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Lipogenesis in pyridoxine deficient rats Song, Gil-Won 1973

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LIPOGENESIS IN PYRIDOXINE DEFICIENT RATS by GIL-WON SONG M.H.Ec, Seoul University, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Divis i o n of Human Nutrition School of Home Economics We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Gil-Won Song Department of Human Nutrition, The University of B r i t i s h Columbia Vancouver 8, Canada D a t e May.. 25, 1973 6 ABSTRACT The purpose of this study was to evaluate the effect of pyridoxine deficiency on lipogenesis in the rat. It i s important i n studies of this type to standardize not only the food intake but also the feeding pattern of the experimental and control animals. Pair-feeding of the control rats with the d e f i c i e n t ones imposes on the former animals a feeding pattern similar to meal-feeding. The l a t t e r pattern e l i c i t s several adaptive changes related to energy u t i l i z a t i o n by the rat. Therefore, an attempt was made to minimize the difference i n the feeding frequency between the deprived and control groups by meal-feeding of the former group. The data were compared with those obtained when only food intake was controlled. Male weanling Wistar rats were used i n the present studies. The deprived rats were allowed food either ad libitum (nibbling) or for 2 hours each day (meal-feeding). The appropriate controls were given a complete diet i n quantities i s o c a l o r i c with the consumption of the deprived groups. Decreased fat storage as well as feed e f f i c i e n c y i n pyridoxine deficient rats were obvious at that time, regardless of the mode of feeding employed. The f a t t y acid content of the epididymal adipose tissue was affected i n the same manner as body f a t . Pyridoxine deprivation also suppressed t o t a l body 14 f a t t y acid synthesis iri vivo from glucose-U- C, whether the animals were meal-fed or nibbling. However, the rates of f a t t y i i i acid synthesis in the epididymal adipose tissue of the meal-fed deprived rats tended to exceed those observed i n the control. The lipogenic capacity of l i v e r s l i c e s from fed nibbling deprived rats exceeded that of the controls, as evidenced by increased f a t t y acid l a b e l l i n g in the presence of glucose-U-"'"^C 14 or acetate-1- C. However, when the nibbling deprived rats were fasted and refed p r i o r to s a c r i f i c e , the incorporation of 14 acetate-1- C into f a t t y acids was lower than i n the controls. No differences in the l a b e l l i n g of l i v e r fatty acids and glyceride glycerol were observed when the meal-fed deprived rats were compared with t h e i r controls. However, pyridoxine deficiency i n meal-fed rats was associated with a decrease in the capacity of the l i v e r to oxidize glucose, as comparison with the controls revealed. Epididymal adipose tissue segments from nibbling deprived rats showed less incorporation of "^C from l a b e l l e d glucose into C0"2, fat t y acids and glyceride glycerol than those from the corresponding controls (expressed on the basis of the deoxyribo-nucleic acid content of the t i s s u e ) . In contrast, increased lipogenic potential of adipose tissue preparation from the deprived meal-fed rats i n the presence of i n s u l i n was observed. In these rats, a decrease in adipocyte size was suggested by lipid/DNA r a t i o lower than that of the controls. Thus, the increased lipogenic capacity observed was possibly due to a decrease in adipocyte siz e , i t s e l f associated with increased s e n s i t i v i t y to i n s u l i n . The a c t i v i t i e s of glucose-6-phosphate dehydrogenase and i i v m a l i c enzyme were depressed i n both the l i v e r and the adipose t i s s u e of the p y r i d o x i n e - d e p r i v e d r a t s . Since these enzymes are concerned with the p r o d u c t i o n of the NADPH needed f o r r e d u c t i v e f a t t y a c i d s y n t h e s i s , the r e s u l t s were c o n s i s t e n t with the i n v i v o f i n d i n g . However, the a c t i v i t i e s of these enzymes d i d not appear to l i m i t the i n v i t r o l i p o g e n i c p o t e n t i a l of the t i s s u e s i n v e s t i g a t e d , s i n c e NADPH produced from glucose-6-phosphate dehydrogenase and m a l i c enzyme alone seemed to be s u f f i c i e n t to support the rat e s of l i p o g e n e s i s seen. The a l t e r a t i o n s i n l i p o g e n e s i s i n p y r i d o x i n e d e p r i v a t i o n observed i n the present and other i n v e s t i g a t i o n could not be ex p l a i n e d on the b a s i s of the known f u n c t i o n s of the p y r i d o x i n e -dependent enzymes. ACKNOWLEDGEMENT I wish to thank my advisor, Dr. Joseph F. Angel for his supervision and support throughout this study. I also would l i k e to express my appreciation to Dr. Melvin Lee and Dr. Samuel W. French for t h e i r valuable discussions and suggestions. This work was supported by the National Research Council of Canada. v TABLE OF CONTENTS Page LIST OF TABLES v i i i LIST OF FIGURES x 1. Introduction 1 2. Review of the Literature_ 3 2.1 General Considerations of Pyridoxine 3 2.1.1 History and Discovery 3 2.1.2 Physiological Role of Pyridoxine 4 2.2 Energy U t i l i z a t i o n i n Pyridoxine Deficiency 6 2.2.1 Basal Metabolism -- 7 2.2.2 Body Temperature 7 2.2.3 Physical A c t i v i t y 8 2.2.4 Absorption of Nutrients -- 8 2.2.5 Fat Metabolism in Pyridoxine Deficiency -- 9 2.2.5.1 Body and Tissue Fat Levels - 9 2.2.5.2 Synthesis and Oxidation of Fat 12 2.2.6 Carbohydrate Metabolism i n Pyridoxine Deficiency -- - - 15 2.2.6.1 General Considerations 15 2.2.6.2 Tissue Glycogen 17 2.2.7 Importance of Considering the Mode of Feeding 18 3. Materials and Methods 21 3.1 Materials 21 3.2 Methods - - 21 v i 3.2.1 Handling of Animals 21 3.2.2 In Vivo Study of Glucose U t i l i z a t i o n 25 3.2.3 In V i t r o Studies 26 3.2.4 Enzyme Studies -- 27 3.2.5 Isotope Counting 28 3.2.6 S t a t i s t i c a l Analysis and Other Calculations 28 4. Experimental and Results 29 4.1 Experiment I : In Vivo Lipogenesis in Pyridoxine Deficiency 29 4.2 Experiment II : I_n Vi t r o Lipogenesis in Pyridoxine Deficiency 35 4.3 Experiment III : A c t i v i t y of NADPH-Producing Enzymes in Pyridoxine Deficiency 42 5. Discussion 49 6. Bibliography 60 APPENDIX A - 77 APPENDIX B -- 81 v i i LIST OF TABLES Number Page 1. Composition of the basal diet 22 2. Composition of the mineral mixture 23 3. Composition of the vitamin mixture 24 4. Cumulative food consumption and body weight gain of pyridoxine-deprived and control rats 30 5. Total food consumption, t o t a l weight gain, and o v e r a l l feed e f f i c i e n c y i n pyridoxine-deprived and control rats .-- 32% 6. Relative sizes of the l i v e r and the epididymal adipose tissue in pyridoxine-deprived and control rats -- 33 7/ Liver, adipose tissue and body fa t t y acid content in pyridoxine-deprived and control rats 34 8. In vivo incorporation of glucose-U-^C into f a t t y acids in pyridoxine-deprived and control rats 36 9. In v i t r o lipogenesis by l i v e r of pyridoxine-deprived and control rats 38 10. In v i t r o lipogenesis by the epididymal adipose tissue of pyridoxine-deprived and control rats -- 40 11. DNA and l i p i d content of the epididymal adipose tissue of pyridoxine-deprived and control rats -- 43 12. A c t i v i t y of glucose-6-phosphate dehydrogenase, 6-phosphogluconage dehydrogenase and malic enzyme in the l i v e r of pyridoxine-deprived and control rats- 45 v i i i A c t i v i t y of glucose-6-phosphate dehydrogenase, 6-phosphate dehydrogenase and malic enzyme in the epididymal adipose tissue of pyridoxine-deprived and control rats Cumulative food consumption and body weight gain in pyridoxine-deprived and control rats Total food consumption, t o t a l weight gain and ov e r a l l feed e f f i c i e n c y i n pyridoxine-deprived and control rats Relative sizes of l i v e r and the epididymal adipose tissue in pyridoxine-deprived and control rats Cumulative food consumption and body weight gain in pyridoxine-deprived and control rats --Total food consumption, t o t a l weight gain and overal l feed e f f i c i e n c y i n pyridoxine-deprived and control rats Relative sizes of l i v e r and the epididymal adipose tissue i n pyridoxine-deprived and control rats ix LIST OF FIGURES Number Page 1. Structural formulae of the various Vitamin B,-o compounds 5 x 1. I n t r o d u c t i o n It has been r e p e a t e d l y shown that the p y r i d o x i n e - d e p r i v e d r a t s t o r e s l e s s body f a t than i t s p a i r - f e d c o n t r o l . However, t h i s d i s c r e p a n c y i n energy storage c o u l d not be t r a c e d back to d i f f e r e n c e s between the two animals i n b a s a l metabolic r a t e , p h y s i c a l a c t i v i t y , a b s o r p t i o n and e x c r e t i o n of n u t r i e n t s . One of the problems encountered when e v a l u a t i n g energy storage i n d e f i c i e n c y s t a t e s i s the need to d i s t i n g u i s h the e f f e c t s of o m i t t i n g a c e r t a i n n u t r i e n t from the consequences of the i n a n i t i o n which accompanies n u t r i t i o n a l d e p r i v a t i o n . P a i r -f e e d i n g , which i s used to e q u a l i z e the food i n t a k e s of the c o n t r o l and the d e f i c i e n t groups, superimposes on the former animals an i n t e r m i t t e n t f e e d i n g p a t t e r n s i m i l a r to meal f e e d i n g . Meal f e e d i n g , the r e s t r i c t i o n of food a v a i l a b i l i t y to a short d a i l y p e r i o d , has been shown to e l i c i t s e v e r a l adaptations r e l a t e d to energy u t i l i z a t i o n i n the r a t . T h e r e f o r e , i t appeared important i n s t u d i e s of t h i s type to s t a n d a r d i z e not only the n u t r i e n t intake of the experimental and c o n t r o l animals, but a l s o t h e i r f e e d i n g p a t t e r n . The present study was c a r r i e d out to evaluate the e f f e c t of p y r i d o x i n e d e f i c i e n c y on l i p o g e n e s i s . An attempt was made to minimize the d i f f e r e n c e s i n f e e d i n g frequency between groups by employing meal-feeding f o r the d e p r i v e d r a t s . The f i n d i n g s were compared with those obtained when only food i n t a k e l e v e l s were c o n t r o l l e d . 1. 2. Data w i l l be presented to show that p y r i d o x i n e d e p r i v a t i o n suppresses o v e r a l l f a t t y a c i d s y n t h e s i s i n i n t a c t r a t s , r e g a r d l e s s of the manner i n which they are f e d . In., a d d i t i o n , i t w i l l be shown that the i m p o s i t i o n of meal-feeding on the d e f i c i e n t animals decreases the magnitude of the d i f f e r e n c e i n l i p o g e n e s i s observed upon comparison with i s o c a l o r i c a l l y - f e d c o n t r o l s . The a c t i v i t i e s of NADPH producing enzymes and the l i p o g e n i c p o t e n t i a l s of l i v e r and adipose t i s s u e p r e p a r a t i o n s w i l l be examined and d i s c u s s e d i n r e l a t i o n to the l i p o g e n i c r a t e s a t t a i n e d by the i n t a c t animals. 2. Review of the Literature 2.1 General Considerations of Pyridoxine"*" 2.1.1 History and Discovery In 1934, Gyorgy established the i d e n t i t y of vitamin as the component of vitamin B complex responsible for curing rat acrodynia. The vitamin was iso l a t e d i n pure form four years l a t e r (Gyorgy, 1938; Ichiba and Michi, 1938; Keresztes.y. and Stevens, 1938; Kuhn and Wendt, 1938; Lepkovsky, 1938). The elucidation of i t s structure (Kuhn e_t al_. , 1939; S t i l l e r e_t a l . , 1939), and i t s synthesis (Harris and Folkers, 1939 a-c) were achieved during the following year. The terms 'pyridoxine', proposed by Gyorgy and Eckhardt (1939), and 'vitamin Bg' then became synonymous u n t i l Snell e_t a l . (1942) demonstrated the existence of one or more substances which had a greater a c t i v i t y than pyridoxine i n promoting the growth of l a c t i c acid bacteria. Subsequently, these substances were i d e n t i f i e d as pyridoxal and pyridoxamine, and their structures were established by Harris et jQ. (1944 a,b). Pyridoxal phosphate was o r i g i n a l l y i d e n t i f i e d as an essential cofactor for the enzymatic decarboxylation of amino acids (Baddiley and Gale, 1945; Gunslaus et a l . , 1944). Other 1. D e f i n i t i o n of terms : The c o l l e c t i v e names are vitamin B or pyridoxine; vitamin Bg alcohol, pyridoxol; vitamin B^ aldehyde, pyridoxal; vitamin B^ amine, pyridoxamine. 3. phosphorylated pyridoxine derivatives, pyridoxamine-5'-phosphate and pyridoxine-5'-phosphate, were also discovered thereafter. The structures of the various vitamin Bg compounds are depicted i n Figure 1. 2.1.2 Physiological Role of Pyridoxine The role of pyridoxine i n the various metabolic events i s well documented (Reviews : Braunstein, 1960, 1964; Meister, 1955, 1962, 1965; Krebs and Fischer, 1964; Mueller, 1964; Sauberlich, 1967; Sherman, 1950; Snell, 1958, 1961). More than 50 pyridoxal phosphate-dependent.enzymes, almost a l l of which are concerned with amino acid metabolism, have been l i s t e d by Sauberlich (1967) Experimental evidence i s consistent with the concept that pyridoxal phosphate affects the s t a b i l i t y of pyridoxal phosphate-dependent enzymes and i s involved in the conformation and the structure of these enzymes. There is no clear evidence of a direct relationship between coenzyme a v a i l a b i l i t y and the rate of biosynthesis of pyridoxal phosphate enzymes at the present time (Chatagner, 1970). Nonetheless, i t may be stated that pyridoxal phosphate i s necessary for almost every stage of amino acid transformations i n l i v i n g organisms. Pyridoxine has been implicated i n the interconversions of polyunsaturated f a t t y acids. However, published data are rather inconsistent (Greenberg and Moon, 1961; Johnston et_ aJ. , 1961; Kirschman and Conigl.io, 1961; Soderjelm, 1962; Swell e_t a l . , 1961 Williams and Scheier, 1962; Witten and Holman, 1952; Reviews : Mueller, 1964; Sherman, 1950). In addition, there has been some evidence of a role for pyridoxal phosphate i n f a t t y acid chain C H 2 O H C H O HO H 3 C C H 2 O H N HO H 3 C C H 2 N H 2 C H 2 O H N HO H 3 C C H 2 O H N P y r i d o x o l P y r i d o x a l P y r i d o x a m i n e C H O HO H 3 C C H 2 N H 2 N C H 2 O . P 0 3 H 2 HO H 3 C C H 2 O . P 0 3 H 2 N P y r i d o x a l - 5 ' - p h o s p h a t e P y r i d o x a m i n e - 5 • - p h o s p h a t e F i g u r e 1. S t r u c t u r a l f o r m u l a e o f t h e v a r i o u s v i t a m i n B 6 compounds 6. elongation (Goswami and Coniglio, 1966; Wakil, 1961), suggesting the involvement of pyridoxine i n fat metabolism. In addition, pyridoxine appears to be related to s t e r o l metabolism, since there have been repeated observations that the rate of l a b e l l e d acetate incorporation into l i v e r cholesterol i s enhanced in pyridoxine d e f i c i e n t rats in v i t r o as well as i n vivo (Goswami and Sadhu, 1960; Hinse and Lupien, 1971; Lupien et_ al. , 1969; Maggi et a l . , 1959; Shah et a l . , 1960; Williams and P e r t e l , 1964). The exact mechanism responsible for increased cholesterogenesis under these conditions i s not clear at the present time. With regard to carbohydrate metabolism, pyridoxal phosphate plays a unique role as far as glycogen phosphorylase is concerned (Baranowski et_ al_. , 1957; Brown and C o r i , 1961; Cori and Illingworth, 1957; Reviews : Fischer and Krebs, 1966; Krebs and Fischer, 1962, 1964). Pyridoxal phosphate functions i n maintaining the tetrameric structure of this enzyme, but i t has not been shown that pyridoxal phosphate plays a c a t a l y t i c role in the glycogenolysis (Jones and Cowgill, 1971). 2.2 Energy U t i l i z a t i o n in Pyridoxine Deficiency Pyridoxine d e f i c i e n t rats have been repeatedly shown to store less energy as body fat than th e i r p air-fed controls (Desikachar and McHenry, 1954; Guggenheim and Diamant, 1957; Huber et a l . , 1964; McHenry and Gavin, 1941; Sure and Easterling, 1949). However, the metabolic alterations leading to t h i s phenomenon have not been c l a r i f i e d adequately. Energy storage under any condition represents the balance between intake and output. In general, energy i s expended to maintain basal metabolism, body temperature, and physical a c t i v i t y . At present, studies dealing with the energy output in pyridoxine d e f i c i e n t animal are limited. Attention was rather directed on the possible e f f e c t of pyridoxine on carbohydrate and fat metabolism. This role for pyridoxine has been often consider ed to be secondary i n nature. 2.2.1 Basal Metabolism Orsini et_ al_. (1942) demonstrated a d e f i n i t e decrease in basal metabolic rate (BMR) i n rats fed pyridoxine d e f i c i e n t rations when compared to ad libitum control animals. However, a p o s s i b i l i t y exists that this effect might have been a direct consequence of i n a n i t i o n , since food intake i n the control rats was not controlled. This p o s s i b i l i t y was tested by Beaton et a l . (1953). The BMR of vitamin B6 d e f i c i e n t rats was comparable with that of the pair-fed controls, but i t was lower than that of ad  libitum controls. Thus, i t appears that Orsini e_t al_. (1942) f a i l e d to d i f f e r e n t i a t e the s p e c i f i c e f f e c t of pyridoxine deficiency from the general effect of i n a n i t i o n . 2.2.2 Body Temperature Heat production for maintaining body temperature constitutes the main component of energy expenditure. Ershoff (1951) found that the survival rate of pyridoxine d e f i c i e n t rats during exposure to cold was less than that of the control animals This phenomenon was attributed to hormonal i n s u f f i c i e n c y , since hypophysectomy, adrenalectomy, and thyroidectomy invariably induce impaired adjustment to cold exposure. 8. Subsequently, Yeh and Weiss (1963) studied the thermo-regulatory behavior of pyridoxine d e f i c i e n t rat exposed to a temperature of 2-3°C. Body temperature of the experimental rats f e l l more rapidly than that of the controls. When trained to press a lever that switched on a heat lamp, the d e f i c i e n t rats did so more frequently than the controls. Thus, the authors suggested that pyridoxine deficiency was associated with either increased heat loss or impaired heat production. Increased loss of heat would possibly account for some of the d i s c r e p a n c y i n energy storage between the pyridoxine-deficient rat and i t s pair-fed control. 2.2.3 Physical A c t i v i t y Measurement of physical a c t i v i t y i n pyridoxine deficiency would y i e l d some insight on the energy expenditure. However, no reports are available. Whether i r r i t a b i l i t y as a consequence of the deficiency results in the increased loss of energy because of physical a c t i v i t y remains to be explored. 2.2.4 Absorption of Nutrients The excretion of faecal nitrogen i n pyridoxine d e f i c i e n t rats receiving controlled amounts of protein was not s i g n i f i c a n t -ly d i f f e r e n t from that of normal animals (Carter and Phizackerley, 1951; Beaton et a l . , 1953). H i s t o l o g i c a l and histochemical examination of the duodenum and upper jejunum from pyridoxine d e f i c i e n t animals showed no difference from control tissues (Asatoor et a l . , 1972). On the high-fat diet, the l i p i d content of faeces from pyridoxine-deficient rats was not d i f f e r e n t from the control 9. samples (Carter and Phizackerley, 1951). This implies that fat absorption i s not impaired as a resu l t of pyridoxine deficiency. These authors also showed that the i n v i t r o absorption of glucose by i n t e s t i n a l preparation (expressed per cm) was unaltered. Thus, i t appears unlike l y that decreased i n t e s t i n a l absorption of nutrients or increased loss of energy by excretion could account for reduced fat storage i n pyridoxine deficiency. 2.2.5 Fat Metabolism i n Pyridoxine Deficiency 2.2.5.1 Body and Tissue Fat Levels It has been repeatedly found that pyridoxine deficiency leads to decreased body fat le v e l s . As early as 1941, McHenry and Gavin observed that carcass fat deposition was diminished i n pyridoxine-deprived rats maintained on a fat free diet containing 96 % casein. These workers suggested that pyridoxine was required for fat synthesis from protein. Similar findings were subsequently reported by Sure and Easterling (1949). Carter and Phizackerley (1951) noted that the difference in body fat stores between the pyridoxine-deficient and the pair-fed control rats was abolished when the l e v e l of dietary fat was increased from 4 to 25 %. Thus, i t was suggested that pyridoxine i s required for the conversion of carbohydrates into fat when low levels of the l a t t e r are included in the diet. However, Beare et_ al_. (1953) observed that, unlike their pair-fed controls, deoxypyridoxol-treated rats f a i l e d to increase t h e i r body f a t t y acids when the l e v e l of dietary fat was increased from 5 to 20 %. Although the l a t t e r findings may be open to question because of the use of deoxypyridoxol, Williams e_t aJ. (1959) reported 10. observations consistent with those of Beare et a l . (1953) even when pyridoxine antagonists were not used. These authors concluded that no general statements could be made regarding the response of body fat to the inte r a c t i o n between pyridoxine and fat levels in the diet. One of the e a r l i e s t responses to n u t r i t i o n a l deficiency is i n a n i t i o n , and i t has been suggested that decreased food intake may account for the reduction of carcass fat i n pyridoxine deprived animals. Beaton e_t a^l. (1956) and Huber e_t aJ. (1964) employed i n s u l i n i n j e c t i o n as a means .of increasing food intake in pyridoxine deprived rats. In both studies, the treated rats exhibited body fat levels almost similar to those of the pair-fed controls. In the studies of the l a t t e r authors, adipose tissue preparations from the deficient untreated rats exhibited a greater s e n s i t i v i t y to i n s u l i n addition. These findings suggeste that impaired i n s u l i n production might account for the decrease i carcass fat in pyridoxine deficiency. This was supported by decreases in the i n s u l i n - l i k e a c t i v i t y of serum and pancreas (Hubef et^ a_l. , 1964) and by the observation of degenerative changes in the 3-cells in the i s l e t s of Langerhans in pyridoxine de f i c i e n t rat (Kuno, 1959). The causes of i n s u l i n i n s u f f i c i e n c y are not clear, but may be related to the abnormal tryptophan metabolism in pyridoxine deficiency. Kotake and Inada (1953) showed that xanthurenic acid may bind i n s u l i n , thereby leading to the exhaustion of the pancreatic 3-cells. The role of pyridoxine in fat metabolism i n r e l a t i o n to food intake was demonstrated i n the rat by Radhakrishnamurty 11. et a l . (1968). After 40 days of pyridoxine depletion, the rats were repleted by i n j e c t i o n with pyridoxol hydrochloride for 5 days. Food was offered either ad_ libitum or i n quantities equal to those eaten by the d e f i c i e n t group. It was found that the l e v e l of body fat increased only when food was provided ad libitum. This response was not related to fat intake, since the observations were similar when dietary fat levels were increased during repletion from 10 % to 25 % and when fat was withdrawn. The effects of pyridoxine deficiency on l i v e r fat levels are less consistent than the response of body f a t . Halliday (1938) observed fa t t y l i v e r in rats deprived of pyridoxine, but this effect was reversed by choline. Engel (1941, 1942) found no changes in l i v e r fat levels over a 6 week deprivation period, but prolonged deficiency for 8-24 weeks doubled the l i v e r fat content. Thus, the length of depletion seems to af f e c t the l e v e l of hepatic l i p i d . However, the results obtained were complicated by a coincident dietary deficiency of i n o s i t o l and b i o t i n , both of which are known to af f e c t l i v e r fat l e v e l . Beare et a l . (1953) also observed fa t t y l i v e r in deoxypyridoxol treated rats. The use of antimetabolite in this experiment does not allow any d e f i n i t e conclusion, since the symptoms induced by vitamin deprivation may d i f f e r from those induced by antagonists (Rosen et_ al_. , 1964). The amount of fat i n the l i v e r may be related to the l e v e l of fat i n the diet. Tulpule and Patwardhan (1950, 1952) showed that pyridoxine deficiency caused an increase in hepatic fat levels in rat fed a diet devoid of f a t . Pyridoxine 12. deficiency alone caused no s i g n i f i c a n t alterations either in the t o t a l l i p i d content or i n the ind i v i d u a l l i p i d fractions in the l i v e r . Furthermore, French (1966) and French and Castagna (1967) demonstrated that pyridoxine deficiency resulted in fat t y l i v e r i f ethanol was also ingested. However, Carter and Phizackerley (1951) could not detect f a t t y l i v e r i n pyridoxine deficiency with varying amount of fat i n the diet. In support of th i s finding, Fidanza and de Cicco (1964) and Pardini and Sauberlich (1964) reported that l i v e r l i p i d levels were not appreciably altered by pyridoxine deficiency.- In the monkey, f a t t y l i v e r with an enlarged and pale appearance was observed in pyridoxine deficiency (Rinehart and Greenberg, 1956; Wizgird e t _ a l . , 1963). The proportion of protein in the diet seems to influence the l e v e l of hepatic l i p i d . Okada and Ochi (1971) observed that l i v e r l i p i d content i n pyridoxine d e f i c i e n t rats was almost doubled when the casein l e v e l i n the diet was increased from 10 % to 70 %. Further analysis of l i v e r fat showed marked increases in glycerides, f a t t y esters, and cholesterol, and a decrease i n phospholipid l e v e l s . At the present time, no general statement on the hepatic l i p i d l e v e l in pyridoxine deficiency i s possible, since the experimental conditions varied considerably among published studies. In any case, l i v e r fat levels are hard to interpret, since they r e f l e c t the outcome of synthesis, oxidation and transport of l i p i d s from and to this tissue. 2.2.5.2 Synthesis and Oxidation of Fat It is reasonable to assume that decreased fat storage i n pyridoxine deficiency 13. r e f l e c t s either depressed fat synthesis or enhanced fat oxidation or both. The rate of synthesis and oxidation of fat i s largely determined by the n u t r i t i o n a l and metabolic state of the animal (Masoro, 1962). Desikachar and McHenry (1954) studied in vivo lipogenesis i n deoxypyridoxol-treated rats kept on a fat free pyridoxine free diet. Administration of glucose-U-^C by stomach tube after 6 weeks resulted i n similar rates of r a d i o a c t i v i t y incorporation into carcass fatty acids in the d e f i c i e n t , ad libitum control and pair-fed control rats. They suggested that rapid u t i l i z a t i o n of fat might be responsible for decreased energy storage i n pyridoxine deficiency. Huber et a l . (1964), using glucose-U-^^C, have shown that the lipogenic a b i l i t y of adipose tissue segments from pyridoxine-depleted rats was greater than that observed in tissues from the pair-fed controls, when i n s u l i n was present. The addition of i n s u l i n to the incubation medium increased net gas exchange i n both the def i c i e n t and the control groups, but the magnitude of the increase was greater i n the former animals. This suggested that i n s u l i n s e n s i t i v i t y of the adipose tissue was enhanced i n pyridoxine deficiency. The addition of pyridoxal phosphate to the medium had no s i g n i f i c a n t e ffect on the incorporation of r a d i o a c t i v i t y into f a t . Williams and Pertel (1964) compared i n v i t r o hepatic lipogenesis i n pyridoxine deficient rats with pair-fed and pair-weighed controls. Liver s l i c e s from the de f i c i e n t animals incorporated more acetate-2- 1 4C into t o t a l l i p i d s than did s l i c e s from the ad_ libitum controls, but less than the s l i c e s from the pair-fed or pair-weighed controls. The greater incorporation of Cx by the s l i c e s from the l a t t e r rats appeared to r e f l e c t the metabolic changes produced by the i r feeding pattern. Pardini and Sauberlich (1967) reported that acetate-l-l^C incorporation into f a t t y acids by adipose tissue, l i v e r , and kidney preparations from de f i c i e n t rats which have been fasted for 48 hours and refed for 48 hours was enhanced when compared with a control group which was fasted s i m i l a r l y and then pair-refed. The variance between the results of these two studies may be attributed mainly to the treatment imposed on the animals p r i o r to s a c r i f i c e . Also the use of acetate as a precursor i n studies of this type may be questioned because the penetration of free acetate into tissues, i t s a c t i v a t i o n to acetyl CoA, and the d i l u t i o n of the l a t t e r with endogenous unlabelled acetyl-CoA can profoundly influence the conversion of exogenous acetate into f a t t y acids (Favarger, 1965). If the view that lipogenesis i s enhanced in pyridoxine deficiency i s correct, the discrepancy between decreased energy storage and increased lipogenic capacity cannot be reconciled unless fat oxidation i s also enhanced. Carter and Phizackerley (1951) demonstrated that l i v e r s l i c e s from pyridoxine depleted animals had a reduced capacity to oxidize l a b e l l e d butanoate and octanoate as compared with tissues from pair-fed controls. Angel and Sabry (1970) demonstrated that diaphragm segments from meal-fed pyridoxine-depleted rats produced more -^CC^ from l a b e l l e d butanoate, octanoate and hexadecanoate than those obtained from c a l o r i c a l l y unrestricted meal-fed controls. Increased f a t t y acid turnover was suggested. Sabo and Gershoff (1971) studied the effect of pyridoxine deprivation for 3 weeks on the lipogenic and l i p o l y t i c a c t i v i t i e s of isolated adipocytes. When incubated with g l u c o s e - l - 1 4 C , c e l l s from the experimental group incorporated more r a d i o a c t i v i t y into fat both with and without the addition of i n s u l i n than those obtained from the control animals. In addition, the s e n s i t i v i t y of adipocytes to epinephrine and theophylline was enhanced i n the defi c i e n t group. The authors suggested that body fat turnover may be increased under these conditions. 2.2.6 Carbohydrate Metabolism i n Pyridoxine Deficiency 2.2.6.1 General Considerations Evidence has accumulated indicating that an abnormality i n carbohydrate metabolism occurs in pyridoxine deficiency. Beaton et_ al_. (1954) found that rats deprived of pyridoxine for 1 week had s l i g h t l y lower fasting blood levels of glucose, lactate, and pyruvate, and lower fasting l i v e r glycogen than their pair-fed controls. Insulin administra-ti o n had a s l i g h t l y more pronounced effect on blood glucose levels i n the def i c i e n t rats than the controls. Guggenheim and Diamant (1957) argued that pyridoxine deficiency i n the rat was not necessarily accompanied by disturbances i n carbohydrate metabolism. Deoxypyridoxol-treated and ad libitum-fed control rats possessed similar glucose and l i v e r glycogen l e v e l s , as well as comparable capacities by l i v e r and diaphragm s l i c e s to synthesize glycogen from glucose. On the other hand, the corresponding pair-fed controls showed higher 16. values than either group. This p a r t i c u l a r study i s an indication of the need to minimize the differences i n the pattern of food intake between the control and the de f i c i e n t rats. Huber e_t a_l. (1962, 1964) reported that pyridoxine-d e f i c i e n t rats were hypoglycaemic when compared with ad libitum control rats in both the fasted and the non-fasted states. Also, impaired glucose tolerance and decreased i n s u l i n - l i k e a c t i v i t y i n serum and pancreas were observed. The simultaneous occurrence of hypoglycaemia, decreased i n s u l i n a v a i l a b i l i t y and increased s e n s i t i v i t y to i n s u l i n suggested the p o s s i b i l i t y of hypophyseal derangement i n pyridoxine d e f i c i e n t rats, since increased i n s u l i n s e n s i t i v i t y and decreased i n s u l i n secretion may be caused by decreased growth hormone production (Engel e_t a l . , 1958). Huber and Gershoff (1965) demonstrated a marked decrease i n growth hormone levels in the hypophysis of pyridoxine d e f i c i e n t rats as compared with pair-fed control. This phenomenon was p a r t l y explained by the fact that the high pyridoxal phosphate of rat hypophysis i s rapidly depleted in pyridoxine deficiency, thus resulting in impaired function. Sabo e_t a l . (1971) determined the l e v e l of several tissue dehydrogenases associated with the t r i c a r b o x y l i c acid and the oxidative metabolism of glucose. Liver homogenates from pyridoxine d e f i c i e n t rats showed decreases in lactate and glucose -6-phosphate dehydrogenase a c t i v i t i e s when compared to both ad  libitum and pair-fed controls. Increases i n lactate, i s o c i t r a t e , malate and glucose-6-phosphate dehydrogenase a c t i v i t i e s were seen i n adipose tissue from d e f i c i e n t animals only when compared 17. to a c l libitum controls. 2.2.6.2 Tissue Glycogen Pyridoxal phosphate i s a component of c r y s t a l l i n e glycogen phosphorylase, but does not seem to pa r t i c i p a t e i n i t s c a t a l y t i c function (Baranowski et a l . , 1957; Illingworth et a l . , 1960; Tiglao and Eisenstein, 1964). The active form of t h i s enzyme i s a tetramer which contains 4 molecules of pyridoxal phosphate attached to e-amino groups of lysine residues on the apoenzyme (Brimacombe and Stacey, 1962; Brown and Cori, 1961; Fischer and Krebs, 1966; Krebs and Fischer, 1962, 1964). Removal .of pyridoxal phosphate inactivates glycogen phosphorylase, apparently due to the d i s s o c i a t i o n of the enzyme into subunits (Fischer, and Krebs, 1966). Pyridoxine deficiency affects the a c t i v i t y of t o t a l glycogen phosphorylase, but the observations on the a c t i v i t y of phosphorylase a are not consistent. In pyridoxine-deficient rats, t o t a l s k e l e t a l muscle phosphorylase levels were reduced to 35 % of those found in control rats (Illingworth e_t al_. , 1960). However, the levels of phosphorylase a, which i s the physiological-ly active form were not decreased in the muscle of the d e f i c i e n t animals. Addition of pyridoxal phosphate to muscle extract f a i l e d to increase a c t i v i t y , suggesting that apoenzyme synthesis i s possibly impaired i n pyridoxine deficiency. Eisenstein (1962) found some decrease i n rat muscle phosphorylase a and in t o t a l phosphorylase a c t i v i t y , but the extent of change was greater for t o t a l phosphorylase. Lyon and Porter (1962) found that phosphorylase a and t o t a l phosphorylase in s k e l e t a l muscle were decreased to the same extent in pyridoxine d e f i c i e n t C^j s t r a i n 18. mice, but not in I s t r a i n . Liver phosphorylase d i f f e r s in some respects from muscle phosphorylase, but performs the same function (Sutherland and Wosilait, 1956). It i s depleted to a lesser degree than muscle phosphorylase i n pyridoxine deficiency (Eisenstein, 1962; Lyon and Porter, 1962). Since pyridoxal phosphate i s needed for phosphorylase a c t i v i t y , one might expect an increase i n the concentration of tissue glycogen when enzyme a c t i v i t y f a l l s i n pyridoxine deficiency. However, this i s not the case (Beaton, 1955; Illingworth et a l . , 1960; Lyon and Porter, 1962). An important consideration would be the requirement for pyridoxal phosphate in transamination and other reactions involved i n gluconeogenesis. Impairment of this process might tend to lower tissue glycogen levels by decreasing the a v a i l a b i l i t y of substrates. Also the amount of food intake and feeding pattern in the ^ deficient and control animals should be considered in the interpretation of the results obtained. 2.2.7 Importance of Considering the Mode of Feeding Inanition i s one of the universal responses to n u t r i t i o n a l deficiency. Therefore, i t i s necessary i n studies of energy u t i l i z a t i o n to distinguish the effects of the lack of a certain nutrient from the consequences of decreased food intake. Pair-feeding has been often used, in order to r e s t r i c t the c a l o r i c intake of the control animals to the l e v e l consumed by the experimental ones. However, another serious problem arises by employing pair-feeding. The deprived rats are allowed access to food at a l l times, and eat small portions of i t throughout the day, p a r t i c u l a r l y during the night (i.e.,nibbling) Once d a i l y , the food intake of the d e f i c i e n t rats i s measured and an i s o c a l o r i c quantity of the complete diet i s offered to the control animals. This amount of food, being smaller than that required to s a t i s f y the appetite of the l a t t e r animals, i s consumed within a r e l a t i v e l y short period of time. Thus, the pair-fed animal becomes an intermittent eater similar to the meal-fed rat. Meal-feeding of the rat has been shown to e l i c i t several adaptive changes in the metabolism, and this subject has been well reviewed by Fabry (1967), L e v e i l l e (1970), and Tepperman and Tepperman (1970). Meal-fed animals develop the machinary to ingest large amount.of food i n an hour or two, to transfer the ingested energy to tissues, and to store i t for further use during the period of fasting between meals. The digestive tract adapts by increasing in s i z e , thereby increasing i t s absorptive area. It has been shown that glucose absorption increased to the same extent as the size of intestine (Leveille and Chakrabarty, 1968). Adaptive hyperlipogenesis, characterized by increases i n the a c t i v i t i e s of l i v e r and p a r t i c u l a r l y adipose tissue enzymes involved in f a t t y acids biosynthesis i s the most noticeable manifestation of the attempt to cope with the problem of temporarily storing the chemical energy ingested (Chakrabarty and L e v e i l l e , 1969; H o l l i f i e l d and Parson, 1962a,b; L e v e i l l e , 1966, 1967a,b; L e v e i l l e and Hanson, 1965, 1966; Tepperman and Tepperman, 1958). Increased s e n s i t i v i t y of adipose tissue to 20. i n s u l i n (Wiley and L e v e i l l e , 1970), increased glycogen deposition (Leveille and Chakrabarty, 1967), an increased fat oxidation (Leveille and Hanson, 1965) have been also reported. Clearly, the pattern of evergy u t i l i z a t i o n i s greatly altered by meal-feeding . On the basis of the foregoing considerations, i t would be reasonable to suspect that the effect of pyridoxine deficiency on the energy u t i l i z a t i o n , i f any, may have been obscured by comparisons with the metabolically d i f f e r e n t pair-fed controls. Therefore, there is a need to re-evaluate the possible role of pyridoxine in fat metabolism under conditions where the c a l o r i c intake and the mode of feeding of both the d e f i c i e n t arid the control animals are standardized. An experimental model in which the deprived animals are adapted to meal-feeding, while the controls are pair-fed would f u l f i l l this requirement. This approach has been employed successfully by Angel and Sabry (1969) to investigate lipogenesis during pyridoxine repletion. 3. Materials and Methods 3.1 Materials Male weanling Wistar rats were obtained from Biobreeding Laboratories of Canada, Ltd., Ottawa, Ontario. The compositions of the basal d i e t , the mineral and the vitamin supplements are shown in Tables 1-3, respectively. Two diets were used : a pyridoxine-free d i e t , and the same diet supplemented with 10 mg pyridoxol•HCl/Kg. Chemicals were Fisher-brand reagent grade, purchased from Fisher S c i e n t i f i c Co., Vancouver, B.C. Biochemicals were obtained from Sigma Chemical Co., St. Louis, Missouri, U.S.A. Radio-chemicals were supplied by Amersham/Searle, Inc., Don M i l l s , Ontario. 3.2 Methods 3.2.1 Handling of Animals The rats were housed singly in screen-bottomed stainless s t e e l cages in an air-conditioned room. Lighting was regulated automatically to provide 12 hours of l i g h t (6:00 a.m. to 6:00 p.m.) and 12 hours of darkness. Water was provided ad libitum. Upon a r r i v a l , the rats were fed the complete diet ad  libitum f o r 3 days. They were then assigned to groups, using tables of random numbers. One group was fed the d e f i c i e n t diet ad libitum (nibblers). Another group was given access to the deficient diet for 2 hours d a i l y , between 9:30 a.m. and 11:30a.m. (meal-fed). One group of control rats was given the complete Table 1. Composition of the basal diet Ingredient g/Kg diet Protein (from vitamin-free casein) 150 Corn o i l 100 Mineral mixture''" , 38 Vitamin mixture 2 5 Sucrose to make 1,000 1. Cf. Table 2. 2. Cf. Table 3. Table 2. Composition of the mineral mixture Ingredient g/Kg mixture CaC0 3 191.071 CaHP04-2H20 376.835 Na 2HP0 4 158.125 KC1 192.373 MgS04 60.586 F e r r i c c i t r a t e 15.522 MnS04-H20 4. 006 ZnC03 1.402 KI0 3 0.080 Table 3. Composition of the vitamin mixture Vitamin g/Kg mixture Thiamine-HC1 0. 200 Riboflavin 0. 400 D-Pantothenic acid, Ca s a l t 0. 800 Nicotinamide 2. 000 D-Biotin 0. 012 F o l i c acid 0. 040 Vitamin #12, 0.11 in mannitol 2. 000 Menadione 0. 008 Choline dihydrogen c i t r a t e 200. 000 Dry alpha-tocopherol acetate, 250 IU/g 17. 624 Dry vitamin A palmitate, 500,000 IU/g 0. 800 Dry vitamin D 2, 500,000 IU/g 0. 080 Non-nutritive c e l l u l o s e 775. 634 1. Pyridoxol-HCl, 0.400 g/Kg was added to the complete diet at the expense of non-nutritive c e l l u l o s e . 25. diet i n r e s t r i c t e d quantities equal to those consumed each day by the nibbling d e f i c i e n t rats, while another one was fed i n quantities equal to those eaten by the meal-fed d e f i c i e n t animals. The controls were given th e i r food at noon each day. The animals were fed the respective diets for 6 weeks. Body weight was recorded weekly. 3.2.2 In Vivo Study of Glucose U t i l i z a t i o n A l l rats were fasted for 22 hours p r i o r to intraperitoneal i n j e c t i o n of 1.25 uCi glucose-U- 1 4C/rat (2.9 mCi/mmole). Immediately thereafter, they were given 5.5g of the appropriate diet each, an amount which a l l animals consumed rapidly. Two hours after glucose administration and the i n i t i a t i o n of feeding, the animals were k i l l e d by ether anaesthesia. Liver, diaphragm, and adipose tissue were quickly excised, blotted with f i l t e r paper, weighed, and s p l i t into proper portions for analysis. Lipids were extracted from l i v e r and adipose tissue according to Folch et aJL. (1957). The l i p i d solution i n chloroform obtained after p u r i f i c a t i o n was evaporated under vacuum at 40-45 °C. After further evaporation under a stream of dry nitrogen, the t o t a l amount of l i p i d s was determined gravi-metrically. For the determination of body f a t t y acids, the i n t e s t i n a l tract was s l i t open and the contents were rinsed out. The carcass (body minus l i v e r and epididymal adipose tissue pads) was then digested with KOH by the method of Jansen el: a l . (1966). The hydrolyzate was made up to volume and a sample was used for i s o l a t i n g the f a t t y acid f r a c t i o n as described below for tissue 26 l i p i d s . A portion of the f a t t y acids obtained was counted in the same manner as tissue f a t t y adids. The l i p i d residue from tissues was saponified by refluxing with 10 ml of freshly prepared 10 % KOH i n 95 % methanol at 85-90 °C for 2 hours. The hydrolyzate was di l u t e d with an equal volume of water, and the non-saponifiable l i p i d s were removed by three successive extractions with 5 ml portions of petroleum ether (boiling range; 38-49.6°C). The remaining solution was a c i d i f i e d with concentrated HC1 and the li b e r a t e d f a t t y acids were extracted s i m i l a r l y . The combined f a t t y acid extracts were dried over anhydrous Na2SO^ and then transferred quantitatively to tared counting v i a l s . The solvent was evaporated with a gentle stream of nitrogen and the residue was dissolved in the s c i n t i l l a t i o n solvent used by L e v e i l l e and Hanson (1965). 3.2.3 In V i t r o Studies When lipogenesis was studied in v i t r o , the animals were fasted for 22 hours, then refed with 5.5 g food. The rats were k i l l e d by decapitation 2 hours l a t e r . In addition, one group of nibbling d e f i c i e n t rats had access to food u n t i l death. The l i v e r and the epididymal adipose tissue pads were excised quickly and weighed. Liver s l i c e s from the l e f t l a t e r a l lobe and adipose tissue segments from the thin peripheries of both pads were prepared immediately. Duplicate samples ( l i v e r : 150-200 mg; adipose tissue : 50-75 mg) were incubated with the radioactive substrates in 25-ml Erlenmeyer fl a s k s . The incubation medium was 3 ml Krebs-Ringer bicarbonate buffer, pH 7.4 (Umbreit et a l . , 1964), containing per ml; either 5 moles glucoe-C- C (0.075 uCi) or 5 moles unlabelled glucose and 10 umoles sodium a c e t a t e - l - 1 4 C (0.04 uCi). In addition, the incubation medium used for adipose tissue contained 0.1 U/ml zinc i n s u l i n . After flushing with 0 2-C0 2 (951 : 5%), the vessels were sealed with rubber serum stoppers equipped with hanging p l a s t i c v i a l s , then were placed for 2 hours i n a Dubnoff water bath shaking at 90 oscillations/minute and maintained at 37°C. At the end of incubation, 0.4 ml of Hyamine Hydroxide were injected into the centre well, followed by the addition of 1 ml of N-H 2S0 4 to the medium. Shaking was continued for another hour to ensure the recovery of ^ C 0 2 . The hanging well was then removed,- wiped c a r e f u l l y on the outside with moistened paper tissue and dropped into a s c i n t i l l a t i o n counting v i a l . One ml of water and 10 ml counting solvent (Patterson and Greene, 1965) were added and the v i a l s were shaken vigorously. The inactivated tissues were allowed to stand i n 10 ml of 2:1 chloroform-methanol for a day, with occasional shaking. Extraction was repeated twice more, and the pooled l i p i d solution was processed as described previously. Deoxyribonucleic acid was extracted from defatted adipose tissue segments by the method of Schneider (1957) and was determined by the diphenylamine reaction (Burton, 1956). 5.2.4 Enzyme Studies After 22 hours of fasting,, the rats were given 5.5g portions of th e i r corresponding d i e t s . Two hours l a t e r , they were k i l l e d by decapitation. Liver and epididymal adipose tissue were excised rapidly, c h i l l e d to 0°C and disintegrated in a 28. glass-teflon homogenizer i n 9 volumes of ice-cold isotonic KC1, pH 7.0. The homogenates were centrifuged at 1,000 g for 15 minutes, then the supernatants were recentrifuged at 15 ,000 g_ at 4 C for 30 minutes. The clear supernatant obtained was used for enzyme assays and for protein determination (Lowry et a l . , 1951). Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, E.C.1.1.1.49) and 6-phosphogluconic acid dehydrogenase (6-phosphogluconate : NADP oxidoreductase, E.C. 1.1.1.44) were assayed according to the method of Glock and McLean (1953). The method of Ochoa (1955) was used to measure malic enzyme (L-malate : NADP oxidoreductase, E.C.1.1.1.40). One unit of enzyme a c t i v i t y was defined as 1 umole of NADPH formed/minute. 3.2.5 Isotope Counting A l l samples were assayed for r a d i o a c t i v i t y in a Picker Nuclear Liquid s c i n t i l l a t i o n counter. Counting e f f i c i e n c y was determined by the channels ratios method using external standardization. A series of -^C-labelled toluene standards, quenched with increasing amounts of acetone were used. Counting e f f i c i e n c y was approximately 80-90% for f a t t y acids and gl y c e r o l , and 70% for C0 2. 3.2.6 S t a t i s t i c a l Analysis and Other Calculations The results were expressed as the mean + standard error of the mean. Differences between groups were evaluated by a standard student ' t ' test (Walpole, 1968). 4. Experimental and Results 4.1 Experiment I : In Vivo Lipogenesis i n Pyridoxine Deficiency This study was conducted in order to observe the effects of pyridoxine deficiency on lipogenesis i n vivo under conditions where food intake and feeding frequency are standardized. In addition, an attempt was made to correlate the findings with those obtained when food intake, but not feeding frequency i s controlled. Weanling rats were fed the deficiency diet either ad  libitum (nibblers) or were given access to food for 2 hours d a i l y (meal-fed). Two groups of control rats were given the complete diet in r e s t r i c t e d quantities equal to those consumed by the experimental groups each day. Some of the rats deprived of pyridoxine manifested marked signs of deficiency such as cutaneous changes on the paws, nose and t a i l at the time of k i l l i n g . After 6 weeks, the animals were fasted for 22 hours, injected with glucose-U-^C and k i l l e d 2 hours l a t e r . Tissue sample were then taken for analysis as described e a r l i e r . The results are summarized in Tables 4-8. The data on cumulative food intake and body weight gain presented in Table 4 show that the pyridoxine-deficient rats, whether meal-fed or nibbling, gained less weight than their respective controls. Over the 6 week depletion period, the cumulative food intake of the nibbling d e f i c i e n t rats was 347 g /rat,and body weight gain was 67 g/rat, with an o v e r a l l feed Table 4. Cumulative food consumption and body weight gain of pyridoxine-deprived and control rats. Values are expressed as g/rat. Time Deprived, meal-fed Control, r e s t r i c t e d ^ (weeks) Food consumed Wt. gain Food consumed Wt. gain 1 24.1±; 1119 5 6.2±0.80 24.1 8.0±1.05 2 62.1± 2.24 10.8+1.45 62.1 14.8il.28 3 100.1± 3.43 20.2±1.91 100.1 27.0±0.76 4 141.6± 5.11 30.5±2.78 141.6 39.6±1.29 5 185.1± 6.91 40.2±2.78 185.1 51.Oil.12 6 228.2± 8.31 ^ 47.0±3.73 228.2 63.5il.28 Time Deprived, nibbler 3 Control, r e s t r i c t e d 4 (weeks) Food consumed Wt. gain Food consumed Wt. gain 1 48. 9± 1.92 22.Oil.34 48.9 25.8±2'.37 2 104.0± 2.08 29.9±2.89 104.0 34.9il.52 3 160.2± 3.66 44.5±2.85 160. 2 53.4±2.43 4 225.5± 6.46 56.2±4.76 225.5 81.2+3.01 5 285.6± 9. 23 62.6+6.68 285.6 98.2±2.52 6 347.3±13.22 67.2±8.36 347. 3 112.1±2.00 1. Meal-fed the pyridoxine-free diet for 6 weeks after weaning. 2. Fed the complete diet at the l e v e l consumed by the deprived-meal-fed rats. 3. Fed the pyridoxine-free diet ad libitum for 6 weeks. 4. Fed the complete diet at the l e v e l consumed by the deprived nibbling rats. 5. Meanistandard error of the mean for 8 rats. 31. e f f i c i e n c y of._0.19g gain/g food consumed (Table 5). The corresponding control gained 112 g, showing an e f f i c i e n c y of 0.31g gain/g food consumed, which was s i g n i f i c a n t l y higher than that attained in the nibbling d e f i c i e n t rats (P <0.001). The magnitude of difference i n weight gain between the meal-fed de f i c i e n t and their pair-fed control rats was small;nevertheless, pyridoxine deficiency reduced feed e f f i c i e n c y s i g n i f i c a n t l y (P <0.001). The data presented i n Table 6 show that pyridoxine deficiency did not aff e c t the r e l a t i v e size (g tissue/lOOg rat) of the l i v e r regardless of the feeding pattern employed. However, a reduction in the r e l a t i v e weight of the epididymal fat pads was observed i n the nibbling d e f i c i e n t rats when compared with th e i r r e s t r i c t e d controls (P <0.05). This difference was not seen when the d e f i c i e n t meal-fed rats were compared with their corresponding controls. Total body, l i v e r and epididymal pad f a t t y acids, expressed i n mg/g tissue, are shown i n Table 7. The nibbling d e f i c i e n t rats possessed less body f a t t y acids than th e i r r e s t r i c t e d controls (P <0.05), but the meal-fed d e f i c i e n t animals had only s l i g h t l y less fatty acids than their r e s t r i c t e d controls. Table 7 also shows a small increase in the l e v e l of hepatic f a t t y acids in the meal-fed d e f i c i e n t rats, as compared with the control animals. However, there was no difference i n this respect between the nibbling d e f i c i e n t rats and th e i r controls. The f a t t y acid content i n the epididymal adipose tissue pads followed a pattern similar to that of body fa t t y acids. Table 5. Total food consumption, t o t a l weight gain and o v e r a l l feed e f f i c i e n c y i n pyridoxine-deprived and control r a t s . Group Food consumption Weight gain Feed e f f i c i e n c y g gain/g food consumed 0.20 ± 0.012 0.27 0.005 <0.001 0.19 ± 0.018 0.31 0.006 <0.001 1. Meal-fed the pyridoxine-free diet for 6 weeks af t e r weaning. 2. Fed the complete diet at the l e v e l consumed by the deprived meal-fed rats. 3. P r o b a b i l i t y of the difference between means occurring by chance. 4. Fed the pyridoxine-free diet ad libitum for 6 weeks. 5. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 6. Mean±standard error of the mean for 8 rats . g/rat g/rat Deprived, meal-fed 1 228 ± 8.36 47.0 ± 3.73 Control, r e s t r i c t e d 2 228 63.5 1.28 p_3 - <0.001 Deprived, n i b b l e r 4 347 ±13.2 67.2 ± 8.36 Control, r e s t r i c t e d 5 347 112.1 2.00 p - . <0.001 Table 6. Relative sizes of the control r a t s . l i v e r and the epididymal adipose tissue in pyridoxine-deprived and Group F i n a l body weight Liver Adipose tissue g/rat g/lOOg body wt. g/lOOg body wt. Deprived, meal-fed"'' 120 ± 5.66 4.25 ± 0.14 0.82 ± 0.08 2 Control, r e s t r i c t e d 135 ± 1.3 3.98 ± 0.09 0.98 ± 0.08 <0. 05 n s 7 ns Deprived, meal-fed^ 133 ± 8.3 4.05 ± 0.06 0.96 ± 0.11 Control, r e s t r i c t e d ^ 184 ± 2.3 3.92 ± 0.10 1.24 ± 0.05 P <0.001 ns <0. 05 1. Meal-fed the pyridoxine-free diet for 6 weeks after weaning. 2. Fed the complete diet at the l e v e l consumed by the deprived meal-fed ra t s . 3. Pr o b a b i l i t y of the difference between means occurring by chance. 4. Fed the pyridoxine-free diet ad_ libi t u m for 6 weeks. 5. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 6. Mean ± standard error of the mean for 8 ra t s . 7. Not s i g n i f i c a n t . Table 7. Liver, adipose tissue and body f a t t y acid content in pyridoxine-deprived and control r a t s . Group Liver Adipose tissue Whole animal mg/g mg/g mg/g Deprived, meal-fed'*" 42 ± 1.7 6 588 ± 17.7 68 ± 5.2 7 Control, r e s t r i c t e d 37 ± 1.1 622 ± 20.9 77 ± 5.7 3 B <0. 05 ns7 ns Deprived, nibbler^ 39 ± 2.1 607 ± 28.5 80 ± 8.1 Control, r e s t r i c t e d ^ 45 ± 2.5 661 ± 12.4 106 ± 7.9 P ns ns <0. 05 1. Meal-fed the pyridoxine-2. Fed the complete diet at free diet for 6 weeks the l e v e l consumed by after weaning, the deprived meal-fed rats . 3. Pr o b a b i l i t y of difference between means occurring by chance. 4. Fed the pyridoxine-free diet ad libitum for 6 weeks. 5. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 6. Mean ± standard error of the mean for 8 rats . 7. Not s i g n i f i c a n t . 35. The incorporation of glucose-U- 1 4C into l i v e r , epididymal adipose tissue, and t o t a l body f a t t y acids i s shown in Table 8. In the nibbling d e f i c i e n t animals, a marked decrease i n the capacity of the l i v e r to incorporate l a b e l l e d glucose into f a t t y acids was apparent upon comparison with the r e s t r i c t e d controls (P <0.001). On the other hand, the capacity of the meal-fed def i c i e n t rats to synthesize f a t t y acids exceeded that of the corresponding controls, but the magnitude of this r i s e did not reach s t a t i s t i c a l s i g n i f i c a n c e . In general, the effects of pyridoxine deprivation on f a t t y acid synthesis by the adipose tissue resembled those observed i n the l i v e r . However, the actual rates of 1 4 C incorporation into adipose tissue f a t t y acids were greater than those observed in the l i v e r . The incorporation of glucose into t o t a l body fatty acids was depressed in the pyridoxine-deprived rats when compared with the respective controls, whether the former rats were nibbling or meal-fed (P <0.001). 4.2 Experiment II : In V i t r o Lipogenesis in Pyridoxine Deficiency The results obtained from the Experiment I suggested that the capacity of the l i v e r and adipose tissue to synthesize f a t t y acid may be increased in the meal-fed pyridoxine-deficient rat, but decreased in the nibbling animal. In order to see i f these changes r e f l e c t alterations in the lipogenic potentials of tissues, and to ascertain that the in_ vivo incorporation data did not result from d i f f e r e n t i a l u t i l i z a t i o n of l a b e l l e d glucose, Table 8. In vivo incorporation of g l u e o s e - U - i n t o f a t t y acids i n pyridoxine-deprived and control ratsT Values are dpm x 10 3/g tissue and are corrected for equal d i l u t i o n . Group Liver Adipose tissue Carcass Whole animal Deprived, meal-fed 2. 149 ± 0. 388 7 12. 619 ± 3. 469 2. 115 ± 0. 257 2. 38 ± 0. 218 Control, r e s t r i c t e d 1. 648 ± 0. 149 7. 687 ± 0. 621 3. 662 ± 0. 237 3. 61 ± 0. 227 4 P ns8 ns <0.001 <0.001 Deprived, nib b l e r ^ 0. 682 ± 0. 074 1. 127 ± 0. 216 1. 066 ± 0. 116 1. 05 ± 0. 113 Control, r e s t r i c t e d ^ 2. 161 ± 0. 277 5. 053 ± 0. 838 2. 175 ± 0. 222 2. 23 ± 0. 216 P <0.001 <0. 001 <0.001 <0 . 001 1. The rats were fasted for 22 hours, then injected with 1.25 uCi glucose-U- C in t r a p e r i t o n e a l l y , arid the corresponding d a i l y meals were given immediately a f t e r the i n j e c t i o n . Rats were k i l l e d 2 hours after i n i t i a t i o n of feeding. 2. Meal-fed the pyridoxine-free diet for 6 weeks afte r weaning. 3. Fed the complete diet at the l e v e l consumed by the deprived meal-fed rat s . 4. Pro b a b i l i t y of difference between means occurring by chance. 5. Fed the pyridoxine-free diet ad libitum for 6 weeks. 6. Fed the complete diet at the l e v e l consumed by the deprived nibbling rats. 7. Mean ± standard error of the mean for 8 rats. 8. Not s i g n i f i c a n t . 37. in v i t r o 1 lipogenesis was studied by using l i v e r s l i c e s and adipose tissue segments.. The rats were fed i n the same manner as i n Experiment I. The data on cumulative food intake and body weight gain, and on r e l a t i v e sizes of the l i v e r and the epididymal adipose tissue are summarized in Appendix A (Tables A1-A3). As "shown in Table 9, the capacity of l i v e r s l i c e s from fed nibbling d e f i c i e n t rats to incorporate r a d i o a c t i v i t y from la b e l l e d glucose into CC^ and f a t t y acids, and from lab e l l e d acetate into f a t t y acids markedly exceeded that of s l i c e s obtained from the r e s t r i c t e d controls (P <0.05; P <0.02; P <0.001, respectively). However, when another group of nibbling d e f i c i e n t rats was fasted and refed in the same manner.as the r e s t r i c t e d control rats, the incorporation of acetate into f a t t y acids was lower than in the controls. No differences in the l a b e l l i n g of l i v e r f a t t y acids and glyceride glycerol were found when the meal-fed de f i c i e n t rats were compared with the corresponding r e s t r i c t e d animals. However, pyridoxine deficiency in meal-fed rats was associated with a decrease i n the a b i l i t y of the l i v e r to oxidize glucose, as comparison with the controls revealed (P <0.025). Table 10 summarizes the data obtained with adipose tissue segments. The values are expressed on the basis of the deoxy-ribonucleic acid "(DNA) content of this tissue. The nibbling deficient rats incorporated less r a d i o a c t i v i t y from glucose into C O 2 , f a t t y acids and glyceride glycerol than th e i r controls (P <0.05; P <0.025; P <0.02, resp e c t i v e l y ) . However, the values Table 9. In v i t r o lipogenesis by l i v e r of pyridoxine-deprived and control r a t s . 1 Values are expressed as nmoles/100 mg tissue/2 hours. Glucose Acetate FStty acids Glyceride- Fatty acids glycerol 2 Deprived, meal-fed 185+ 17 8 12 .3 ± 3. 72 35 .1 ± 1. 38 210 .6* 50. 03 3 Control, r e s t r i c t e d 246 ± 16 15 .3 ± 3. 15 38 .6 ± 1. 95 321 .4 ± 78. 34 4 P <-:0. 02 5 n s9 ns ns Deprived, n i b b l e r ( f e d ) 5 253 ± 34 70 .2± 17. 74 37 .5 ± 4. 00 661 .2 ± 8 4 . 48 Deprived, nibbler - - - 58 . 3 ± 7. 11 7 Control, r e s t r i c t e d 172 ± 12 5 .1+ 1. 39 33 .1 ± 2. 24 134 .8 ± 28. 00 p [Deprived (fed) vs. Control] < 0.05 < 0.02 ns < 0.001 p [Deprived vs. Control] - - - < 0.02 1. The rats were fasted for 22 hours, then refed and were k i l l e d 2 hours a f t e r i n i t i a t i o n of feeding. 2. Meal-fed the pyridoxine-free diet for 6 weeks a f t e r weaning. 3. Fed the complete diet at the l e v e l consumed by the deprived meal-fed r a t s . 4. Probabi l i t y of difference between means occurring by chance. 5. Fed the pyridoxine-free diet ad libitum for 6 weeks and had access to food u n t i l death. (continued next page) Table 9. (Continued) 6. Fed the pyridoxine-free diet ad libitum for 6 weeks. 7. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 8. Mean ± standard error of the mean for 8 rats. 9. Not s i g n i f i c a n t . to Table 10. In v i t r o lipogenesis by the epididymal adipose tissue of pyridoxine-deprived and control rats. J- Values are expressed as nmoles/ug DNA/2 hours. Group Glucose CO. Fatty acids Glyceride glycerol Acetate Fatty acids Deprived, meal-fed 3 Control, r e s t r i c t e d 4 P Deprived, nibbler (fed)^ Deprived, n i b b l e r ^ 7 Control, r e s t r i c t e d p [Deprived(fed) vs. Control] p .[Deprived vs. Control]-46.3-'± 6.448 46.3 ± 3.68 30.1 ± 2.90 24.1 ± 3.74 <0.05 <0.02 19.4 ± 3.79 19.2 ± 4.84 31.3 ± 3.57 35.5 ± 4.16 <0.05 <0.025 9.6 ± 1.00 6.1 ± 1.48 ns 9 5.5 ± 0.82 9.5 ± 1.15 <0.02 28.8 ± 3.03 26.2 ± 5.41 ns 21.7 ± 3.86 17.4 ± 2.44 27.1 ± 3.10 ns <0. 05 1. The rats were fasted for 22 hours, then refed and were k i l l e d 2 hours a f t e r i n i t i a t i o n of feeding. 2. Meal-fed the pyridoxine-free diet for 6 weeks afte r weaning. 3. Fed the complete diet at the l e v e l consumed by the deprived meal-fed r a t s . 4. P r o b a b i l i t y of difference between means occurring by chance. 5. Fed the pyridoxine-free diet ad libitum for 6 weeks and had access to food u n t i l death. (continued next page) Table 10. (Continued) 6. Fed the pyridoxine-free diet ad libitum for 6 w.eeks. 7. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 8. Mean ± standard error of the mean for 8 rats. 9. Not s i g n i f i c a n t . observed i n the meal-fed deprived group were greater than those obtained i n the corresponding r e s t r i c t e d control group (CG^, P <0.05; fatty acids, P <0.02; gl y c e r o l , ns); When la b e l l e d acetate was added to the incubation medium along with unlabelled glucose, neither the fed nibbling nor the meal-fed de f i c i e n t animals exhibited any significant, differences from th e i r respective controls. However, the fasted-refed nibbling d e f i c i e n t rats exhibited s i g n i f i c a n t l y decreased acetate incorporation into f a t t y acids (P <0.05) when compared with the controls. DNA and fat were measured in the thin peripheries of the epididymal adipose tissue which were used i n studying in v i t r o lipogenesis. As Table 11 shows, there was a marked increase i n the amount of DNA/g adipose tissue i n the pyridoxine-deficient animals as compared with t h e i r respective controls (meal-fed, P <0.01; nibbling, P <0.001). The l i p i d content of this tissue was unaltered when the d e f i c i e n t rats were meal-fed, but was decreased below the values observed i n the r e s t r i c t e d controls when the d e f i c i e n t rats were fed in a nibbling fashion. 4.3 Experiment III : A c t i v i t y of NADPH-Producing Enzymes in Pyridoxine Deficiency The enhanced lipogenic capacity of tissues in meal-fed pyridoxine-deficient rats observed suggested the p o s s i b i l i t y that this phenomenon could be associated with the increased NADPH production to support f a t t y acid synthesis. Thus, glucose-6-phosphate dehydrogenase, 6-phosphogluconate Table 11. DNA and l i p i d content of the epididymal adipose tissue of pyridoxine-deprived and control rats.1 Group DNA Total l i p i d Hg/g mg/g 2 Deprived, meal-fed 740 ± 113.77 642 ± 19.9 3 Control, r e s t r i c t e d 449 ± 47.9 698 ± 18.7 P 4 <0. 05 n s 8 Deprived, n i b b l e r 5 515 ± 50.2 672 ± 18.3 Control, r e s t r i c t e d 232 ± 22.1 750 ± 14.8 P <0.001 <0.01 1. Tissue samples were taken from the thin peripheries of both epididymal f a t pads. 2. Meal-fed the pyridoxine-free diet for 6 weeks af t e r weaning. 3. Fed the complete diet at the l e v e l consumed by the deprived meal-fed rats. 4. P r o b a b i l i t y of difference between means occurring by chance. 5. Fed the pyridoxine-free diet ad libitum for 6 weeks. 6. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 7. Mean ± standard error of the mean for 8 r a t s . 8. Not s i g n i f i c a n t dehydrogenase and malic enzyme, which supply most of the NADPH required, were measured. Enzyme assays were conducted on samples from animals fasted and refed in the same manner described previously (Experiment I). The data on cumulative food intake and body weight gain , and on r e l a t i v e sizes of the l i v e r and the epididymal adipose tissue are presented in Appendix B (Tables B1-B3). As Table 12 shows, pyridoxine deficiency led to decreases in the a c t i v i t i e s of l i v e r glucose-6-phosphate dehydrogenase and malic enzymes, whether the animals were meal-fed (P <0.001 and P <0.005, respectively) or nibbling (P <0.01 and P <0.001, respectively). The a c t i v i t y of 6-phosphogluconate dehydrogenase remained unaltered i n the meal-fed deprived animals, but was s i g n i f i c a n t l y lowered i n the nibbling rats (P <0.025), as -comparison with the appropriate r e s t r i c t e d controls revealed. The a c t i v i t i e s of the adipose tissue enzymes are presented in Table 13. As comparison with the control groups showed, pyridoxine deficiency was associated with decreases i n the a c t i v i t i e s of a l l of the enzymes measured, whether the animals were meal-fed or nibbling. In general, the same patterns of l i v e r and adipose tissue enzymes were obtained when enzyme a c t i v i t y was expressed in units/g protein, units/g fresh tissue and units/100 g rat. Therefore, only the f i r s t method of expression was used herein. Table 12. A c t i v i t y of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malic enzyme i n the l i v e r of pyridoxine-deprived and control rats. Values are expressed as units/g soluble protein . Group Glucose-6-phosphate 6-phosphogluconate Malic enzyme dehydrogenase dehydrogenase 3 Deprived, meal-fed 20. 25 ± 4.1188 42. 86 ± 1.636 39. 59 ± 1.888 Control, r e s t r i c t e d ^ 41. 53.'+ 5.710 48. 92 ± 2.719 54. 38 ± 3.159 p5 <0.001 n s 9 <0.005 Deprived, nibbler^ 14. 50 ± 3.270 36. 96 ± 3.332 11. 83 ± 0.985 7 Control, r e s t r i c t e d 31. 83 ± 4.428 49. 10 ± 3.362 45. 50 ± 4.561 P <0.001 <0.025 <0.001 1. The rats were fasted for 22 hours, then refed and were k i l l e d 2 hours a f t e r i n i t i a -t i on of feeding. 2. One unit of enzyme a c t i v i t y i s defined as lumole of NADPH formed/minute at room temperature. 3. Meal-fed the pyridoxine-free diet for 6 weeks af t e r weaning. 4. Fed the complete diet at the l e v e l consumed by the deprived meal-fed rat s . 5. P r o b a b i l i t y of difference between means occurring by chance. 6. Fed the pyridoxine-free diet ad libitum for 6 weeks,, (continued next page) Table 12. (Continued) 7. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 8. Mean ± standard error of the mean for 8 r a t s . 9. Not s i g n i f i c a n t . i -p. Table 13. A c t i v i t y of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and , malic enzyme i n the epididymal adipose tissue of.pyridoxine-deprived and control r a t s . Values are expressed as units/g soluble protein. Group Glucose-6-phosphate dehydrogenase 6-phosphogluconate dehydrogenase Malic enzyme 3 Deprived, meal-fed 104. ,73 ± 9. 905 8 53. 71 ± 4. 178 162. ,18 ± 22. , 128 Control, r e s t r i c t e d ^ 131. ,76 ± 7. 339 69. 25 ± 3. 272 214, .64 ± 30. ,997 p5 <0 . 05 <0. 02 ns 9 Deprived, nibbler 78. ,37 ± 7. 683 44. 41 ± 3. 976 55. , 76 ± 6. , 019 7 Control, r e s t r i c t e d 195. ,56 ±14. 638 101. 76 ± 5. 014 195. ,80 ±23. ,686 P <0 . 001 <0. 001 <0. 001 1. The rats were fasted for 22 i n i t i a t i o n of feeding. hours, then refed and were k i l l e d \ 1 hours a f t e r 2. One unit of enzyme a c t i v i t y i s defined as lymole of NADPH formed/minute at room temperature. 3. Meal-fed the pyridoxine-free diet for 6 weeks afte r weaning. 4. Fed the complete diet at the l e v e l consumed by the deprived meal-fed rat s . 5. P r o b a b i l i t y of difference between means occurring bypchance. 6. Fed the pyridoxine-free diet ad_ li b i t u m for 6 weeks. (Continued next page) Table 13. (Continued) 7. Fed the complete diet at the l e v e l consumed by the deprived nibbling rats 8. Mean ± standard error of the mean for 8 rats. 9. Not s i g n i f i c a n t . -i> GO 5. Discussion Pyridoxine deficiency has long been suspected to a l t e r fat metabolism, but the nature of this effect and i t s s p e c i f i c i t y remain u n c l a r i f i e d . The basic a l t e r a t i o n i s a decrease i n energy storage as body fat i n the pyridoxine-deficient rat i n comparison to i t s pair-fed control (McHenry and Gavin, 1941; Carter and Phizackerley, 1951; Desikachar and McHenry, 1954; Beaton et a l . , 1956; Huber et a l . , 1964). One serious d i f f i c u l t y encountered i n studies of t h i s type arises from the need to distinguish between the effects of the nutrient deficiency and the consequences of the concomittant inan i t i o n . This problem i s usually circumvented by pair-feeding. Under these conditions, the deprived rat eats ad libitum, while the control i s offered i t s allotment of food once or twice d a i l y . This amount, being smaller than that required to s a t i s f y the appetite of the l a t t e r animal, i s consumed within a short period of time. Thus, pair-feeding imposes on the control rat an intermittent pattern of food intake analogous to meal-feeding. It i s now evident that intermittent feeding has i t s metabolic consequences which render comparison between the nibbling d e f i c i e n t rat and i t s pair-fed control of rather doubtful value. Meal-feeding of the rat e l i c i t s several adaptive responses related to energy u t i l i z a t i o n and storage; these include : increased f a t t y acid synthesis and oxidation, increased glycogen deposition, decreased voluntary physical a c t i v i t y and increased 49. 50. e f f i c i e n c y of food u t i l i z a t i o n (Braun and Fabry, 1969; Fabry, 1964; Fabry and Braun, 1967; Fabry and Tepperman, 1970; L e v e i l l e , 1967 a-d; L e v e i l l e and Chakrabarty, 1967; L e v e i l l e and Hanson, 1965; L e v e i l l e and O'Hea, 1967). Therefore, i t i s important i n studies related to energy u t i l i z a t i o n not only to equalize the food intakes, but also to standardize the feeding pattern of the experimental and pair-fed control groups. There are two general methods by which pair-feeding can be performed without introducing gross differences between the feeding patterns of the d e f i c i e n t and the control animals. The controls may be fed their d a i l y allotment of food at predetermin-ed intervals throughout the day in order to simulate the feeding pattern of the nibbling experimental rats. The process can be f a c i l i t a t e d by using a variety of automatic feeding devices (Lee e_t al_. , 1962; Quarterman e_t al_. , 1970; Cohn and Joseph, 1967). However, this method 'is cumbersome and requires consider-able investment in equipment, especially when large numbers of animals are to be pair-fed. Another method consists of imposing a meal-feeding pattern on the deprived rats, thereby minimizing the difference in feeding frequency between these and the r e s t r i c t e d control animals. This approach was used i n the present investigation in p a r a l l e l with pair-feeding performed i n the usual manner with the deprived rats being fed ad libitum. In the studies reported herein, the animals were deprived of pyridoxine for 6 weeks after weaning. This length of time i s known to be adequate for the development of pyridoxine deficiency in nibbling (Radhakrishnamurty and Sabry, 1968) and meal-fed (Mellor, unpublished data) rats, as evidenced by reductions in the a c t i v i t i e s of l i v e r and erythrocyte alanine and aspartate aminotransferases. The data on growth and food consumption are consistent with the concept that pyridoxine deficiency i s associated with a decrease in feed e f f i c i e n c y (Sure and Easterling, 1949). However, the magnitude of the difference i n weight gain between the meal-fed and control animals was considerably smaller than that observed between the nibblers and the i r controls. Since intermittent feeding elevates feed e f f i c i e n c y i n normal rats (Leveille and Hanson, 1965), the present results imply that the reduction of this parameter in pyridoxine deficiency is not en t i r e l y due to differences i n the mode of feeding between the deprived and the control r a t s , since a decrease was seen even when feeding frequency was equalized. The discrepancy i n energy u t i l i z a t i o n between the pyridoxine deprived rat and i t s c a l o r i c a l l y r e s t r i c t e d control was i l l u s t r a t e d in the present studies by measurement of body f a t t y acid l e v e l s . Although the animals were fed equal amounts of food, the deprived nibbling rats had a lower percentage of body f a t t y acids than the controls. This was in agreement with previous observations (Beaton e_t al_. , 1956; Carter and Phizackerley, 1951; Desikachar and McHenry, 1954; Guggenheim and Diamant, 1957 ; Huber e_t al_., 1964). The difference in f a t t y acid percentages was not seen when the rats were meal-fed. Neverthe-less, the t o t a l amount of fat t y acids stored was c l e a r l y reduced, since the meal-fed d e f i c i e n t rats were smaller than their 52. corresponding controls. As with feed e f f i c i e n c y , these observations imply that pyridoxine deficiency exerts an effect on fat storage that i s unrelated to the mode of feeding employed. As expected, the f a t t y acid content of the adipose tissue resembled that of the whole animal, although the differences between groups did not always a t t a i n s t a t i s t i c a l s i gnificance. The responses of l i v e r f a t t y acids are more d i f f i c u l t to interpret, because l i v e r fat levels at any time represent the net outcome of synthesis, degradation and mobilization to and from this organ. Thus, while l i v e r fat was unaltered i n the deprived nibbling rats, i t was s l i g h t l y increased i n the meal-fed ones, as comparison with the corresponding controls revealed. The responses of the nibbling rats i n the present studies agree with the findings of Carter and Phizackerley (1951) and Fidanza and de Cicco (1964). On the basis of t h e i r studies with rats fed d i f f e r e n t levels of f a t , Williams ejt a l . (1959) concluded that generalizations regarding the e f f e c t of pyridoxine deficiency on l i v e r fat are d i f f i c u l t to make. This may be due to the fact that fatty metamorphosis of the l i v e r i n pyridoxine d e f i c i e n t rats appears to be f o c a l i n d i f f e r e n t l i v e r lobes -v^Van increase in fat can be missed by taking just one lobe for measurement (French and Marsh, 1964). A decrease i n body fat stores may r e f l e c t a similar a l t e r a t i o n in synthesis. Sullivan et a l . (1971) showed that hepatic lipogenesis reached a maximum 5 hours after meal-i n i t i a t i o n and a minimum after 24 hours. In the present study, lipogenesis was tested over a 2-hour period s t a r t i n g with the i n i t i a t i o n of feeding. This may not coincide with the maximum lipogenic rates expected at the 5-hour point. However, i t was assumed that, a l l other effects being equal, any differences observed between the deprived and the control rats would be due to pyridoxine. In vivo measurement of lipogenesis from l a b e l l e d glucose in the present investigation supports the contention that pyridoxine deprivation suppresses o v e r a l l f a t t y acid production in the intact r a t , regardless of the manner i n which i t i s fed. However, i t should be emphasized that the effects of the deficiency were more pronounced in the nibbling animals than in the meal-fed ones. It i s possible that the nibbling animals ate more of the d e f i c i e n t d i e t , thus exhibiting the e f f e c t of deficiency to a greater extent. Furthermore, the imposition of intermittent feeding appeared to have diminished the differences between the deprived and the control rats i n the degree of f a t t y acid l a b e l l i n g in the l i v e r and the epididymal adipose tissue. Indeed, the rates of -^^ C incorporation into the f a t t y acids of these tissues exhibited a tendency to exceed those observed i n the controls when the d e f i c i e n t rats were meal-fed. This raises serious questions regarding the v a l i d i t y of using tissues from certain locations as indicators of t o t a l lipogenesis in the intact animal, since variations among tissues i n innervation, blood supply and enzyme concentration may be important factors to consider, especially in the adipose tissue (Shafrir and Wertheimer, 1965). The present observations on in vivo lipogenesis i n the nibbling pyridoxine-deprived rats are not i n agreement with 54. those of Desikachar and McHenry (1954). These authors found that 1 4 C incorporation into carcass f a t t y acids proceeded at equal rates i n deoxypyridoxol-treated and pair-fed control rats following intubation with glucose-U-"^C. However, the use of a fat - f r e e diet and the antimetabolite renders the interpretation of the data of Desikachar and McHenry (1954) in r e l a t i o n to the present study rather d i f f i c u l t . On the other hand, part of the present findings i s in accord with the results of Williams and Pertel (1964), who reported that the incorporation of la b e l l e d acetate into the f r a c t i o n of l i v e r l i p i d s not precipitated by dig i t o n i n ( t r i g l y c e r i d e s and f a t t y acids) was considerably decreased i n the nibbling pyridoxine-deprived r a t s , as comparison with pair-fed and pair-weighed controls revealed. A reduction in t o t a l f a t t y acid synthesis i n the intact animal may not necessarily r e f l e c t a decrease i n the a b i l i t y of i t s tissues to synthesize f a t . In v i t r o measurements are valuable in this respect, because they provide a f a i r l y complete inventory of the enzymes present (Favarger, 1965). Therefore, the effects of pyridoxine deficiency on lipogenesis by l i v e r s l i c e s and epididymal adipose tissue segments were also investigated. On the basis of observations made on l i v e r s l i c e s from fasted-refed nibbling rats, i t may be stated that pyridoxine deficiency decreases the lipogenic p o t e n t i a l of th i s tissue. This conclusion agrees with the in_ vivo data obtained in the present studies and with the in v i t r o and i n vivo measurements reported by Williams and Pertel (1964) and by Sabo et_ a l . (1971). On the other hand, i f i t i s assumed that the actual status of the nibbling animals i s better represented by l i v e r s l i c e s from rats that had access to food u n t i l death, i t would appear that pyridoxine deprivation leads to the stimulation of hepatic f a t t y acid synthesis to levels greater than those observed in the c a l o r i c a l l y r e s t r i c t e d controls. This contradiction seems to be due to the difference i n feeding frequency between the deprived and the control animals, since the imposition of meal-feeding on the former rats resulted i n lipogenic rates comparable with tho.se observed in the l a t t e r . In normal rats, L e v e i l l e (1970) observed that the lipogenic superiority of l i v e r tissues from the meal-fed animals was evident only i f the nibbling ones were fasted and refed. In any case, the quantitative contribution of the l i v e r to t o t a l f a t t y acid production i n rodents i s small compared with that of the adipose tissue (Jansen_et _a l . , 1966; L e v e i l l e , 1967a). Thus, i t can be calculated from the results of Experiment I that the contribution of the l i v e r to t o t a l f a t t y acid synthesis did not exceed 5-10% of the f a t t y acid r a d i o a c t i v i t y recovered from the whole animal. According to Hartman et a l . (1971), the metabolic a c t i v i t y of adipose tissue preparations should be expressed in terms of the number of adipocytes present, rather than the fresh weight of the tissue or i t s l i p i d content.. This i s especially true when comparisons are made between tissues from animals i n d i f f e r e n t n u t r i t i o n a l states, since the l i p i d content and/or the adipocyte number per unit weight may be altered. Although the DNA content of the adipose tissue does not necessarily represent a t a l l y of the adipocytes present (Hubbard and Matthew, 1971; Rodbell, 1964), i t s t i l l offers more meaningful information than fresh tissue weight. Therefore, the rates of lipogenesis observed in the present studies were expressed i n r e l a t i o n to the DNA content of the adipose tissue preparations used. Pyridoxine deficiency i n nibbling rats led to the suppression of lipogenesis in adipose tissue preparations incubated with glucose and i n s u l i n , as comparison with the r e s t r i c t e d controls showed. This was in agreement with the results obtained in intact rats. The data are not shown here i n d e t a i l , but the expression of results i n terms of fresh tissue weight would lead to the opposite conclusion, which would then agree with the findings of Huber et a l . (1964) in nibbling rats. Clearly, this r e f l e c t s the fact that the DNA content of the tissue obtained from the deprived rats was almost doubled, with the li k e l i h o o d of the number of adipocytes per unit weight being increased, too. This does not necessarily imply that the t o t a l number of adipocytes i n the epididymal adipose tissue pads i s increased in pyridoxine deficiency; an apparent increase i n c e l l number per unit weight may result from a decrease i n c e l l size (decreased fat content) alone. In any case, the apparent depression of lipogenesis i n the nibbling pyridoxine-deficient rats may be due to differences i n the mode of feeding between these and the c a l o r i c a l l y r e s t r i c t e d controls. This view i s supported by the observed increase in lipogenesis from glucose in adipose tissue from meal-fed deprived animals. While differences between the deprived and the control 57. goups were readi l y seen when adipose tissue preparations were incubated with glucose, this was not the case with acetate. Favarger (1965) considered data obtained with the l a t t e r compound of lesser value than those arrived at with glucose, because of differences in the rates of exogenous acetate entry into tissues, i t s a c tivation into acetyl CoA and the d i l u t i o n of the l a t t e r with endogenous acetyl G.oA . Furthermore, the p o s s i b i l i t y that the size of tissue acetate pool(s) may be decreased i n pyridoxine deficiency has been raised (Lupien et a l . , 1969). A reduction in the size of the endogenous acetate pool(s) would lead to decreased isotope d i l u t i o n and thus increased apparent lipogenesis from exogenous la b e l l e d acetate. It i s appropriate at t h i s point to speculate on the causes of the observed increase in the lipogenic potential of adipose tissue preparations obtained from the meal-fed pyridoxine deprived rats. As the data presented in Table 11 show, the DNA l e v e l of this tissue was considerably elevated i n the deficiency state, while i t s l i p i d content was unaltered. The r a t i o of l i p i d to DNA, which provides a rough indicat i o n of c e l l size, was 0.87 mg/ug in the deprived rats and 1.56 mg/ug in their controls. This suggests that adipocyte size may well be smaller in the d e f i c i e n t rat than in the control. This would increase the s e n s i t i v i t y of c e l l s to the i n s u l i n which was added to the incubation medium used in the present work (Salans and Dougherty, 1971; Salans et_ a l . , 1968). An increase i n the s e n s i t i v i t y of adipose tissue preparations from the nibbling pyridoxine def i c i e n t rats to i n s u l i n was previously observed by Huber e_t al_. 58. (1964). Because of the dependence of reductive fa t t y acid synthesis on the a v a i l a b i l i t y of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) (Ba l l , 1966; Masoro, 1962), the a c t i v i t y of key enzymes concerned with the production of t h i s cofactor were evaluated i n the l i v e r and the adipose tissue. The a c t i v i t i e s of glucose-6-phosphate dehydrogenase and malic enzyme, which are considered v a l i d indicators of lipogenesis ( L e v e i l l e , 1972), were decreased i n both the l i v e r and the adipose tissue of pyridoxine-deprived rats. This was true whether the animals were nibbling or meal-fed, although the magnitude of the decrease below the controls was smaller in the l a t t e r than in the former animals. These findings are in agreement with published data on nibbling rats showing a general tendency for tissue dehydrogenase a c t i v i t y to decrease i n pyridoxine de f i c i e n t rats (French and Marsh, 1964; Sabo et a l . , 1971). The results observed in the present study are consistent with the o v e r a l l suppression of lipogenesis seen in vivo(Experiment I ) . It should be remembered, however, that enzyme determinations in v i t r o represent maximal rates that may not exist i n intact tissues, since enzyme a c t i v i t y i n animals i s modulated by a variety of factors. Furthermore the observed rates of glucose-6-phosphate dehydrogenase and malic enzyme alone seem to be capable of generating enough NADPH to sustain the lipogenic a c t i v i t y observed i n v i t r o (Experiment I I ) . On the basis of the o v e r a l l findings of the present investigation, i t seems clear that pyridoxine deficiency suppresses lipogenesis i n the intact rat. This conclusion i s borne out by measurement of lipogenesis from glucose i n vivo, as well as the a c t i v i t i e s of key enzymes involved with NADPH production. The data also emphasize the importance of considering the differences i n the mode of feeding when tissue from deficient and control rats are compared. It i s not clear yet why pyridoxine deficiency affects lipogenesis i n the manner seen i n this and i n other investigations, and this phenomenon cannot be explained i n r e l a t i o n to the known functions of pyridoxal phosphate enzymes. Whether the response of lipogenesis to pyridoxine deprivation i s brought about by changes i n i n s u l i n a v a i l a b i l i t y (Huber e_t a l . , 1964) or not i s contingent upon consideration of the feeding pattern of the control animals used, since meal-feeding i s known to increase i n s u l i n production in normal rats (Wiley and L e v e i l l e , 1970). Therefore, further studies on this aspect and on adipose tissue c e l l u l a r i t y and adipocyte size are warranted. 60. 6. Bibliography Angel,J.F. and Sabry,Z.I. 1969. Lipogenesis in pyridoxine repletion. Proc. Can. Fed. B i o l . 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Wizgird,J.P., Greenberg,L.D. and Moon,H.D. 1963. Hepatic lesions in pyridoxine-deficient monkeys. Fed. P r o c , 22:483. Yeh,S.D. and Weiss,B. 1963. Behavioral thermoregulation during vitamin Bfi deficiency. Am. J. Physiol., 205:857. APPENDIX A (EXPERIMENT II.) Table A l . Cumulative food consumption and body weight gain of pyridoxine-deprived and control rats. Values are expressed as g/rat. Time Deprived, meal -f ed'*' Control, r e s t r i c t e d 2 (weeks) Food consumed Wt. gain Food consumed Wt. gain 1 23.3± 1.535 16.3±3.79 23.3 23.7±3.98 2 62.1± 3.44 14.7±3.61 62.1 19.3+2.56 3 95.1± 4.94 22.913.67 95.1 27.1±1.86 4 131.2± 8.52 32.9±4.42 131. 2 34.9±2.31 5 178.2± 11.28 41.4±6.68 178.2 41.011.56 6 210.6± 15.02 46.0+3.99 210.6 53.6±2.01 Time Deprived, 3 nibbler Control, 4 r e s t r i c t e d (weeks) Food consumed Wt. gain Food consumed Wt. gain 1 57.»7± 2.60 24.1±2.81 57.7 28.4±4.31 2 126.3± 4.68 39.0±2.15 126.3 39.6±2.85 3 168.2± 6.11 49.6±2.45 •168. 2 57.7±2.82 4 220.0± 8.36 51.1±3.98 220. 0 66.113.50 5 240.4±22.39 56.9±3.27 240.4 74.7+3.29 6 308.4±11.22 57.6±3.86 308.4 93.912.61 1. Meal-fed the pyridoxine-free diet for 6 weeks after weaning. 2. Fed the complete diet at the l e v e l consumed by the deprived-meal-fed rats. 3. Fed the pyridoxine-free diet ad libitum for 6 weeks. 4. Fed the complete diet at the l e v e l consumed by the deprived nibbling rats. 5. Mean standard error of the mean for 8 rats. Table A2. Total food consumption, t o t a l weight gain and o v e r a l l feed e f f i c i e n c y i n pyridoxine-deprived and control rats. Group Food consumption Weight gain Feed e f f i c i e n c y g/rat g/rat g gain/g food consumed Deprived, meal-fed 1 211 ± 15.0 6 46.0 ± 7.44 0.24 ± 0.010 2 Control, r e s t r i c t e d 211 53.6 ± 2. 01.: 0.25 ± 0.010 - 7 ns ns Deprived, n i b b l e r 4 308 ± 11.2 57.6 ± 3.86 0.19 ± 0.012 Control, r e s t r i c t e d 5 308 93.9 ± 2.61 0.30 ± 0.008 P - <0.001 <0.001 1. Meal-fed the pyridoxine-free diet for 6 weeks after weaning. 2i Fed the complete diet at the l e v e l consumed by the deprived meal-fed rats. 3. P r o b a b i l i t y of the difference between means occurring by chance. 4. Fed the pyridoxine-free diet ad_ libibum for 6 weeks. 5. Fed the complete diet at the l e v e l consumed by the deprived nibbling rats. 6. Mean ± standard error of the mean for 8 rats . 7. Not s i g n i f i c a n t . Table A3 Relative sizes of the l i v e r and the epididymal adipose tissue in pyridoxine-deprived and control r a t s . Group F i n a l body weight Liver Adipose tissue g/rat g/lOOg body wt g/lOOg body wt. Deprived, meal-fed 1 190 ± 4.56 3.65 ± 0.13 0.50 ± 0.08 2 Control, r e s t r i c t e d 98 ± 1.4 3.52 ± 0.06 0.50 ± 0.02 3 P ns 7 ns ns Deprived, n i b b l e r 4 100 ± 4.1 4.56 ± 0.41 0.58 ± 0.05 Control, r e s t r i c t e d 5 137 ± 2 . 7 3.37 ± 0.10 0.90 ± 0.06 P <0.001 <0. 02 <0. 01 1. Meal-fed the pyridoxine-free diet for 6 weeks afte r weaning. 2. Fed the complete diet at the l e v e l consumed by the deprived meal-fed rat s . 3. Probabil i t y of the difference between means occurring by chance. 4. Fed the pyridoxine-free diet ad libitum for 6 weeks. 5. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 6. Mean ± standard error of the mean for 8 rats. 7. Not s i g n i f i c a n t . APPENDIX B (EXPERIMENT III.) 82. Table Bl Cumulative food consumption and body weight gain of pyridoxine-deprived and control rats. Values are expressed as g/rat. Time (weeks) Deprived, meal- fed 1 Control, 7 r e s t r i c t e d Food consumed Wt. gain Food consumed Wt. gain 1 20. 2± 0.92 -1. 4± 0.96 20. 2 -2.1+0.58 2 54. 3± 1.91 11. 6± 1. 43' 54. 3 13.2±0.86 3 92. 9± 3.15 24. 2± 1.52 92^9 • 23.8±1.08 4 131.8± 4.83 32. i±: If 86 131. 8 31.6±1.28 5 169.8± 6.83 39. 1± 2. 56 169. 8 40.5±1.29 6 209.0± 7.67 44. 7'± 1.84 209.0 48.6±1.98 Time (weeks) Deprived, 3 nibbler Control, 4 r e s t r i c t e d Food consumed Wt. gain Food consumed Wt. gain 1 44.6± 1.51 27.8± 0.98 44.6 25.2±0.70 2 99.2± 4.40. 39,9± 1.82 99. 2 45.0±0.93 3 153.2± 7.71 50.6± 2.64 153. 2 58.6±1.02 4 212.0±10.52 62. 2± 3. 38 212. 0 79.0±0.65 5 260.8±11.79 63.4± 2.06 260.8 87.0±0.89 6 297.3±12.6 64.4± 3.17 297. 3 9 3 . O i l . 4 3 1. Meal-fed the pyridoxine-free diet for 6 weeks after weaning. 2. Fed the complete diet at the l e v e l consumed by the deprived-meal-fed rats. 3. Fed the pyridoxine-free diet ad_ libitum for 6 weeks. 4. Fed the complete diet at the l e v e l consumed by the deprived nibbling rats. 5. Mean±standard error of the mean for 8 rats. Table B2. Total food consumption, t o t a l weight gain and o v e r a l l feed e f f i c i e n c y i n pyridoxine-deprived and control rats. Group Food consumption Weight gain Feed e f f i c i e n c y g/rat g/rat g gain/g food consumed Deprived, meal-fed 1 209 ± 7.76 44.7 ± 1.84 0.2 2 ± 0.041 2 Control, r e s t r i c t e d 209 48.6 ± 1.98 0.23 ± 0.010 3 P - n s7 ns 4 Deprived, nibbler 297 ±12.6 64.4 ± 3.17 0.20 ± 0.017 Control, r e s t r i c t e d 5 297 93.0 ± 1.43 0.30 ± 0.007 P - <0.001 <0.001 1. Meal-fed the pyridoxine-free diet for 6 weeks after weaning. 2. Fed the complete diet at the l e v e l consumed by the deprived meal-fed rat s . 3. Pr o b a b i l i t y of the difference between means occurring by chance. 4. Fed the pyridoxine-free diet ad libitum for 6 weeks. 5. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 6. Mean ± standard error of the mean for 8 rats . 7. Not s i g n i f i c a n t . Table B3. Relative sizes of the l i v e r control r a t s . and the epididymal adipose tissue i n pyridoxine-deprived and Group F i n a l body weight Liver Adipose tissue g/rat g/lOOg body wt. g/lOOg body wt. Deprived, meal-fed"*" 108 ± 2.66 3. 3 ± 0.05 .0.78 ± 0.05 2 Control, r e s t r i c t e d 114 ± 2.9 3.4 ± 0.09 0.83 ± 0.05 3 P ns7 ns ns Deprived, n i b b l e r ^ 114 ± 4.2 3.4 ± 0.10 0.64 ± 0.05 Control, r e s t r i c t e d ^ 142 ± 2.8 3v0 ± 0.06 1.14 ± 0.12 P <0.001 <0.01 <0.01 1. Meal-fed the pyridoxine- free diet for 6 weeks after weaning. 2. Fed the complete di e t at the l e v e l consumed by the deprived meal-fed r a t s . 3. P r o b a b i l i t y of the difference between means occurring by chance. 4. Fed the pyridoxine-free diet ad libitum for 6 weeks. 5. Fed the complete diet at the l e v e l consumed by the deprived nibbling r a t s . 6. Mean ± standard error of the mean for 8 ra t s . 7. Not s i g n i f i c a n t . 

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