333

Biochimica et Biophysica Acta, 451 (1976) 333--341 © Elsevier/North-Holland Biomedical Press

BBA 28086

METABOLISM OF PYRIDOXINE IN THE LIVER OF VITAMIN B-6-DEFICIENT RATS

GEORGE P. TRYFIATES and FRANK L. SAUS

Department of Biochemistry, West Virginia University School of Medicine, Morgantown, W. Va. 26506 (U.S.A.) (Received May 10th, 1976)

Summary The metabolism of [6-3H]pyridoxine • HC1 was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding ad libitum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 #Ci of [6-3H]pyridoxine • HC1 and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H *) columns and the percent radioactivity for each vitamin c o m p o u n d was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days. In vitamin B-6-deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5'-P and pyridoxal 5'-P reached 36 and 44% levels, respectively. Pyridoxine 5'-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5'-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5'-P was falling, pyridoxamine 5'-P was linearly synthesized reaching a plateau level (62%) in 5 days which also remained unchanged through 7 days. Some pyridoxal was also formed (7% at 1 h) which by 12 h had dropped to a plateau low level {3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5'-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [3H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an Abbreviation:

[6 -3 H] pyridoxine, 3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridine.

334 extended label retention period, (b) a rapid pyridoxal 5'-P synthesis, and (c) a continunous synthesis (and accumulation) of pyridoxamine 5'-P which is not utilized or further metabolized.

Introduction The absence of vitamin B-6 or its derivatives from the diet of animals results in characteristic deficiency symptoms which lead to premature death. During vitamin B-6 deficiency the concentration of the cofactor in liver is decreased by at least 2.5 times [1] and enzymatic activities as well as hormonal enzyme induction are altered [1--7]. Investigators as early as 1943 reported inhibition of tumor growth in the absence of dietary pyridoxine [8,9]. It was further shown that the growth of a spectrum of tumors was impaired by pyridoxine depletion [10], and vitamin antagonists (i.e., 4-deoxypyridoxine) alone or in combination with other agents exerted marked inhibitory effects on tumor growth [11--14]. Recent studies in our laboratory have shown that vitamin B-6 is also needed for the growth of several lines of Morris hepatomas. Tumors grown in pair-fed control Buffalo rats were 2--3 times heavier than those grown in animals fed the same diet without pyridoxine [7,15--17]. Attempts to learn of the causative factors regarding these observations led us to studies on the interconversions of tritiated pyridoxine in the liver of vitamin B-6 deficient animals. Methods Buffalo strain, 32-days-old female rats were placed upon arrival in individual cages in a windowless, air-conditioned room with constant temperature (74 ° F) in the animal quarters. The lights were off from 8.00 p.m. to 8.00 a.m. Vitamin B-6 deficiency was induced by feeding a diet lacking pyridoxine but otherwise optimal [18]. Animals of the same weight and age were matched in pairs. One animal of each pair was fed the deficient diet ad libitum while the other (control animal) received daily 6 g of the same diet supplemented with pyridoxine [16]. Caloric intake was restricted to this a m o u n t because of previous experimental observations. Similar growth patterns (Fig. 1) and daily food consumption were seen if the a m o u n t of diet given daily the pair-fed " p a r t n e r " was based on the total consumed the previous week by the deficient " p a r t n e r " divided by seven [1,7]. Diets were prepared commercially according to French [19] by contract with Teklad Mills, Inc., Madison, Wisc. Animals were injected intraperitoneally with 33 pCi (~4 pg) of [6-3H]pyri doxine • HC1, specific activity 1.7 Ci/mmol. They were sacrificed by decapitation at intervals of 2, 5, 15, 30 and 60 min; at 3, 6, 12 and 24 h, and at 3, 5, and 7 days following injection and after 55 days on the respective diets (Fig. 1).

Extraction of vitamin B-6 compounds Since analysis of a large number of samples was required livers, when necessary, were frozen and stored in vacuum and under a nitrogen atmosphere at

335

lo@

i:ii/ I 20r

I I

01 30

I 40

] I 60 60 Age (doys)

L 70

1 80

1 90

Fig. 1. G r o w t h c u r v e s of Buffalo rats fed ad l i b i t u m a diet d e f i c i e n t in p y r i d o x i n c or pair-fed t h e s a m e d i e t s u p p l e m e n t e d w i t h p y r i d o x i n e . O p e n circles, d e f i c i e n t animals.

--20°C. Storage for several weeks under these conditions did not result in breakdown of the vitamin compounds [20]. Upon removal, livers were washed with cold water, blotted and vitamin B-6 compounds extracted according to the method of Johansson et al. [21].

Separation of vitamin B-6 compounds Radioactively labeled vitamin B-6 compounds were separated using Dowex 50W-X8(H ÷) ion exchange columns, 100--200 mesh. The resin was washed with 3 M HC1, water, and then with 0.05 M ammonium formate buffer, pH 4.25, until the pH fell to 4.25. Columns of 40.5 × 1.1 cm (internal diameter) were washed with 100 ml of formate buffer (above) prior to sample application. 10 ml of clear filtrate were layered onto each column and, after adsorption, pyridoxal 5'-P and pyridoxine 5'-P were eluted using 100 ml of formate buffer. The other vitamin compounds were separated using linear gradient elution of increasing pH as described by Johannson et al. [21]. Radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer using 1 ml from each fraction and 10 ml of Bray's solution [22]. A typical elution run is shown in Fig. 2 {dotted line radioactivity). Recoveries of 97--99% were obtained routinely. Standard solutions of vitamin B-6 compounds were chromatographed individually and also as a mixture on Dowex-(H*) columns. Separation of all six compounds is shown in Fig. 2. Further identification of each peak eluting was made using high voltage electrophoresis (Savant) [23] and thin-layer chromatography in silica gel [24]. Pyridoxal 5'-P was also identifed by its yellow color at pH 4 . 2 5 .

336

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200

300

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500

600

700

rlqi

Fig. 2. C h r o m a t o g r a p h i c s e p a r a t i o n of a m i x t u r e o f s t a n d a r d v i t a m i n B 4 c o m p o u n d s a n d a r a d i o a c t i v e l y l a b e l e d fiver s a m p l e on a D o w e x - [ H ÷] c o l u m n . T h e g r a p h s h o w s r e p r e s e n t a t i v e results. T h e liver s a m p l e was a c i d - e x t r a c t e d 5 rain a f t e r the i n j e c t i o n of 6 [ 3 H ] p y r i d o x i n e • HCI t o d e f i c i e n t a n i m a l s (see also Fig. 3). 10 m l of s a m p l e w e r e c h r o m a t o g r a p h e d as d e s c r i b e d in M e t h o d s . T h e c o n c e n t r a t i o n of e a c h v i t a m i n B-6 c o m p o u n d ( s t a n d a r d ) was 3 0 p g p e r ml. E l u t i o n o f v i t a m i n c o m p o u n d s w a s a s follows (in t h e o r d e r of a p p e a r a n c e of each p e a k , f r o m the left): p y r i d o x a l 5'-P; p y r i d o x i n e 5'-P; p y r i d o x a m i n e 5'-P; p y r i d o x a l ; p y r i d o x i n e ; p y r t d o x a m i n e . O t h e r details in M e t h o d s section. - - , standards; ...... , radioactivity.

Determination of enzyme activities Liver activity levels of pyridoxal kinase (ATP:pyridoxal 5'-phosphotransferase, EC 2.7.1.35) and pyridoxine 5'-phosphate (or pyridoxamine 5'-phosphate) oxidase (pyridoxamine 5'-phosphate: O5 oxidoreductase (deaminating), EC 1.4.3.5) of vitamin B-6 deficient and control rats were measured as follows. Livers were quickly removed, washed with cold water, blotted, minced with scissors and homogenized in two volumes of 0.075 M potassium phosphate buffer, pH 6.8. The homogenate was filtered through four layers of cheesecloth and the filtrate centrifuged for 30 min at 27 000 X g and 4 ° C in the Sorval centrifuge. The clear supernatant was diluted with one volume of cold distilled water (made to pH 6.8 with 0.1 M KOH) and used for enzyme activity and protein determinations. Pyridoxal kinase activity was measured as described by Neary and Diven [25]. Pyridoxine 5'-phosphate, (pyridoxamine 5'-phosphate) oxidase activity was measured by the method of Wada and Snell [26]. The a m o u n t of pyridoxal 5'-P formed was determined by the phenylhydrazine reaction, as described [26]. One unit of activity of pyridoxal kinase is that amount of protein which catalyzes the formation of 1 nmol of pyridoxal 5'-P per min at 37°C [25]. A unit of oxidase activity is that amount of protein which catalyzes the formation of 1 nmol of pyridoxal 5'-P per h at 37°C using pyridoxine 5'-P as substrate [26]. Protein was measured by the Biuret procedure [27]. Materials Pyridoxal • HCI, pyridoxamine • 2HC1 and pyrodixal 5'-P (95% purity) were purchased from Sigma Chemical Company, St. Louis, Mo. Pyridoxine • HC1

337 (98% purity) and pyridoxamine 5'-P monohydrochloride (97%) were purchased from Aldrich Chemical Company, Milwaukee, Wisc. Pyrodoxine 5'-P was prepared as described by Peterson and Sober [28] from pyridoxamine 5'-P monohydrochloride and sodium nitrite. After standing overnight in the presence of urea for destruction of nitrous acid pyridoxine 5'-P was separated on a Dowex(H*) column as described above. [6-3H]Pyridoxine • HC1 was purchased from Amersham/Searle, Arlington Heights, Ill. The resin was obtained from BioRad Laboratories, Rockville Centre, N,Y. Buffalo strain rats were purchased from Simonsen Laboratories, Gilroy, Calif. Results

Fig. 1 shows growth curves of animals fed the pyridoxine deficient and supplemented diets. The growth of animals fed the deficient diet reached a plateau approximately after 3 weeks while those fed the sufficient diet began to level off after 5 weeks. Similar growth patterns were also reported earlier [1]. Fig. 2.shows representative elution patterns of (a) a mixture of standard vitamin B-6 compounds and (b) a radioactively labeled liver sample extracted 5 rain following the administration of [6-3H]pyridoxine to deficient animals (see also Fig. 3). The graph illustrates the separation of all six vitamin compounds.

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Fig. 3. T i m e - c o u r s e o f specific i n c o r p o r a t i o n of t r i t i a t e d p y r i d o x i n e a n d its m e t a b o l i t e s b y rat liver cells. (Since i n c o r p o r a t i o n o f labeled p y r i d o x i n e is e x p r e s s e d p e r m l o f liver e x t r a c t a n d t h e acid e x t r a c t i o n was b a s e d on the w e i g h t o f t h e Uver ( M e t h o d s ) , the i n c o r p o r a t i o n s h o w n is specific i n c o r p o r a t i o n ) . F o l l o w ing the i n t r a p e r i t o r ~ a l a d m i n i s t r a t i o n of [ 6 - 3 H ] p y r t d o x i n e . HC1 • v i t a m i n B-6 c o m p o u n d s w e r e acide x t r a c t e d at t h e i n d i c a t e d t i m e p e r i o d s a n d r a d i o a c t i v i t y in e a c h s a m p l e m e a s u r e d ( M e t h o d s ) . c p m p e r m l of e x t r a c t ( s a m p l e ) are s h o w n . S a m p l e s w e r e e x t r a c t e d at 2, 5, 15, 30, a n d 6 0 rain; 3, 6, 12, 24 h; 3, 5 a n d 7 d a y s f o l l o w i n g label i n j e c t i o n . T h e n a t u r a l l o g a r i t h m s (In) o f t i m e intervals are used in the graphs. • -', pair-fed a n i m a l s ; × × , d e f i c i e n t animals.

338 The correspondence of elution of standard vitamin B-6 compounds (solid line) with the elution of the extracted radioactively labeled liver vitamin forms (dotted line) is shown. It was mentioned (Methods) that individual peaks were further identified by other methods [23,24] and by chromatographing on a Dowex-H ÷ each compound separately. Pyridoxal 5'-P was also identified by its reaction with phenyl hydrazine [26]. Fig. 3 is a time-course graph showing specific incorporation of tritiated pyridoxine and its metabolites by liver cells of vitamin B-6 deficient and control animals. Lack of dietary pyridoxine resulted in slightly delayed, approximately two-fold greater label uptake by the deficient animals. Further, practically all of the incorporated specific radioactivity (interconverted, Fig. 4) was retained by the deficient animals. However, the incorporated label in control animals began to drop after 30 min reaching low values, even at 3 hours. Maximal uptake by control and deficient animals occurred after 30 and 60 min, respectively, following injection of [ 6-3H ] pyridoxine. Fig. 4 shows the metabolic interconversions of tritiated pyridoxine in the liver of deficient animals. Lack of dietary pyridoxine caused rapid disappearance {conversion) of labeled pyridoxine, rapid synthesis of pyridoxine 5'-P and

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Fig. 4. M e t a b o l i c i n t e r c o n v e r s i o n s o f [6 -3 II] p y r i d o x i n e • HCI in t h e liver o f v i t a m i n B - 6 - d c f i c i e n t B u f f a l o rats: T i m e - c o u r s e o f d i s t r i b u t i o n of s p e c i f i c r a d i o a c t i v i t y . V i t a m i n B-6 c o m p o u n d s w e r e e x t r a c t e d f r o m liver at t h e i n d i c a t e d t i m e i n t e r v a l s a n d c h r o m a t o g r a p h i c a l l y s e p a r a t e d o n D o w e x - [ H ÷] c o l u m n s . P e r c e n t r a d i o a c t i v i t y o f e a c h s e p a r a t e d p e a k ( c o m p o u n d ) at t h e i n d i c a t e d t i m e (see Fig. 3) w a s c a l c u l a t e d f r o m t h e t o t a l c p m u n d e r the p e a k ( f r a c t i o n s c o m p r i s i n g e a c h p e a k w e r e p o o l e d ) a n d t h e t o t a l r a d i o a c t i v i t y c h r o m a t o g r a p h e d . R e c o v e r i e s o f 9 7 - - 9 9 % w e r e r o u t i n e l y o b t a i n e d . S a m p l e s of 10 m l w e r e c h r o m a t o g r a p h e d . T o t a l r a d i o a c t i v i t y c h r o m a t o g r a p h e d at e a c h t i m e i n t e r v a l m a y be c a l c u l a t e d b y r e f e r r i n g t o t h e d a t a o f Pig. 3. O t h e r d e t a i l s in t h e M e t h o d s s e c t i o n a n d also F i g u r e 3. S y m b o l s : X . . . . . . X, [ 3 H | p y r i doxine: ao, [ 3 H ] p y r i d o x i n e 5'-P; :: c., [ 3 H ] p y r i d o x a l 5'-P: rj ~;, [ 3 H ] p y r i d o x a m i n e 5'-/'; . . . . . . . . , [311]pyridoxal;tJ ...... r: [3H]pyridoxamine.

339

TABLE

I

ItEPATIC ACTIVITY OF DEFICIENCY

LEVELS

OF ENZYMES

INVOLVED

IN VITAMIN

B-6 M E T A B O L I S M .

EFFECTS

A v e r a g e v a l u e s -+ S . D . a r e g i v e n . N u m b e r in parentheses is n u m b e r o f l i v e r s a s s a y e d . A v e r a g e l i v e r w e i g h t s w e r e 5.1 -+ 0 . 8 g a n d 4 . 6 -+ 0 . 5 g f o r t h e c o n t r o l and deficient animals, respectively. ........................................................................ Animal Pyridoxal kinase group units/rain per mg protein ..............................................................................

Pyridoxine 5'-phosphate u n i t s / h p e r m g protein

Control

9 . 2 +- 0 . 8 ( 3 ) 1 4 . 0 +- 1.1 ( 4 )

1.9 +- 0 . 3 ( 5 ) 0 . 6 *- 0 . 1 ( 4 ) .......................................................................

Deficient

oxidase

practically simultaneous synthesis of pyridoxal 5'-P. Synthesis of these two phosphorylated vitamin derivatives was so fast that by 2 min pyridoxal 5'-P had reached a level of 44%, pyridoxine 5'-P had dropped to 36% and 93% of the injected label was metabolized. Pyridoxine 5'-P dropped to a minimal, steady level (3%) by 15 min while, on the other hand, pyridoxal 5'-P rose proportionally at this time to a high of 79%. It then began to drop in a linear fashion, reaching a constant level of 29% by 5 days. Synthesis of pyridoxamine 5'-P began early (8% at 2 min) increasing continually and practically in proportion to the rate of disappearance of pyridoxal 5'-P. Some pyridoxal was also synthesized reaching a high level of 8% at 60 min and decreasing thereafter to low levels. Lack of dietary pyridoxine effected an accumulation and retention of pyridoxamine 5'-P which was not further metabolized. Hepatic activity levels of enzymes involved in vitamin B-6 metabolism are shown in Table I. The level of pyridoxal kinase (the enzyme responsible for the phosphorylation of ingested dietary pyridoxine) is over 3 times higher in control than in deficient animals while that of pyridoxine 5'-phosphate oxidase is 1.5 times greater in the state of deficiency. These results show that, although the kinase level in the state of deficiency is lower, the animal endeavors to convert the available pyridoxine to a utilizable form, i.e., pyridoxal 5'-P. The coenzyme is further metabolized (Fig. 4) to pyridoxamine 5'-P presumably via transaminase reactions [26,29]. Discussion

The metabolic interconversions of tritiated pyridoxine in the state of vitamin B-6 deficiency may be better understood in terms of the following scheme [26, 29,30l: I

2

3

Pyridoxine -+ pyridoxine 5'-P -~ pyridoxal 5'-P ~ pyridoxamine 5'-P 4 $5 pyridoxal The data presented demonstrate that synthesis of pyridoxal 5'-P (reaction 2) from pyridoxine 5'-P via reaction 1 is of prime importance to the deficient animal. Both reactions 1 and 2 are very fast. Reaction 3 (pyridoxamine 5'-phosphate oxidase) does not appear to occur in the deficient state (Fig. 4), at least during the experimental period studied. Conversion of pyridoxal 5'-P to pyri-

340 doxamine 5'-P (reaction 4) occurs in practically linear fashion following maximal (80%) synthesis of the former {Fig. 4). An equilibrium between these two vitamin B-6 forms was reached after 5 days with pyridoxamine 5'-P predominating in a ratio of 2 : 1. The reverse was reported for normal {non-deficient) mice [30]. In agreement with this report, higher pyridoxamine 5'-P levels were also found in the liver of deficient mice [20]. Johannson et al. [21] reported on the metabolic interconversions of pyridoxine in liver and carcass of normal, non-deficient, mice using tritiated pyridoxine. Maximal synthesis of pyridoxal 5'-P (4 h) followed the early synthesis of pyridoxine 5'-P (40%; 10 min). Appreciable synthesis of pyridoxamine 5'-P occurred at 24 h, increasing slowly up to 7 days. Colombini and McCoy [23[ reported similar findings [21] but with high levels of pyridoxal and pyridoxine [23]. More recently, Johannson etal. [30] reported on the metabolic interconversions of different forms of vitamin B-6 in mouse liver and carcass following the administration of labeled pyridoxamine, pyridoxine 5'-P and pyridoxal 5'-P intravenously. Results obtained with these precursors confirmed previous findings by the same workers [21]. In contrast to the results with control animals [21,23,30], observations with pyridoxine depleted animals show that (a) the metabolic interconversions of tritiated pyridoxine are highly accelerated; {b) the order of synthesis of the vitamin B-6 compounds (i.e., order of appearance of label) is not altered, and (c) pyridoxamine 5'-P is accumulated {Fig. 4). The high pyridoxal kinase level found in control animals (Table I; ref. 37) presumably reflects the need of the animal to convert available dietary pyridoxine to a (phosphorylated) utilizable form. The finding of a high oxidase level in the state of deficiency may represent a case of product inhibition in vivo. Wada and Snell [26] reported inhibition of the oxidase by high pyridoxal 5'-P levels. The higher liver level of this coenzyme seen in pair-fed control animals [1 ] may in effect exercise an in vivo control over the activity of the oxidase. In comparison with the aforementioned findings [21,23], phosphorylation of labeled pyridoxine was very fast in spite of the reduced kinase level (Fig. 4; Table I). Formation of pyridoxamine 5'-P presumably occurs via reaction 4 or by the reversibility of reaction 3 which has yet to be demonstrated in vivo [26,29,30]. The data strongly suggest that the equilibrium of reaction 4 is well to the right, i.e., to the synthesis of pyridoxamine 5'-P {Fig. 4). This reaction is known to occur with the bound coenzymes during enzymatic transamination [31,32]. In the state of vitamin B-6 deficiency the levels of L-amino acids in blood and liver are increased while liver keto acid levels remain the same [33,34]. Due to unbalanced metabolism in the direction of amino acid catabolism the emergence of an acute need for increased transamination is very likely. Increased transamination could conceivably occur with or without increased apoenzyme levels. It should, however, be mentioned that during progressive vitamin B-6 deficiency the activity of hepatic tyrosine aminotransferase remained significantly high and its response to steroid administration increased linearly as the period of depletion progressed [1]. It is herewith proposed that, because dietary pyridoxine is not supplied, the animal must regenerate pyridoxal 5'-P as rapidly as {enzyme bound) pyridoxamine 5'-P is formed during enzymatic transamination that the process could continue. It is possible that the regeneration of pyridoxal 5'-P from enzymatically bound pyridoxamine 5'-P is not as rapid.

341 In that case, a higher level of the latter coenzyme would be expected upon extraction (Methods) of the vitamin B-6 forms from liver homogenates [35,36]. The slow regeneration (or lack there-of) of pyridoxal 5'-P from (enzymatically bound) pyridoxamine 5'-P could be the causative factor in the observed inhibition of tumor growth in the absence of dietary pyridoxine [7--17].

Acknowledgements This study was supported by USPHS Grant CA13759 from the National Cancer Institute. Acknowledgement is herewith given to Dr. Harold P. Morris for his active support and interest in this work. Thanks are due to Sister Ernestine Davis for typing the manuscript.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

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