r o b i o l o g i c a l a s s a y and R u d z k i e t a l . (1976) c o n c l u d e d t h a t t h e m i c r o b i o l o g i c a l a s s a y 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 l e v e l s i n serum. I n a l l c a s e s , N^CH^FH^ was used as s t a n d a r d f o r t h e r a d i o a s s a y 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 used f o r p r e p a r a t i o n o f t h e m i c r o b i o l o g i c a l s t a n d a r d c u r v e . - 37 -I t can be seen from t h e r e s u l t s o b t a i n e d i n t h i s e x p e r i m e n t t h a t t h e growth o f L_. c a s e i and t h e m i l k p r o t e i n b i n d i n g a b i l i t y a r e dependent, 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 system. I t i s t o be 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 methods o f a s s a y u s i n g P t e G l u as 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 and N^CH^FH^ as s t a n d a r d f o r t h e r a d i o a s s a y w i l l y i e l d d i f f e r e n t r e s u l t s . S i n c e P t e G l u and N^CH^FH^ r e a c t d i f f e r e n t l y i n each system o f a s s a y , i t may 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 p r e p a r e d f r o m a s i n g u l a r pure f o r m o f 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 o f 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. I t has been w e l l documented t h a t N^CH^FH^ i s t h e predominant f o r m o f f o l a t e f o und i n serum ( H e r b e r t e t a l . , 1962). However, forms o f f o l a t e s o t h e r t h a n N^CH„FH, 3 4 may a l s o be 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 a f f i n i t y f o r t h e m i l k p r o t e i n o r cause a g r e a t e r growth r e s p o n s e o f L. c a s e i . T h i s may p a r t i a l l y e x p l a i n why Shaw et^ a l . (1974) o b t a i n e d a poor c o r r e l a t i o n c o e f f i c i e n t f o r serum f o l a t e l e v e l s measured by b o t h methods even though N^CH„FH. was used as s t a n d a r d f o r b o t h a s s a y s . 3 4 T h i s may a l s o be 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 r e s u l t s o b t a i n e d when baby f o o d was a s s a y e d f o r f o l a t e by b o t h methods. Food f o l a t e s have been a s s a y e d u s i n g b o t h _L. c a s e i (Hoppner, 1971; Hoppner e t a l . , 1972; 1973; B u t t e r f i e l d and C a l l o w a y , 1972; and o t h e r ) and S_. f a e c a l i s ( L i n e t a l . , 1975; Dong and Oace, 1973 and o t h e r s ) . However, th e r e s p o n s e t o t h e d i f f e r e n t forms o f f o l a t e v a r y among t h e s e organisms ( S t o k s t a d and Koch, 1967; Dong and Oace, 1973). - 38 -O t h e r 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 f o o d s y s t e m 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 method i s used . Many p r o t e i n s have been shown t o e x h i b i t f o l a t e b i n d i n g p r o p e r t i e s . B i n d e r s 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 i n m i l k ( G h i t i s , 1967; Metz e t a l . , 1968) and many o t h e r b i o l o g i c a l m a t e r i a l s ( Rothenberg e t a l . , 1977). Indeed, t h e a b i l i t y t o b i n d f o l a t e forms t h e b a s i s o f t h e r a d i o a s s a y 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 method. Thus t h e p r e s e n c e o f o t h e r p r o t e i n s w i t h f o l a t e b i n d i n g a b i l i t y i n t h e system t o be a s s a y e d , may cause e r r o r s u n l e s s c o r r e c t i o n s a r e made f o r t h e i r s p e c i f i c f o l a t e b i n d i n g a b i l i t y . F o r t h i s r e a s o n , T a j u d d i n and Gardyna (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 t o c o r r e c t f o r 3 n o n - s p e c i f i c b i n d i n g a b i l i t y o f H P t e G l u t o t h e a s s a y m a t e r i a l . F o r d e t a l . , (1969) s t a t e d t h a t h e a t i n g m i l k f o r 10 m i n u t e s a t 100°C r e l e a s e d t h e p r o t e i n bound f o l a t e and d e s t r o y e d t h e p r o t e i n b i n d i n g a b i l i t y . Thus, Dunn and F o s t e r (1973) e x t r a c t e d t h e i r serum samples by b o i l i n g i n a w a t e r b a t h f o r 15 m i n u t e s i n 0.05 M l y s i n e b u f f e r , pH 10.5. T h i s p r o c e d u r e d e n a t u r e d t h e serum b i n d e r and r e l e a s e d t h e bound f o l a t e . ( 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 r e d u c i n g agent was added t o t h e i r serum e x t r a c t s d u r i n g h e a t i n g ) . I n t h e two e x p e r i m e n t s u s i n g f o o d m a t e r i a l s d e s c r i b e d h e r e , t h e e x t r a c t i o n p r o c e s s i n v o l v e d h e a t i n g t h e f o o d samples f o r 10 m i n u t e s i n a b o i l i n g w a t e r b a t h and a s s a y i n g an e x t r a c t b ackground 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 d e n a t u r e d by h e a t i n g were compensated f o r i n t h e back g r o u n d sample. I n a l l f o o d s t e s t e d w i t h t h e e x c e p t i o n o f t h e mixed v e g e t a b l e s , t h e e x t r a c t b ackground c o u n t s were f o u n d t o be g r e a t e r t h a n 20% o f t h e e x t r a c t sample c o u n t s ; i n d i c a t i n g t h a t t h e h e a t i n g p r o c e s s was n o t s u f f i c i e n t t o d e s t r o y a l l t h e p r o t e i n b i n d i n g a c t i v i t y . T h e r e f o r e , 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 H PGA bound t o each f o o d sample. H i g h e x t r a c t b ackground c o u n t s however, t e n d t o d e c r e a s e t h e v a l i d i t y o f t h e a s s a y . O t h e r f a c t o r s s u c h as pH, m e t a l c a t a l y s t s and t h e p r e s e n c e o f p r o - o r a n t i - o x i d a n t s w i t h i n t h e f o o d system may a l s o a f f e c t t h e v a l i d i t y o f t h e a s s a y . The r e c o v e r y o f N^CH^FH^ from a f o o d system does i n d i c a t e however, t h a t p a r t i c u l a r l y a t l o w e r c o n c e n t r a t i o n s o f f o l a t e t h e e x p e r i m e n t a l p r o c e d u r e i s v a l i d . As shown i n t h i s e x p e r i m e n t ^ a t h i g h c o n c e n t r a t i o n s o f f o l a t e , 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 b i n d a l l t h e f o l a t e p r e s e n t i n t h e sample t h u s d e c r e a s i n g t h e v a l i d i t y o f 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 h e r e i n d e s c r i b e d i s a f a s t e r , s i m p l e r and more a c c u r a t e method f o r m e a s u r i n g l e v e l s o f N^CH^FH^ t h a n theJL,. c a s e i m i c r o b i o l o g i c a l method. When N^CH^FH^ s t a n d a r d c u r v e s 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^ as d e t e r m i n e d by 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 by t h e 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 . When f r e e and 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 baby f o o d s were a s s a y e d r e s u l t s o b t a i n e d by t h e two methods d i d n o t compare w e l l . When t h e r a d i o a s s a y method o f f o l a t e a s s a y i s c a r r i e d o u t a t pH 7.3 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 o f t h e m i l k p r o t e i n t o b i n d P t e G l u and N^CH^FH^. 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 growth r e s p o n s e t o t h e two forms o f f o l a t e . The 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 ut by t h e methods 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 -m e a s u r i n g s i n g u l a r forms o f f o l a t e u s i n g s t a n d a r d c u r v e s c o n s t r u c t e d f r o m d a t a o b t a i n e d u s i n g t h e same f o l a t e . N^CH^FH^ and P t e G l u a r e j u s t two of t h e many forms o f f o l a t e t h a t have been i d e n t i f i e d . O t h e r forms may show g r e a t e r o r l e s s e r b i n d i n g a f f i n i t i e s and 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 growth 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 t o g e t h e r i n any one m a t e r i a l may a l s o cause v a r i a t i o n s . 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 known t o c o n t a i n many forms o f f o l a t e c annot 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 methods d e s c r i b e d u n t i l 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, forms 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 e ach o t h e r and w i t h b i o l o g i c a l m a t e r i a l s i s o b t a i n e d . 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 f o o d f o l a t e l e v e l s can 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. I t s h o u l d a l s o be r e c o g n i z e d 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 a v a i l a b l e a t t h i s t i m 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. - 41 -CHAPTER I I I . KINETIC STUDIES INTRODUCTION Q u a l i t y d e t e r i o r a t i o n i n t h e f o r m o f n u t r i t i o n a l d e g r a d a t i o n can o c c u r i n any f o o d d u r i n g h e a t p r o c e s s i n g . The r a t e s o f t h e 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 s u c h f a c t o r s as t e m p e r a t u r e 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 oxygen p r e s e n t and p r e s e n c e o r absence o f o t h e r c h e m i c a l s s u c h as p r o - o r a n t i -o x i d a n t s . The p r e s e n c e o r absence o f o t h e r c h e m i c a l s makes t h e p r e d i c t i o n o f 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 f o o d systems > t h e r e a r e many s u b s t a n c e s p r e s e n t w h i c h i n f l u e n c e t h e r a t e o f d e g r a d a t i o n o f t h e v a r i o u s n u t r i e n t s . V e r y l i t t l e r e s e a r c h has been c o n d u c t e d on t h e e f f e c t o f 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 . C h e l d i n e t a l . ( 1 9 4 3 ) , S c h w e i g e r t e t a l . ( 1 9 4 6 ) , S t o k s t a d ^ t a l . ( 1 9 4 7 ) , Hanning and M i t t s ( 1 9 4 9 ) , G h i t i s , ( 1 9 6 6 ) , H e r b e r t (1967, 1968b), F o r d e t a l . ( 1 9 6 8 ) , S c h r o e d e r (1971) and M a l i n (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 on t h e n u t r i t i o n a l s t a b i l i t y o f f o l a t e s w i t h i n d i f f e r e n t f o o d systems and have f o u n d them t o be 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 by o x i d a t i v e h e a t i n g and by t h e a c t i o n o f u l t r a v i o l e t l i g h t . H e r b e r t (1968b) e s t i m a t e d t h a t as much as 95% o f t h e i n i t i a l f o l a t e s i n f o o d 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 p r o c e s s e s . F o r d a l . ( 1 9 6 8 ) , i n v e s t i g a t i n g t h e e f f e c t s o f UHT p r o c e s s i n g and subsequent s t o r a g e o f m i l k on t h e v i t a m i n c o n t e n t , c o n c l u d e d t h a t v a r i a t i o n i n t h e s t a b i l i t y o f f o l a t e s was r e l a t e d d i r e c t l y t o t h e p r e s e n c e o f oxygen i n t h e m i l k , t h e d e a e r a t i o n o f 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 t h e 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 be 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 by v a r i o u s means (Waters and M o l l i n , 1961). G h i t i s (1966) found a h e a t l a b i l e f r a c t i o n p r e s e n t i n m i l k r e s e m b l i n g N^CH^FH^. The f r a c t i o n c o u l d be p r o t e c t e d f r o m o x i d a t i o n by t h e p r e s e n c e o f a s c o r b i c a c i d a l t h o u g h t h e a u t h o r found t h a t l e v e l s o f a s c o r b a t e n a t u r a l l y p r e s e n t 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 f o l a t e f r a c t i o n d u r i n g b o i l i n g . 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 m i c r o b i o l o g i c a l a s s a y 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 N^CH^FH^ l e v e l s . The c o m p l e x i t y o f f o o d s i n w h i c h f o l a t e l e v e l s have been measured, makes i t d i f f i c u l t t o p r e d i c t t h e t h e r m a l d e g r a d a t i o n c h a r a c t e r i s t i c s o f f o l a t e w i t h i n a f o o d s y s t e m o t h e r t h a n t h e one t e s t e d . S e v e r a l r e c e n t 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. Examples o f such s t u d i e s a r e t h o s e o f t h i a m i n e d e g r a d a t i o n c a r r i e d o u t by 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 by h e a t i n g i n oxygen was c a r r i e d o ut u s i n g a s i m p l e model sys t e m o f N^CH FH i n phosphate b u f f e r . The r a d i o a s s a y method as d e s c r i b e d j 4 i n C h a p t e r I I was used f o r 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 s . S i n c e t h e 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 was t h o u g h t t o be an o x i d a t i o n r e a c t i o n , a n a t t e m p t was made t o i d e n t i f y t h e r e a c t i o n mechanism by r e p e a t i n g t h e above e x p e r i m e n t i n t h e absence o f any r e d u c i n g agent.N^CH^FH^ was a s s a y e d , t h e r e f o r e , i n t h e absence o f m e r c a p t o e t h a n o l . S i n g h e t a l . (1976) have 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 d e g r a d a t i o n and oxygen u p t a k e f o l l o w s second o r d e r r e a c t i o n , k i n e t i c s . The d e g r a d a t i o n o f N^CH^FH^ i n t h e p r e s e n c e o f a l i m i t e d oxygen s u p p l y was t h e r e f o r e a l s o s t u d i e d . - 43 -I n o r d e r t o s t u d y t h e e f f e c t s o f a h e a t t r e a t m e n t , 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 t e m p e r a t u r e and, a f t e r a h o l d i n g t i m e a t t h e r e q u i r e d t e m p e r a t u r e , 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 . E r d t s i c k and Beumer (1976) d e v e l o p e d a new 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 o f 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. They found t h a t t h e come-up 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 t h e r m a l d e a t h t i m e t u b e . Thus, t h e t h e r m a l d e a t h t i m e pouch was used i n t h e i n i t i a l k i n e t i c s t u d i e s r e p o r t e d h e r e . METHODS P r e p a r a t i o n o f N 5CH FH S o l u t i o n s S o l u t i o n s o f N CH^FH^ were made up i n 0.1 M phosphate b u f f e r pH 7.3. The a p p r o x i m a t e s t a r t i n g c o n c e n t r a t i o n was 35 ug/ml f o r a l l k i n e t i c s t u d i e s . 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 s t a n d a r d s o l u t i o n s . K i n e t i c S t u d i e s U s i n g Thermal Death Time Pouches Pouches were 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 L e c i t h i n Company, A t l a n t a , USA) a m a t e r i a l n o r m a l l y used f o r making meat and ag a r sausages. 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 pouches (4 cm x 6 cm between s e a l s ) were made. Each e n v e l o p e c o n t a i n e d 1.5 ml o f N^CH^FH^ s o l u t i o n . A f t e r f i l l i n g , t h e e n v e l o p e s were s e a l e d and used f o r t h e - 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 . The pouches were s t r e t c h e d f l a t i n a v e r t i c a l p o s i t i o n by w e i g h t s a t t a c h e d t o t h e l o w e r s e a l ( 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 h e pouch t h i c k n e s s was found t o be l e s s t h a n 1 mm. 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 pouches w e r e d e t e r m i n e d p r i o r t o n u t r i e n t d e s t r u c t i o n s t u d i e s (Appendix C ) . Pouches c o n t a i n i n g N^CH^FH^ s o l u t i o n were h e a t e d t o 100°C i n a w a t e r b a t h . 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 . 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 A g l a s s f l a s k was p l a c e d i n an a g i t a t e d w a t e r b a t h . A condenser was 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 an arm ( F i g . 9 ) . Samples o f N^CH^FH^ i n 0.1 M phosphate b u f f e r (pH 7.3) were p l a c e d i n t h e f l a s k and oxygen was b u b b l e d c o n s t a n t l y t h r o u g h t h e sample w h i l e i t was 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 . A l l a p p a r a t u s were k e p t i n d i f f u s e l i g h t t o m i n i m i z e N^CH^FH^ d e g r a d a t i o n due t o u l t r a v i o l e t r a d i a t i o n . D u p l i c a t e 100 ;ul samples were removed f r o m t h e f l a s k , a t r e g u l a r i n t e r v a l s and a s s a y e d by t h e r a d i o a s s a y method. N 5 C H 3 F H 4 samples were h e a t e d t o 100°, 90°, 75°, 60°, 55° and 40°C. P r e l i m i n a r y s t u d i e s s i m i l a r t o t h o s e o u t l i n e d i n App e n d i x 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 e x p e r i m e n t . - 45 -Figure 8. Thermal death time pouch w i t h weight attached. - 46 -condenser oxygen i water bath sample Figure 9. Apparatus used f o r k i n e t i c studies of the n u t r i t i o n a l degradation of 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 A s s a y System  Samples o f N^CH^FH^ were t r e a t e d i n an i d e n t i c a l manner t o t h a t d e s c r i b e d i n t h e p r e v i o u s e x p e r i m e n t . I n t h e a s s a y system, however, no m e r c a p t o e t h a n o l was used i n t h e b u f f e r , t h e m i l k b i n d i n g p r o t e i n o r t h e t r i t i a t e d P t e G l u . A l l r e a g e n t s were k e p t on i c e and i n d i f f u s e l i g h t t h r o u g h o u t t h e e x p e r i m e n t t o m i n i m i z e d e g r a d a t i o n . N^CH^FH^ samples were h e a t e d t o 90, 75, and 40°C i n t h i s e x p e r i m e n t . K i n e t i c S t u d i e s U s i n g L i m i t e d Oxygen S u p p l y S i n c e a d i s s o l v e d - o x y g e n meter (Model 57 Y e l l o w S p r i n g s I n s t r u m e n t Co., Ohio) was used f o r m e a s u r i n g changes i n oxygen c o n c e n t r a t i o n , N^CH^FH^ samples were d i s s o l v e d i n t r i s / N a C l b u f f e r (pH 7.3) f o r t h i s e x p e r i m e n t . 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 g i v e i n i t i a l oxygen c o n c e n t r a t i o n s o f 6.3, 9.6 and 14.4 ppm by volume. N^CH^FH^ was t h e n added t o each b u f f e r and t h e oxygen 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 hypodermic s y r i n g e s w h i c h 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 w a t e r b a t h a t 20°C. N^CH^FH^ and oxygen c o n c e n t r a t i o n s were measured a t i n t e r v a l s . The r a d i o a s s a y 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 was used 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 . A sample o f N^CH„FH. i n t r i s / N a C l 3 4 J 4 b u f f e r a t 20° C w i t h an u n l i m i t e d s u p p l y o f oxygen was a l s o a s s a y e d o v e r t h e same t i m e p e r i o d . - 48 -THEORETICAL CONSIDERATIONS F i r s t O r d e r R e a c t i o n s E x p e r i m e n t a l r e s u l t s i n d i c a t e d t h a t t h e d e g r a d a t i o n o f N^CH^FH^ ( 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 ) f o l l o w s a f i r s t o r d e r mechanism. The r e a c t i o n scheme a t a c o n s t a n t t e m p e r a t u r e and p r e s s u r e may t h u s be w r i t t e n : -> P r o d u c t (1) (N CH 3FH 4) ( d e g r a d a t i o n p r o d u c t ) where k i s t h e r a t e c o n s t a n t . Thus t h e r a t e o f r e a c t i o n may be e x p r e s s e d as: - d = .k' d t £ A J ng ml min (2) w h i c h may be i n t e g r a t e d and r e a r r a n g e d t o I n L A J o . -1 m m (3) where | A e q u a l s t h e c o n c e n t r a t i o n a f t e r t m i n u t e s i s t h e i n i t i a l c o n c e n t r a t i o n o f N CH„FH. and o 3 4 Al - 49 -However, should oxygen be involved i n the degradation of N^CH„FH, then the reaction must be considered to be pseudo f i r s t order. 3 4 J  This i s the case since the concentration of oxygen i s very much greater than that of N^CH^FH^ and therefore the change i n i t s concentration i s n e g l i g i b l e compared with the change i n concentration of the other reactant present. Thus, the o v e r a l l order i s reduced. This reaction scheme i s written: k A + B » Product (4) (N5CH FH ) (0 ) (degradation product) where k i s the rate constant. The rate of reaction may be expressed as [product] d L Pro ,dt ng ml mxn (5) If the oxygen concentration i s e s s e n t i a l l y unchanged throughout the reaction compared to the concentration of N^CH^FH^, then i t i s regarded as a constant and the rate expression i s written d I ProductJ dt = k' -.-1 . -1 ng ml mm (6) where k' = k m m -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 o r d e r k i n e t i c s a l t h o u g h i t i n v o l v e s 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 t o a c c o r d i n g l y as a pseudo f i r s t o r d e r r e a c t i o n . The A r r h e n i u s E q u a t i o n A r r h e n i u s (1889) f o u n d t h a t t h e e x p e r i m e n t a l l y o b s e r v e d v a r i a t i o n o f t h e r a t e c o n s t a n t (k) w i t h a b s o l u t e t e m p e r a t u r e (T) can be e x p r e s s e d : k = A e " E / R T (8) 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 f o r 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 . B o t h A and E a r e d e t e r m i n e d 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 . Once A and E a r e known f o r a r e a c t i o n , i t s r a t e may be c a l c u l a t e d f o r any t e m p e r a t u r e f r o m t h e above e q u a t i o n . Second Order R e a c t i o n s E x p e r i m e n t a l r e s u l t s i n d i c a t e d t h a t t h e d e g r a d a t i o n o f N^CH^FH^ ( i n t h e p r e s e n c e o f a l i m i t e d oxygen s u p p l y ) c o u l d f o l l o w a second o r d e r mechanism. I n t h i s c a s e , t h e r e a c t i o n scheme i s as f o l l o w s : ,.k A + B » P r o d u c t (9) (N^CH^FH^) (oxygen) ( d e g r a d a t i o n p r o d u c t ) where k i s t h e r a t e c o n s t a n t . - 51 -I f t h e d i s s o l v e d oxygen, B , undergoes a second o r d e r r e a c t i o n o f f i n i t e speed 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 o f r e a c t i o n can be e x p r e s s e d a s : -d A d t = k B J ng ml ^ min ^ (10) L e t | V J and £BJ ^ be t h e i n i t i a l 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 £AJ t and^BJ ^ 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 be t h e amount o f A o r B t h a t has r e a c t e d a t t i m e ' t ' . Then a t any t i m e t A . J o [ X (ID '] t - H o - M (12) T h e r e f o r e , -44 d t d t (13) S u b s t i t u t i n g i n t o t h e r a t e e q u a t i o n g i v e s -d [ A ] T d [x] d t d t k(W o - M)(H O - H] < 1 4 ) I n t e g r a t i o n from t = 0 g i v e s 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 should be noted that throughout these experiments, an 3 increase i n the b i n d i n g of H PteGlu to the m i l k p r o t e i n i n the radioassay system i s r e f e r r e d to as a decrease i n the c o n c e n t r a t i o n of N^CH^FH^ as determined from the radioassay standard curve. Thus, the assumption i s made th a t the degradation product of N^CH^FH^ does not bind to the m i l k p r o t e i n . K i n e t i c Studies Using Thermal Death Time Pouches 5 o The degradation curve f o r N CH^FH^ at 100 C i n phosphate b u f f e r (pH 7.3) i s shown i n F i g . 10 as a semilog p l o t of N^CH^FH^ r e t a i n e d versus heating time at constant temperature. Because i t was d i f f i c u l t to e l i m i n a t e a l l a i r from the pouches during s e a l i n g , and because oxygen i s able to d i f f u s e s l o w l y through nylon i t was found to be impossible to ac c u r a t e l y monitor the amount of oxygen present i n the system throughout - 53 -- 54 -t h e h e a t i n g p e r i o d . S i n c e oxygen i s t h o u g h t t o be i m p o r t a n t i n t h e 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 pouches were 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 degrades 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 S u p p l y 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 5 C H 3 F H 4 a t 100, 90, 75, 60, 55 and 40°C i n phosphate b u f f e r (pH 7.3) (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 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 be seen i n F i g u r e 11. Two s t a g e s t o t h e r e a c t i o n a r e a p p a r e n t . The f i r s t p a r t of t h e r e a c t i o n o c c u r s a l o n g t h e h o r i z o n t a l p o r t i o n o f t h e c u r v e (shown as X-Y f o r 40°C) where 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 (N^CH^FH c o n c e n t r a t i o n ) i s o c c u r r i n g . The second p a r t o f t h e r e a c t i o n o c c u r s a l o n g t h e s l o p e p o r t i o n of t h e c u r v e (shown as Y-Z f o r 40°C) where 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 as shown by a d e c r e a s e i n f o l a t e - c o n c e n t r a t i o n . The H o r i z o n t a l X-Y The l e n g t h o f t i m e f r o m X t o Y was found t o i n c r e a s e as t h e t e m p e r a t u r e o f r e a c t i o n d e c r e a s e d . A p l o t o f t e m p e r a t u r e o f r e a c t i o n 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 and i n d i c a t e s t h a t t h e s e i n t e r v a l s a r e t e m p e r a t u r e dependent. - 55 -Figure 11. Degradation of N CH^FH^ heated to v a r i o u s temperatures i n an u n l i m i t e d oxygen supply. - 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 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 N^CH^FH^, t h e d a t a used 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 o r d e r k i n e t i c e q u a t i o n f o r each t e m p e r a t u r e . 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 i n F i g . 1 3 . 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 ( T a b l e V 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 k i n e t i c s . As 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 as 'pseudo f i r s t o r d e r ' s i n c e oxygen was p r e s e n t i n an abundant s u p p l y t h r o u g h o u t t h e r e a c t i o n and t h u s i t s change 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 compared t o t h e change i n 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 p r e s e n t . 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 f o r t h i s e x p e r i m e n t a r e r e p o r t e d i n T a b l e V I . A p l o t o f t h e r a t e c o n s t a n t s (k') a g a i n s t t e m p e r a t u r e o f r e a c t i o n i s shown i n F i g u r e 14. S i n c e t h e r a t e c o n s t a n t was found t o i n c r e a s e as t h e t e m p e r a t u r e o f t h e r e a c t i o n was r a i s e d , an A r r h e n i u s - t y p e p l o t was c o n s t r u c t e d ( F i g u r e 15). The s t r a i g h t l i n e o b t a i n e d d e m o n s t r a t e s t h e v a l i d i t y o f t h e A r r h e n i u s e q u a t i o n i n t h i s r e a c t i o n . From t h e s e d a t a , A ( t h e f r e q u e n c y f a c t o r ) was found t o be 1440 min w h i l e E ( t h e a c t i v a t i o n energy) was found t o be 7.10 k c a l mole \ Thus t h e complete r a t e e x p r e s s i o n may be w r i t t e n . -7100 RT k' = 1440 e m i n " 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 o r d e r r e a c t i o n . - 58 -3-5 0 25 50 75 100 125 150 TIME (minutes) Figure 13. Regression analyses performed on data presented i n Figure 11 according to pseudo f i r s t order r e a c t i o n equation (6). T a b l e V I . 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 ' j , o f N CH^FH^ d e g r a d a t i o n i n an a b u n d a n t . s u p p l y o f oxygen. Rate C o e f f i c i e n t I n t e r c e p t C o n s t a n t o f d e t e r m i n a t i o n (min 1 ] f r 2 ] 100 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 Temperature o f R e a c t i o n - 60 -- 61 -Figure 15. P l o t of data i n Figure 14 according to Arrhenius equation (8). - 62 -K i n e t i c Studies Using U n l i m i t e d Oxygen Supply and a Mercaptoethanol - Free Assay System  The r a t e of degradation curves f o r N~*CH„FH, at AO, 75 and 90°C 3 4 i n phosphate b u f f e r (pH 7.3) measured i n a mercaptoethanol - f r e e assay are shown as a semilog p l o t of N^CH^FH^ r e t a i n e d versus heating time at constant temperature i n Figure 16. The degradation o f N^CH^FH^ i s observed to occur immediately when samples are heated to 90° and 75°C i n the presence of an u n l i m i t e d oxygen supply. Degradation occurs a f t e r 90 minutes i n the sample heated to 40°C. A pseudo f i r s t order . k i n e t i c equation was a p p l i e d t o a l l the data of the samples heated to 75° and 90°C and to data recorded a f t e r 90 minutes i n the sample heated to 40°C. L i n e a r r e g r e s s i o n analyses of the data are shown i n Figure 17. The high c o e f f i c i e n t s of determination obtained (Table VII) i n d i c a t e that the r e a c t i o n f o l l o w s pseudo f i r s t order r e a c t i o n . k i n e t i c s . The c a l c u l a t e d pseudo f i r s t order r a t e constants (k') are reported i n Table V I I and are observed to be very s i m i l a r to the values obtained when the N^CH^FH^ was assayed i n a system c o n t a i n i n g mercaptoethanol. K i n e t i c Studies Using a L i m i t e d Oxygen Supply The r a t e of degradation curves, f o r N^CH^FH^ at 20°C i n phosphate b u f f e r (pH 7.3) and i n the presence of a l i m i t e d oxygen supply are shown i n F i g ure 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 which d i s s o l v e d oxygen was maintained at 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 90 TIME (minutes) 180 270 F i g u r e 16. Degradation of N CH^FH^ heated to v a r i o u s temperatures i n an u n l i m i t e d oxygen supply and assayed using a mercaptoethanol-free system. - 64 -v 4 0 ° • 7 5 ° o 9 0 ° 0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0 T I M E (minutes) Figure 17. Regression analyses performed on data presented i n Figure 16 according to pseudo f i r s t order r e a c t i o n equation (6) s. T a b l e V I I . 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') of 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 a s s a y system. Temperature o f R e a c t i o n lo. I n t e r c e p t R a t e C o e f f i c i e n t C o n s t a n t . -1 m m o f D e t e r m i n a t i o n 21 40 0.060 0.018 0.96 75 0.062 0.057 0.94 S i 90 -0.012 0.077 0.96 - 66 -o-oo -115 -2 -30 C o n c e n t r a t i o n • U n l i m i t e d • 14-4 ppm 0 9 - 6 p p m A 6 -3 ppm - 3 - 4 5 0 6 12 T IME (hours) 18 24 Figure 18. Influence of d i s s o l v e d oxygen content on N^CH^FH^ degradation at 20°C. - 67 -As 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^ i s e s s e n t i a l l y c o n s t a n t 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 f i r s t s i x t o e i g h t h o u r s o f t h e e x p e r i m e n t ( 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 r a d i o a s s a y s y s t e m ) . 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 e x p e r i m e n t , however, i n d i c a t e s t h a t oxygen c o n c e n t r a t i o n 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 6 t o 8 h o u r s t h a n d u r i n g t h e r e m a i n d e r o f t h e e x p e r i m e n t ( F i g u r e 1 9 ) . T h i s i s i n d i c a t i v e o f an o x i d a t i o n r e a c t i o n t a k i n g p l a c e 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 b u t t h e r a t e o f o x i d a t i o n d e c r e a s e s a f t e r t h e f i r s t 6 t o 8 h o u r s . The d e g r a d a t i o n o f N^CH^FH^ appears t o depend on t h e c o n c e n t r a t i o n o f d i s s o l v e d oxygen. T h i s i s o b s e r v e d from F i g u r e 18 where 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 s f a s t e r f o r h i g h e r i n i t i a l d i s s o l v e d oxygen c o n c e n t r a t i o n . I t i s a l s o a p p a r e n t t h a t i n t h e p r e s e n c e o f a l i m i t e d oxygen s u p p l y 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 on s e m i l o g c o o r d i n a t e s . 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 second o r d e r k i n e t i c s , t h e d a t a f r o m t h e s l o p i n g p o r t i o n o f e a c h c u r v e were p l o t t e d a c c o r d i n g t o a second o r d e r k i n e t i c e q u a t i o n ( F i g u r e 2 0 ) . The u n i t s o f v i t a m i n c o n c e n t r a t i o n (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 (B) were m g / l i t e r and p a r t s p e r m i l l i o n , r e s p e c t i v e l y . S i n c e oxygen was p r e s e n t i n a g r e a t e r c o n c e n t r a t i o n t h a n t h e v i t a m i n , t h e i n t e g r a t e d second o r d e r r a t e e q u a t i o n was r e v e r s e d t o a v o i d t h e use o f n e g a t i v e v a l u e s . P l o t s o f t h e i n t e g r a t e d second o r d e r r a t e e q u a t i o n f o r samples c o n t a i n i n g i n i t i a l d i s s o l v e d oxygen c o n c e n t r a t i o n s o f 14.4 and 9.6 p a r t s p e r m i l l i o n a r e shown i n F i g u r e 20. The c a l c u l a t e d second o r d e r r a t e c o n s t a n t s f o r t h e two i n i t i a l 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 s i m i l a r as i l l u s t r a t e d i n T a b l e V I I I . - 68 -0 6 12 18 24 T I M E (hours) F i g u r e 19. Change i n oxygen 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 . - 69 -0 4 8 12 16 T I M E (hours) Figure 20. Regression analyses performed on par t of the data presented i n F i g ure 18 according to second order r e a c t i o n equation (16). T a b l e V I I I . Second 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 oxygen a t 20°C. I n i t i a l d i s s o l v e d cone. ppm1 I n t e r c e p t Rate C o n s t a n t |mg 1 _ h r C o e f f i c i e n t of d e t e r m i n a t i o n 2\ 9.6 14.4 0.028 -0.011 0.036 0.038 0.92 0.94 The r a t e c o n s t a n t s a r e based on d u p l i c a t e t r i a l s . - 71 -DISCUSSION F o l i c a c i d has been shown by many w o r k e r s t o be v e r y s u s c e p t i b l e t o d e g r a d a t i o n by o x i d a t i v e h e a t i n g . ( C h e l d i n e t a l . , 1943; S c h w e i g e r t eit a l . , 1946; S t o k s t a d e t a l . , 1947; Hanning and M i t t s , 1949; G h i t i s , 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 , 1971; M a l i n , 1977). Methods o f measurement o f f o l i c a c i d d e g r a d a t i o n i n a l l t h e s e e x p e r i m e n t s 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 bas e d on 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 t h e growth o f t h e t e s t m i c r o o r g a n i s m s . Throughout 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. I n i t i a l e x p e r i m e n t s u t i l i s i n g t h e t h e r m a l d e a t h t i m e pouches showed t h a t , when N^CH^FH^ i n a pho s p h a t e b u f f e r was 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 , a change 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 t h a t i t no l o n g e r had t h e a b i l i t y t o 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 . S i n c e oxygen was th o u g h t 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 oxygen was c o n s t a n t l y b u b b l e d t h r o u g h t h e sample. D e g r a d a t i o n c u r v e s i n d i c a t e d an i n i t i a l p e r i o d o f t i m e , ( w h i c h i n c r e a s e d as t h e t e m p e r a t u r e o f h e a t i n g d e c r e a s e d ) , when t h e N^CH^FH^ c o n c e n t r a t i o n , as measured by t h e r a d i o a s s a y method, d i d n o t appear t o change. T h i s was f o l l o w e d by a r a p i d 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 . T h i s r e a c t i o n appeared t o f o l l o w pseudo f i r s t o r d e r k i n e t i c s and t h e pseudo f i r s t o r d e r r a t e c o n s t a n t s were shown t o d e c r e a s e as t h e t e m p e r a t u r e o f r e a c t i o n d e c r e a s e d . - 72 -C o n f o r m i t y w i t h t h e A r r h e n i u s e q u a t i o n 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 energy f o r t h e t h e r m a l breakdown o f N^CH^FH^ i n 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 s u p p l y . Gupta and Huennekens (1967) have shown t h a t o x i d a t i o n o f N^CH^FH^ y i e l d s a d i h y d r o 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 . T h i s compound was shown by t h e a u t h o r s t o be r e d u c e d r e a d i l y back t o N^CH^FH^ upon 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 o f t h e r a d i o a s s a y method, i t was s p e c u l a t e d t h a t t h i s may be i n f l u e n c i n g t h e f i r s t p a r t o f t h e d e g r a d a t i o n r e a c t i o n where no a p p r e c i a b l e change i n N^CH^FH^ c o n c e n t r a t i o n was t a k i n g p l a c e . 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 e l i m i n a t e d f r o m t h e a s s a y s y s t e m and t h e d e g r a d a t i o n o f N^CH^FH^ was examined as i n t h e p r e v i o u s e x p e r i m e n t . The 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 d e c r e a s e t h e p e r i o d 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 change i n N^CH^FH^ c o n c e n t r a t i o n was o b s e r v e d . However, samples h e a t e d t o 40° C s t i l l e x h i b i t e d an i n i t i a l t i m e p e r i o d b e f o r e N^CH^FH^ c o n c e n t r a t i o n changes were d e t e c t e d . The absence 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 t h e r a t e c o n s t a n t s f o r t h e d e g r a d a t i o n r e a c t i o n . When t h e d e g r a d a t i o n o f N^CH^FH^ was o b s e r v e d i n a 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 appeared t o f o l l o w second o r d e r r e a c t i o n k i n e t i c s . The r a t e c o n s t a n t s 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 element o f c a u t i o n , however. Oxygen c o n c e n t r a t i o n s , a l t h o u g h l i m i t e d , were s t i l l i n r e l a t i v e l y l a r g e amounts compared t o t h e N^CH^FH^ c o n c e n t r a t i o n s . Thus, t h e r e a c t i o n c o u l d s t i l l be r e g a r d e d as pseudo f i r s t o r d e r a l t h o u 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 , f o r t h e r e g r e s s i o n l i n e s - 73 -o b t a i n e d when 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 t h e d a t a were n o t a s h i g h a s when a second 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 . G r e a t e r i n i t i a l 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 i n o r d e r t o a c c u r a t e l y a s c e r t a i n t h e r a t e c o n s t a n t s f o r t h i s r e a c t i o n . I t does a p p e a r , however, t h a t t h e p r e s e n c e 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 d e c r e a s e 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 s t a g e s o f t h e r e a c t i o n i n d i c a t e s t h a t o x i d a t i o n i s t a k i n g p l a c e a l t h o u g h t h i s i s n o t d e t e c t e d by a change i n N^CH^FH^ c o n c e n t r a t i o n s . Thus, i t a p p e a r s t h a t t h e m e r c a p t o e t h a n o l p r e s e n t i n t h e a s s a y s y s t e m i s r e d u c i n g 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^ d u r i n g 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 . 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 o f an u n l i m i t e d oxygen 3 4 s u p p l y can be d e s c r i b e d by a pseudo f i r s t o r d e r r e a c t i o n . 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 c r e a s e as t h e t e m p e r a t u r e o f r e a c t i o n i n c r e a s e s and 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 e q u a t i o n . I n t h e p r e s e n c e o f a l i m i t e d oxygen s u p p l y t h e o v e r a l l r e a c t i o n appears t o be second o r d e r i n n a t u r e . Oxygen c o n c e n t r a t i o n s d e c r e a s e t h r o u g h o u t 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 i s p r e s e n t i n t h e a s s a y system, 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 o b s e r v e d immediately.When m e r c a p t o e t h a n o l i s e l i m i n a t e d from t h e a s s a y , N^CH^FH^ c o n c e n t r a t i o n s were o b s e r v e d t o d e c r e a s e e a r l i e r . No change i n t h e d e g r a d a t i o n r a t e c o n s t a n t f o r each t e m p e r a t u r e was d e t e c t e d . - 74 -The degradation of J^CH^FH^ i s th e r e f o r e thought to be an o x i d a t i o n r e a c t i o n probably to the N"* methyl d i h y d r o f o l a t e . This compound does not bind to the f o l a t e b i n d i n g p r o t e i n i n the radioassay method. The o x i d a t i o n product i s r e a d i l y reduced back to N^CH^FH^ i n the presence of mercaptoethanol. Care must be taken, t h e r e f o r e , to exclude the presence of mercaptoethanol i n the assay system when attempting to measure the r a t e of breakdown of N^CH^FH^ s i n c e , i n the i n i t i a l stages of the r e a c t i o n , no degradation w i l l be observed i f i t i s used. The presence of other reducing agents i n a food system may have a s i m i l a r e f f e c t on the r a t e of degradation of N 5CH^FH A. - 75 -CHAPTER IV. IDENTIFICATION OF THE DEGRADATION PRODUCT AND THE EFFECT OF ASCORBIC ACID ON RATE OF DEGRADATION OF N 5 METHYLTETRAHYDROFOLIC ACID INTRODUCTION N^CH^FH^ has been shown t o be s u s c e p t i b l e t o d e g r a d a t i o n by h e a t i n t h e p r e s e n c e o f oxygen. There a r e s e v e r a l p u b l i c a t i o n s 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 p r o d u c t . O ' B r d i n e t a l . (1975) r e p o r t e d t h a t N^CH^FH^ was " n u t r i t i o n a l l y v e r y u n s t a b l e " 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 a u t h o r s s t a t e d however, t h a t t h e r e l a t i v e l y r a p i d n u t r i t i o n a l i n a c t i v a t i o n o f t h i s compound was u n l i k e l y t o be due t o c l e a v a g e o f t h e Cg - N^Q bond, s i n c e t h i s f o rm o f t h e v i t a m i n b e i n g 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 e x p e c t e d t o be r e s i s t a n t t o s u c h r e a c t i o n s . D o n a l d s o n and K e r e s z t e s y (1962) have 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 by 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 as 5 m e t h y l 5, 6, d i h y d r o f o l i c a c i d . 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 . I n t h i s p a r t o f t h e r e s e a r c h an a t t e m p t was made t o i d e n t i f y 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^ u s i n g u l t r a v i o l e t s p e c t r o s c o p y . D o n a l d s o n and K e r e s z t e s y (1962) showed t h a t v a r i o u s r e d u c i n g a g e n t s a r e r e q u i r e d f o r t h e c o n v e r s i o n o f N^CH^FH^ t o 5, 10 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 by 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 a c i d r e d u c t a s e . The a u t h o r s s p e c u l a t e d t h a t t h e r o l e o f t h e r e d u c i n g agent was t o p r e v e n t t h e d e g r a d a t i o n o f N^CH^FH^ t o t h e dehydro-compound. Ho m o c y s t e i n e , m e r c a p t o e t h a n o l and a s c o r b i c a c i d were a l l f ound t o be - 76 -e f f e c t i v e r e d u c i n g a g e n t s . 0 ' B r a i n e t a l . (1975) s t a t e d t h a t a s c o r b i c a c i d i n phosphate b u f f e r a f f o r d e d b e t t e r p r o t e c t i o n a g a i n s t o x i d a t i o n t h a n m e r c a p t o e t h a n o l a t l o w e r c o n c e n t r a t i o n s o f f o l a t e . The p r e v i o u s c h a p t e r i n d i c a t e d t h a t , m e r c a p t o e t h a n o l was a b l e t o r e d u c e 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 oxygen back t o t h e N^CH^FH^ compound. I t was s u g g e s t e d t h a t t h e p r e s e n c e 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 system may have an e f f e c t on t h e r a t e o f o x i d a t i o n o f N^CH^FH^. A s c o r b i c a c i d i s a r e d u c i n g agent commonly found i n f o o d s . To o b s e r v e i t s e f f e c t on 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 added t o a f o o d , t h e f o o d was h e a t e d and t h e d e g r a d a t i o n o f N^CH^FH^ was measured u s i n g t h e r a d i o a s s a y method. METHODS U l t r a v i o l e t S p e c t r o s c o p y A sample o f N^CH^FH^ i n phosphate b u f f e r (pH 7.3 0.1M) was h e a t e d t o 70°C i n a w a t e r b a t h . Oxygen was b u b b l e d t h r o u g h 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 by b o t h u l t r a v i o l e t s p e c t r o s c o p y and t h e r a d i o a s s a y method e v e r y 15 m i n u t e s . No m e r c a p t o e t h a n o l was used i n t h e r a d i o a s s a y method. Samples 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 p r e v i o u s l y used were t h e n h e a t e d t o 100°C f o r 2 h o u r s . Oxygen was b u b b l e d t h r o u g h t h e sample t h r o u g h o u t t h e h e a t i n g p e r i o d . The r e s u l t i n g sample was t h e n a n a l y z e d by u l t r a v i o l e t s p e c t r o s c o p y . - 77 -The E f f e c t of A s c o r b i c A c i d on The Degradation o f N CH FH^ i n a Food A baby food was chosen f o r t h i s experiment s i n c e t h i s negated a blending process p r i o r to the assay. Care was a l s o taken to ensure t h a t the food chosen contained l i t t l e or no a s c o r b i c a c i d (according to manufacturer's n u t r i e n t content t a b l e s ) . Two lOOg samples of "bacon and egg b r e a k f a s t " baby food were placed i n f l a s k s . 15 mg of a s c o r b i c a c i d were added to one sample. The f l a s k s were then placed i n a b o i l i n g water bath and oxygen was bubbled through the samples. Both samples were c o n s t a n t l y s t i r r e d throughout a two-hour heating p e r i o d . Care was taken to ensure that very l i t t l e evaporation of the sample took p l a c e . Samples were removed every 15 minutes f o r the f i r s t hour of heating and every 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 bath samples were assayed f o r f r e e and t o t a l f o l a t e by the radioassay method described 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 bsorption 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 analyzed. No other absorption peaks were detected at the concentrations t e s t e d . The decrease i n absorption peak height w i t h l e n g t h of time of heating the N^CH^FH, sample i n phosphate b u f f e r i s shown i n Figure 21. A f t e r - 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 peak was no l o n g e r d e t e c t a b l e ( c a r e was t a k e n t o e n s u r e t h a t samples were n o t exposed t o u l t r a v i o l e t l i g h t f o r t o o l o n g a p e r i o d p r i o r t o o b t a i n i n g an u l t r a v i o l e t s c a n as i t was found t h a t t h e a b s o r p t i o n peak d i s a p p e a r e d v e r y r a p i d l y upon e x p o s u r e t o l i g h t a t t h i s w a v e l e n g t h ) . C o r r e s p o n d i n g r a d i o a s s a y r e s u l t s shown i n T a b l e IX i n d i c a t e 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 i m m e d i a t e l y . A f t e r h e a t i n g t h e sample f o r 45 m i n u t e s , a 54 p e r c e n t d e c r e a s e i n c o n c e n t r a t i o n was o b s e r v e d . 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 a s c o r b a t e p r e s e n t i n t h e sample p r o t e c t e d t h e N^CH^FH^ from o x i d a t i o n d u r i n g 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 . Samples 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 t h o s e used i n t h e p r e v i o u s 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 peaks a t 290 and 249 nm a f t e r h e a t i n g 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 an u n l i m i t e d oxygen s u p p l y . T h i s i s shown i n F i g u r e 22. The E f f e c t 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 o f N^CH^FH^ i n a Food A s t a n d a r d c u r v e c o n s t r u c t e d u s i n g N^CH^FH^ samples was used f o r c a l c u l a t i n g f o o d f o l a t e c o n c e n t r a t i o n s . 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 f o l a t e ( a s measured by t h e r a d i o a s s a y method) i n a f o o d system, 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 w i t h an u n l i m i t e d s u p p l y o f oxygen a r e shown i n F i g u r e 23. F r e e 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 two f o o d samples a r e shown. The f r e e f o l a t e c o n c e n t r a t i o n o f t h e f o o d sample c o n t a i n i n g no a s c o r b a t e i s shown t o d e c r e a s e d u r i n g t h e f i r s t 15 m i n u t e s o f h e a t i n g . A s i m i l a r d e c r e a s e i n t h e f r e e f o l a t e c o n c e n t r a t i o n o f t h e sample c o n t a i n i n g 15 mg a s c o r b i c a c i d p e r 100 ml o f f o o d does n o t o c c u r u n t i l a f t e r 30 m i n u t e s o f h e a t i n g . I t i s o f i n t e r e s t t o n o t e t h a t v e r y l i t t l e - 79 -F i g u r e 21. D e c r e a s e 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. T a b l e I X . E f f e c t o f h e a t i n g on N CH FR, s t a b i l i t y . Change i n C o n c e n t r a t i o n (%) 0 27.31 0 15 26.70 2 30 27.54 0 45 12.64 54 60 5.23 81 Time C o n c e n t r a t i o n o f o f H e a t i n g N 5 C H 3 F H 4 (mins) (ng/ml) - 82 -Figure 22. U l t r a v i o l e t absorption spectrum of N m e t h y l d i h y d r o f o l i c a c i d . ^- max = 290 nm (€ = 31.0 x 10 3 M _ 1 cm"1) - 84 -120 TIME (minutes) Figure 23. The influence of ascorbic a c i d on the degradation of free and t o t a l f o l a t e i n a food heated to 100°C i n the presence of an unlimited oxygen supply. - 85 -d i f f e r e n c e i s o b s e r v e d i n t h e t o t a l f o l a t e c o n t e n t o f t h e two samples. 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 f o l a t e 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 C o n s u l t a t i o n w i t h C l i n i c a l A s s a y s ( M a s s a c h u s e t t s ) c o n f i r m e d t h a t t h e peak a t 265 nm o b s e r v e d when N^CH^FH^ samples were s u b j e c t e d t o u l t r a v i o l e t s p e c t r o s c o p y was due 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 added t o t h e N^CH^FH^ p r i o r t o l y o p h i l i z a t i o n . The p r e s e n c e o f t h i s peak 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 u l t r a v i o l e t 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^ sample. D o n a l d s o n and K e r e s z t e s y (1962) and Gupta and Huennekens (1967) have i n d i c a t e d t h a t 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 1 0 3 M _ 1 c m " 1 ) . The p r e s e n c e o f a s c o r b i c a c i d i n t h e sample i s shown t o a f f e c t 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 t would appear t h a t a s c o r b i c a c i d 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 o x i d i z e d 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 a b s o r b u l t r a v i o l e t l i g h t . Hence, t h e a b s o r p t i o n peak a t 265 nm i s o b s e r v e d t o d e c r e a s e when t h e sample c o n t a i n i n g N^CH^FH^ i s h e a t e d . The p r e s e n c e o f a s c o r b i c a c i d i n t h e N^CH^FH^ samples e x p l a i n s why t h e sample h e a t e d t o 40°C and 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 s ystem ( 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 . T h i n l a y e r and column chromatography o f N^CH^FH^ samples h e a t e d f o r s h o r t p e r i o d s o f t i m e 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 t h u s - 86 -making t h e i d e n t i f i c a t i o n o f t h e d e g r a d a t i o n p r o d u c t i m p o s s i b l e . The u l t r a v i o l e t a b s o r p t i o n peaks a t 290 and 249 nm o b s e r v e d when h i g h e r c o n c e n t r a t i o n s o f sample were used c o u l d o n l y be d e t e c t e d when t h e a b s o r p t i o n peak f o r a s c o r b i c a c i d had d i s a p p e a r e d . These a b s o r p t i o n peaks c o n f i r m t h a t t h e p r o d u c t o f N^CH^FH^ o x i d a t i o n i s t h e dihydro-compound. S i n c e a s c o r b i c a c i d i s an e f f e c t i v e r e d u c i n g agent i n t h i s 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 r a t e s i n f o o d s i s of i n t e r e s t . G h i t i s (1966) and F o r d et: a l . (1968) have o b s e r v e d t h a t t h e p r e s e n c e o f a s c o r b a t e i n m i l k i s n e c e s s a r y t o s t a b i l i z e f o l a t e r e t e n t i o n d u r i n g h e a t i n g p r o c e s s e s . G h i t i s (1966) s t a t e d , however, t h a t t h e a s c o r b i c a c i d c o n t e n t n o r m a l l y 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 p r o t e c t N^CH^FH^ from d e g r a d a t i o n d u r i n g b o i l i n g . The a u t h o r s u g g e s t e d t h a t i f a s c o r b i c a c i d i s used t o e n r i c h p r o c e s s e d m i l k s t h e n i t s h o u l d be 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 from o x i d a t i o n d u r i n g h e a t i n g . 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 i n a s c o r b i c a c i d . L i v e r and g r e e n v e g e t a b l e s a r e examples o f s u c h f o o d s . However, o t h e r f o o d s s u c h as eggs, w h i c h a r e r e l a t i v e l y r i c h i n f o l a t e s , do n o t c o n t a i n a s c o r b i c a c i d . 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 d e g r a d a t i o n d u r i n g h e a t i n g . The t o t a l f o l a t e c o n t e n t o f t h e f o o d examined d i d n o t change d u r i n g two h o u r s o f h e a t i n g i n a b o i l i n g w a t e r b a t h . - 87 -CONCLUSIONS When N^CH^FH^ i s heated i n the presence of oxygen, the degradation product i s the d i h y d r o f o l a t e compound. This confirms the work of Donaldson and Keresztesy (1962) and Gupta and Hennekens (1966). The degradation of N^CH^FH^ by heating i n oxygen i s delayed by the presence of a s c o r b i c a c i d . The a s c o r b i c a c i d a c t s as a reducing agent, converting the dihydro - compound back to the tetrahydro - compound. The presence of 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 th e r e f o r e of great importance i f the food i s to be subjected to heating . Foods which are r i c h i n a s c o r b i c a c i d may be heated f o r longer periods than foods which c o n t a i n l i t t l e or no a s c o r b i c a c i d before e x h i b i t i n g a decrease i n f o l a t e contentJ If foods are to be enriched w i t h ascorbate, then l e v e l s should be s u f f i c i e n t to pr o t e c t the f o l a t e from o x i d a t i o n . Future s t u d i e s should be d i r e c t e d towards the determination of the r e s i s t a n c e of other f o l a t e compounds to food processing operations. The e f f e c t s of other food c o n s t i t u e n t s on the r a t e of f o l a t e degradation and the determination of reducing agent l e v e l s necessary to p r o t e c t f o l a t e s during heat processing should a l s o be s t u d i e d . 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Serum f o l a t e l e v e l s : Comparison of m i c r o b i o l o g i c assay and r a d i o i s o t o p e k i t methods. Am. J . C l i n . Path. 66:746. Waters, A.H. and D.L. M o l l i n . 1961. Studies on the f o l i c a c i d a c t i v i t y of human serum. J . C l i n . Path. 14:335. Waxman, S., C. Schreiber and V. Herbert. 1971. R a d i o i s o t o p i c assay f o r measurement of serum f o l a t e l e v e l s . Blood 38:219. Waxman, S. and C. Schreiber. 1973. Measurement of 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 JHPGA radioassay. Blood 42:281. - 94 -Zettn e r , A. and P.E. Duly. 1974. P r i n c i p l e s of competitive b i n d i n g assays ( s a t u r a t i o n analyses) I I Sequential s a t u r a t i o n . C l i n . Chem. 20:5. Zettn e r , A. and P.E. Duly. 1975. R e l a t i v e e f f i c a c y of se p a r a t i o n of 'fr e e ' and 'bound' (3' 5' - -%) pteroylglutamate by cha 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 P hosphate 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 as s u p p l i e d by 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 ph o s p h a t e b u f f e r ( N a ^ P C y N a H ^ O ^ O ) pH 7.3 i n t h e r a d i o a s s a y method. S t a n d a r d c u r v e s o b t a i n e d u s i n g t h i s method d i d 3 n o t d i f f e r s i g n i f i c a n t l y as shown by t h e v a l u e s o f p e r c e n t H P t e G l u bound f o r each s t a n d a r d used i n T a b l e A - l . 3 T a b l e 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. C o n c e n t r a t i o n o f S t a n d a r d (ng/100 u l ) 3 H P t e G l u bound (%) ( T r i s / N a C l ) 3 H P t e G l u bound (%) ( P h o s p h a t e ) * 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 C o r r e c t i o n L i q u i d s c i n t i l l a t i o n c o u n t i n g i s a method o f d e t e c t i n g r a d i a t i o n and i s used m a i n l y f o r l o w energy b e t a e m i t t e r s . The method i s b a s e d on t h e energy t r a n s f e r f r o m t h e i o n i z i n g p a r t i c l e 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 ( f l u o r ) w h i c h e m i t s some o f t h e e n e r g y as l i g h t . The l i g h t i s d e t e c t e d by t h e p h o t o m u l t i p l i e r tube w h i c h r e s p o n d s by p r o d u c i n g a c h a r g e p u l s e w h i c h when a m p l i f i e d , i s c o u n t e d . 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 be d e t e c t e d . However, i n l i q u i d s c i n t i l l a t i o n 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 r e d u c e t h e e f f i c i e n c y o f energy t r a n s f e r and 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 a t a r e d e t e c t e d . T h i s i s known as 'quenching'. A number o f methods have been used t o c o r r e c t 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 . I n 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 was u t i l i z e d . A s t a n d a r d c u r v e r e l a t i n g c o u n t i n g e f f i c i e n c y t o t h e r a t i o o f t h e n e t c o u n t r a t e s o f a sample i n two d i f f e r e n t c h a n n e l s i s c o n s t r u c t e d . 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 samples con-t a i n i n g known amounts o f r a d i o a c t i v i t y i n two a p p r o p r i a t e c h a n n e l s . Unknown samples o f t h e same r a d i o n u c l e i d e can t h e n be c o u n t e d i n t h e s e same two 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 e f f i c i e n c y c o r r e s p o n d i n g t o t h e o b s e r v e d c h a n n e l s r a t i o i s o b t a i n e d . Such i n f o r m a t i o n i s n e c e s s a r y i n c r o s s - c o m p a r i n g t h e c o u n t i n g r e s u l t s o f samples. A s e r i e s o f quenched samples were 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 T a b l e B - l . - 99 -T a b l e B - l . C o m p o s i t i o n of quenched samples. Sample No. B u f f e r ( u l ) S c i n t i v e r s e (ml) H P t e G 3 u ( u l ) C h l o r o f o r m ( i l l ) 1 2 3 4 5 6 500 12 50 0 100 200 300 400 500 A l l samples were c o u n t e d i n t h e N u c l e a r C h i c a g o Isocap, 300 L i q u i d s c i n t i l l a t i o n c o u n t e r u s i n g t h e t r i t i u m program^ The'A/B c h a n n e l s r a t i o f o r each sample was a u t o m a t i c a l l y c a l c u l a t e d by t h e c o u n t e r . The c o u n t i n g e f f i c i e n c y f o r each o f t h e samples was t h e n c a l c u l a t e d . C o u n t i n g e f f i c i e n c y v e r s u s c h a n n e l s r a t i o was t h e n p l o t t e d f o r a l l t h e samples. The p l o t so c o n s t r u c t e d i s shown i n F i g u r e , B l . From a l i n e a r r e g r e s s i o n p e r f o r m e d on t h i s p l o t i t was t h e n p o s s i b l e t o c a l c u l a t e t h e c o u n t i n g e f f i c i e n c y f o r any sample g i v e n i t s c h a n n e l s r a t i o . The 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 c o u l d t h e n be c a l c u l a t e d f o r each sample. The m a j o r i t y o f t h e samples c o u n t e d t h r o u g h o u t t h i s r e s e a r c h had c h a n n e l s r a t i o v a l u e s of .50 - .75. - 100 -Figure Bl. 0 Quench c o r r e c t i o n curve. - 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 Lag Times f o r Thermal Death Time Pouches 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) were used f o r m e a s u r i n g 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 d e a t h t i m e pouches. The t h e r m o c o u p l e s were i n c o r p o r a t e d i n t o t h e end s e a l . A D i g i t e c 128 d a t a l o g g e r was used f o r t e m p e r a t u r e r e c o r d i n g . Pouches ( w i t h t h e r m o c o u p l e s i n c o r p o r a t e d i n t o t h e end s e a l ) were f i l l e d w i t h 1.5 ml 0.1M p h o s p h a t e b u f f e r and, w h i l e e l i m i n a t i n g as much a i r a s p o s s i b l e , t h e ends were h e a t s e a l e d . Each pouch was 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 t e m p e r a t u r e was r e c o r d e d a t one second i n t e r v a l s u s i n g t h e m i l l i v o l t c h a n n e l o f t h e r e c o r d e r . When t h e i n t e r n a l pouch t e m p e r a t u r e r e a c h e d t h e t e m p e r a t u r e o f t h e b o i l i n g w a t e r b a t h , t h e pouch was removed and i m m e d i a t e l y p l a c e d i n a c o l d w a t e r b a t h . Once a g a i n , t h e i n t e r n a l pouch t e m p e r a t u r e was r e c o r d e d u n t i l i t r e a c h e d t h e t e m p e r a t u r e o f t h e w a t e r b a t h . Ten s e r i e s of 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 c o o l i n g . A f t e r c o n v e r s i o n o f m i l l v o l t r e a d i n g s t o °C i t was f o u n d t h a t 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 f o r pouches c o n t a i n i n g 1.5 m l 0.1M phosphate b u f f e r were o f t h e o r d e r o f 11 seconds + 2 s e c o n d s . L a g t i m e s of 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 under i n v e s t i g a t i o n . 

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