vitamin B-12-methionine-deficient diet

225

Biockimica et Biophysica Acta, 497 (1977) 225--233 © Elsevier/North-Holland Biomedical Press

BBA 28183

E F F E C T OF METHIONINE ON THE METABOLISM OF FORMATE AND HISTIDINE BY RATS FED FOLATE/VITAMIN B-12-METHIONINEDEFICIENT DIET

FLORENCE CHIAO and E.L.R. STOKSTAD

Department of Nutritional Sciences, University of California, Berkeley, Calif. 94 720 (U.S.A.) (Received August 25th, 1976)

Summary The metabolism of formate and histidine were compared in rats and in perfused livers of rats on diets deficient in vitamin B-12, methionine, and folic acid. Excretion of formate and formiminoglutamic acid, and the oxidation of [ 2-14C]histidine and [ 14C]formate to 14CO2 were measured. Liver folate levels decreased to 40% of normal on the vitamin B-12- and methionine-deficient diets but the rate of oxidation of histidine to CO2 in the whole animal decreased to 15% of normal. This indicated a reduction in the metabolic activity of the liver folates in vitamin B-12 deficiency. Comparison of formate and histidine catabolism in folic acid deficiency showed that the oxidation of histidine was decreased to 5% of normal but formate oxidation was decreased to only 30% of normal. This indicates that 25% of formate oxidation normally proceeds by a non-folate-dependent pathway.

Introduction Elevated excretion of FIGlu and formate occurs in rats fed a diet low in folate [1,2] or low in both vitamin B-12 and methionine [3--6]. In the case of vitamin B-12 deficiency, the elevated FIGlu excretion has been shown to be due to a decreased oxidation of the metabolite and this can be restored to normal by supplementation with methionine in the absence of vitamin B-12 [ 5,6]. The methionine effect is believed to be due to inhibition by S-adenosylmethionine [7,8] of methylenetetrahydrofolate reductase, the enzyme responsible for the synthesis of methyltetrahydrofolate. This results in an increased proportion of non-methyltetrahydrofolate derivatives and these can participate in folateAbbreviation:

FIGIu, formiminoglutarnicacid.

226 dependent reactions such as the metabolism of FIGlu to glutamic acid. Oxidation of formate can occur via two different pathways. The first involves dehydrogenation of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate by 10-formyltetrahydrofolate: NADP oxidoreductase [9 ]. This enzyme provides a mechanism for the observation that formate oxidation is decreased in folic acid deficiency [10,11]. Evidence also exists showing that formate may be oxidized by catalase [12--14]. In a model system, the oxidation of hypoxanthine by xanthine oxidase produced H202 which in combination with catalase oxidized formate [12,14]. It was also found that catalase levels in liver and kidney were reduced in folate deficiency, and this paralleled the reduction of in vitro oxidation of formate by homogenates of these tissues [15]. The purpose of this investigation was to study the effect of vitamin B-12 and folic acid deficiencies on formate metabolism and to assess the proportion of formate oxidation that occurred via folate-dependent pathways. Experimental Procedure Animals and diets. Male, weanling Sprague-Dawley rats, weighing 50--70 g, were individually housed in stainless steel screen-bottom metabolic cages in a constant temperature room and were given feed and water ad libitum. The soy protein vitamin B-12-free and methionine-low (0.2%) diet had the following composition (g/kg): soy assay protein, 200; glucose monohydrate, 714; corn oil with vitamins A, D, and E, 40; salt mixture [15], 35; watersoluble vitamin premix in glucose monohydrate, 10; choline chloride, 1. The vitamins were supplied in the following amounts (per kg): vitamin A, 15 000 I.U.; vitamin D (viosterol), 2000 units; a-tocopherol acetate, 50 mg; biotin, 0.2 mg; thiamine • HC1, 15 rag; pyridoxine • HC1, 15 mg; calcium pantothenate, 50 mg; niacin • HC1, 50 mg; riboflavin, 15 mg; menadione, 10 mg. The casein-based folic acid-deficient diet had the same composition except that 200 g of casein was used and 10 g of succinylsulfathiazole was added to inhibit intestinal bacterial synthesis of folic acid. When these diets were supplemented, folic acid was added at 5 mg, vitamin B-12 at 100 pg, and methionine at 15 g/kg of diet. Analytical methods. Formiminoglutamic acid was determined by the enzymatic m e t h o d of Tabor and Wyngarden [16], and formate by the enzymatic m e t h o d of Rabinowitz and Pricer [17 ]. Liver folates, after hydrolysis with hog kidney 7-glutamyl carboxypeptidase, were determined by the microbiological m e t h o d of Waters and Mollin [18] using Lactobacillus casei as test organism. Vitamin B-12 was determined by the method of Hutner and Bach [19] using Euglena gracilis. Liver catalase was estimated by the procedure of Beers and Sizer [20], as described in the Worthington Enzyme Manual [21], in which the decomposition of H202 was followed by the decrease in absorbance at 240 nm in a recording spectrophotometer; 1 unit of activity corresponds to 1 pmol of H202 decomposed per min at 25°C. In vivo metabolic studies. The metabolism of [14C]formate and [14C]histidine to respiratory ~4CO2 was studied in animals that were vitamin deficient, as evidenced by a plateau in their excretion of formate and FIGlu. This occurred after 8--12 weeks on the deficient diets.

227 For the study of histidine metabolism, rats were injected intraperitoneally with 2 pCi L-[2-14C]histidine (0.36 gmol in 0.5 ml saline) and then placed in 25-cm diameter glass dessicators which served as metabolic chambers. The chambers were flushed with air (200 ml per min) and 14CO2 was collected by bubbling the air through 20 ml of ethanolamine/ethyleneglycol monomethylether (1 : 1, v/v). Radioactivity in aliquots (0.5 ml) of this solution was determined by scintillation counting. [~4C]Formate oxidation was studied in a similar manner. In this case, rats were fasted overnight before injection with 1 pCi [14C]formate (0.74 mmol in .0.6 ml saline). In some experiments, rats were preinjected with L-methionine (250 pmol) 15 min prior to injection with labeled compounds. Perfusion studies. The liver perfusion method employed in the present study was the same as that described by Buehring et al. [22]. The perfusion media, prepared according to the m e t h o d of Hems et al. [23], contained 2.5% bovine serum albumin, 1 0 0 mg glucose per dl, Krebs-Henseleit buffer, and 2.5% hemoglobin supplied as washed human red cells. In the studies on [14C]formate metabolism, 100 ml perfusion medium containing 500 pmol or 1000 pmol of sodium formate together with 1 gCi or 2 pCi [~4C]formate (0.05 Ci/1.8 g) was used. In some experiments, L-methionine was added to the perfusion medium in the following manner: 250 pmol (37 mg) added at 0 and 60 min, and 125 pmol (18.5 mg) added at 30 and 90 min during the 2 h perfusion. Radioactive COs was aspirated from the liver chamber into 30 ml of ethanolamine/ethyleneglycol m o n o m e t h y l e t h e r (1 : 1, v/v) and trapping solution, and an aliquot was counted in a scintillation counter. At the end of each perfusion, the liver was removed from the chamber, weighed, and homogenized with one volume of water. Liver protein was precipitated with 10% sulfosalicyclic acid (10 ml) and removed by centrifugation. An aliquot of the supernatant was counted for radioactivity and the remainder was stored at --20°C in preparation for chromatography. The perfusate was deproteinized by the addition of 10 ml 10% sulfosalicyclic acid, and the precipitate removed by centrifugation. Chromatography o f formate metabolites. Metabolites of formate in the liver and perfusate extracts were chromatographed on AG-50 W-X8 (Bio Rad) columns. Columns (0.9 X 60 cm) were packed to 55 cm and were equilibrated with two volumes 0.2 M sodium citrate buffer, pH 3.25. The equivalent of 5 g liver or 10 ml of perfusate was applied to each column. Elution was carried out with 100 ml of 0.2 M citrate buffer (I), pH 3.25, 100 ml 0.2 M citrate buffer (II), pH 4.25, 100 ml 0.2 M citrate buffer (III), pH 5.25, and finally with 100 ml 0.1 M NaOH. 5-ml fractions were collected and aliquots of the fractions measured for radioactivity. Radioactive compounds. [14C]Formate (0.05 Ci/1.8 g ) a n d L-[2-~4C]histi dine (55 Ci/mol) were obtained from Amersham Searle, Des Plaines, Ill. Results

In vivo experiments on formate and histidine metabolism in vitamin B-12- and fola te-deficien t an imals The first two experiments {Table I) involved a study of the effects of vitamin

228 TABLE I E F F E C T O F V I T A M I N B-12, M E T H I O N I N E , A N D F O L I C A C I D O N U R I N A R Y E X C R E T I O N OF F O R M A T E A N D F O R M I M I N O G L U T A M I C A C I D ( E X P T . I) A N D E F F E C T O F M E T H I O N I N E P R E I N J E C T I O N O F R E S P I R A T O R Y P R O D U C T I O N OF 14CO2 F R O M I N J E C T E D [ 1 4 C ] F O R M A T E A N D L - [ 2 - 1 4 C ] H I S T I D I N E ( E X P T . n ) IN R A T S Diet No.

Diet c o m p o s i t i o n Protein

Vitamin B-12

Methionine b

Folic acid b

Soy

2

Soy

3

Soy

4

--

Liver folate (pg]g)

methio-

nine (g/kg)

added

1

Total

Body weight (g)

--

+

2

4 0 6 +- 33 e

+

+

17

4 1 4 -+ 31

14.0

+

--

+

2

4 2 0 + 34

n.d. f

Soy

+

+

+

17

4 4 7 -+ 33

14.2

5

Casein g

+

--

--

6

350

16

0.8

6

Casein

+

--

+

6

4 5 4 .+- 23

n.d.

-+

5.7

a T i m e on d i e t : E x p t . I, 12 w e e k s ; E x p t . II, 8 w e e k s . 5 a n i m a l s p e r g r o u p . b W h e r e i n d i c a t e d b y " + " , t h e f o l l o w i n g ' l e v e l s w e r e a d d e d : v i t a m i n B-12, 1 0 0 p g / k g ; folic acid, 5 m g / k g ; m e t h i o n i n e , 15 g]kg of diet. c 2 5 0 p m o l L - m e t h i o n i n e i n j e c t e d i n t r a p e r i t o n e a l l y i n t o e a c h a n i m a l 15 rain p r e c e d i n g dose of form a t e or histidine. d A m o u n t i n j e c t e d : [ 14C] f o r m a t e , 1 pCi; L - [ 2 -l 4 C ] h i s t i d i n e , 2 pCi. e Standard deviation. f Not determined. g 1% s u c c i n y l s u l f a t h i a z o l e a d d e d .

B-12, methionine, and folic acid on the excretion of formate, FIGlu, and on the oxidation o f [ '4C] formate and ['4C ] histidine to 'aCO2. FIGlu excretion by rats on the +B-12 +Met soy protein diet (diet 4) was 0.6 pmol/kg b o d y weight per day. This increased to 9.3 with vitamin B-12 alone and to 8.0 with methionine alone and to 360 on the --B-12 --Met diet (diet 1). This shows that neither methionine nor vitamin B-12 alone is as effective as a combination o f the two in pr om ot i ng the metabolism o f FIGlu and t hereby decreasing its excretion. Fo r mate excretion was also increased but the relative difference between the --B-12 --Met group and the +B-12 +Met group was n o t as great as with FIGlu excretion. Formate excretion on the --B-12 --Met diet (diet 1) was 30 times greater than that on the +B-12 +Met diet (diet 4) while the corresponding value for FIGlu excretion was 600 times greater. The folate-deficient casein diet had a more marked effect on f o r m a t e and FIGlu excretion than it did on the metabolism o f f o r ma t e and histidine to CO2. Folate deficiency increased form at e and FIGlu excretion by approximately 1000-fold, while the oxidation of histi-

229

Liver vitamin B-12 (rig/g)

23.6 + 12.2

Experiment I a Metabolite excretion per kg body weight per day

Experiment Methionine injection c

Formate (pmol)

FIGlu (#mol)

5 9 0 -+ 2 9 0

360 + 100

II a

Percent of injected dose d respired as 1 4 C O 2 i n 1 h Formate

Histidine

-+

12.5 + 2.0 15.6 + 1.3

0.50 + 0.26 3.31 + 0.45

-+

1 7 . 4 + 3.7 2 9 . 1 +- 3 . 0

3.42 +0.74 2.59 + 0.40

31.6

-+ 1 6 . 2

35-+

8

8.0+-

2.8

222

+ 64

29 ±

5

9 . 3 +-

1.3

n.d.

n.d.

407

+ 64

20 ±

4

0 . 6 +-

0.8

n.d.

n.d.

n.d.

n.d.

3 3 3 0 +- 4 3 5

~1

460 + 116

<~0.2

-+

7.1 +- 0 . 5 8.2 + 0.7

0 . 1 2 -+ 0 . 0 3 0.55 ± 0.15

-+

2 3 . 5 + 5.7 38.4 + 4.3

2.26 ± 1.34 2.47 ± 1.31

dine to CO~ was decreased by a factor of 19 and the oxidation of formate decreased by a factor of 3. These oxidation rates would appear to provide a b e t t er measure of relative activities of the folate e n z y m e system involved. Vitamin B-12 deficiency pr oduced a 40-fold increase in FIGlu excretion but decreased histidine oxidation to CO2 by a fact or of approx. 7.0 (diet 1 compared to diet 3). In the case of vitamin B-12 deficiency, injection of methionine just prior to administration of histidine p r o d u c e d the same rate of CO2 respiration (3.3%) as the addition of m e t hi oni ne to the diet (3.4%). This compares with the rate of 0.5% for 14CO2 respiration on the --B-12 --Met diet (diet 1). Injection of methionine also stimulated the oxidation of histidine on the casein diet b u t restored it to only a b o u t one-fifth (0.55%) of the normal level (2.3%) obtained on the folate-supplemented casein diet. It should also be n o t e d t hat the effect on f o rm at e oxidation was n o t as marked as that on histidine. On the --B-12 --Met diet (diet 1), histidine conversion to 14CO2 was reduced to 15% of t h a t on the methionine-supplemented diet (diet 2) while f o r m a t e oxidation was correspondingly reduced to only 72%. Similarly, on the casein diet, folate

230 deficiency reduced histidine oxidation to 5% of the folate-supplemented group but decreased formate oxidation to 30% of the supplemented animals.

Perfusion experiments When 250 or 500 pmol of formate was added to the perfusion fluid, approx. 40% of the formate was oxidized and no effect of methionine on formate oxidation was observed. When the a m o u n t of formate was increased to 1000 pmol in order to overload the formate oxidation system, the addition of methionine to the perfusate increased oxidation of formate to CO2 by about 3-fold (Table II). The average oxidation rate in two perfused livers was 5.6% without methionine and 16.3% with methionine added to the perfusate. The a m o u n t of formate remaining in the perfusate accounted for almost all of the formate which had not been oxidized to CO2. Very little formate had been retained by the liver. Chromatography of the perfusate and liver extracts after perfusion showed that essentially all the radioactivity was present as formate. The value of 5.6% oxidation of formate in the absence of methionine and 16.3% in the presence of methionine may be compared with the values of 1% without methionine and 20% with methionine in the oxidation of L-[2-14C]histidine in the perfused liver under similar conditions as reported by Buehring et al. [22]. It thus appears that in the absence of methionine there is a complete shutoff of histidine and FIGlu metabolism in the perfused liver while formate oxidation is reduced only by two-thirds. Also, when smaller a m o u n t s of formate were used (250 gmol), methionine addition produced no increase. This suggests that part of the formate metabolism proceeds through a folate-insensitive pathway.

Effect o f methionine and folic acid on catalase activity As catalase has been reported to be involved in the oxidation of formate [12--14], this enzyme was assayed in the livers of rats on the soy protein and casein diets. The results given in Table III show that addition of methionine to

T A B L E II EFFECT OF METHIONINE ON THE METABOLISM OF [14C]FORMATE IN PERFUSED F R O M R A T S O N D I E T S D E F I C I E N T I N M E T H I O N I N E A N D V I T A M I N B-12 Rat No.

1 2 3 4

Urinary FIGIu p r i o r t o sacrifice (#mol/kg body weight)

Methionine a

398 178 300 395

--+ +

in perfusate

Formate b metabolized to 1 4 C O 2 in 90 m i n e (%)

LIVERS

Formate b

remaining in perfusate (%)

6.4 4.7 19.0 13.5

88 90 72 78

a L - M e t h i o n i n e w a s a d d e d to t h e p e r f u s i o n m e d i u m in t h e f o l l o w i n g m a n n e r : 2 5 0 /~mol a d d e d a t 0 and 6 0 m i n , a n d 1 2 5 p m o l a d d e d a t 3 0 a n d 9 0 m i n . b 1 0 0 0 p m o l ( 6 8 r a g ) s o d i u m f o r m a t e t o g e t h e r w i t h 1 p C i [ 14C] f o r m a t e ( s p e c i f i c a c t i v i t y , 0 . 0 5 Ci/ 1.8 g) w a s a d d e d to t h e p e r f u s i o n m e d i u m at t h e b e g i n n i n g o f e a c h p e r f u s i o n . c C O 2 r e c o v e r y v a l u e s are f o r t h e 3 0 - - 1 2 0 min period. 14CO 2 p r o d u c t i o n in t h e f i r s t 3 0 r a i n w a s o m i t t e d t o a l l o w d e p l e t i o n of r e s i d u a l t i s s u e m e t h i o n i n e a n d to p e r m i t c o m p a r i s o n w i t h t h e d a t a o f B u e h r i n g e t al. [ 2 2 ] .

231 T A B L E III E F F E C T O F D I E T A R Y V I T A M I N B-12, M E T H I O N I N E , A N D F O L A T E C A T A L A S E IN R A T L I V E R Dietary composition

ON T H E A C T I V I T Y OF

Nos. animals

Catalase a c t i v i t y ( u n i t b / # g w e t tissue)

Protein

Vitamin B-12 a

Methionine a

Folic a acid

Soy

--

--

+

3

4 4 . 5 -+ 9 . 1 c

Soy

--

+

+

3

3 5 . 2 +- 2 . 2

Casein d

+

_

--

4

2 6 . 6 -+ 1 . 8 e

Casein

+

--

+

4

3 8 . 3 -+ 3 . 5

a S u p p l e m e n t s a d d e d a t f o l l o w i n g levels w h e r e i n d i c a t e d b y " + " : v i t a m i n B-12, 1 0 0 pg; folic acid, 5 rag; a n d m e t h i o n i n e , 1 5 g/kg o f diet. b One u n i t = 1 p m o l H 2 0 2 d e c o m p o s e d / m i n a t 2 5 ° C . c S t a n d a r d deviation. d S u c c i n y l s u l f a t h i a z o l e a d d e d a t 1 0 g/kg of diet. e S i g n i f i c a n t a t P <: 0 . 0 0 5 .

the soy protein diet produced a 20% decrease (not statistically significant) in catalase level, while in the in vivo metabolic tests methionine addition to the diet increased formate metabolism to '4CO2 by 51%. Folate deficiency in.the casein diet decreased hepatic catalase activity by 30% (significant at P < 0.005). Formate oxidation in the in vivo experiments was reduced by 68% by folate deficiency. Thus the changes in catalase levels were smaller than the changes in formate oxidation on a folate-deficient diet. Discussion The effect of vitamin B-12 deficiency in increasing FIGlu and formate excretion may be explained by the methyl trap theory. The essence of this is that, in the absence of vitamin B-12, methionine synthesis is decreased and methyltetrahydrofolate accumulates. This creates a deficiency of tetrahydrofolic acid which is the active form capable of participating in the metabolism of FIGIu, and in the oxidation of formate to CO2 by the dehydrogenation of 10formyltetrahydrofolate pathway [9]. The action of methionine in decreasing FIGlu and formate excretion in the absence of added vitamin B-12 appears to be due to the conversion of methionine to S-adenosylmethionine. This exerts a feedback control by inhibiting methylenetetrahydrofolate reductase, thereby decreasing the formation of methyltetrahydrofolate. The methyl trap theory can also account for the effect of vitamin B-12 deficiency in lowering hepatic levels of folate and decreasing the proportion of pteroyl oligo-7-L-glutamates (folate polyglutamates). This is based on the observation that methyltetrahydrofolate cannot serve as a substrate for folic acid polyglutamate synthetase (ref. 24 and Brody, T., personal communication). The reduced synthesis of polyglutamates, which are preferentially retained by tissues [25], prevents folate from accumulating in the liver. The observations here are consistent with this hypothesis. Liver folate levels on the --B-12 --Met diet were reduced, being 40% of that on methionine alone, or with vitamin B-12 plus methionine. These

232 are similar to previously published values [26,27]. If we use the metabolism of [2-14C]histidine to 14CO2 in the whole animal as a relative measure of folate activity, we find that the formiminotransferase system in rats on the --B-12 --Met diet is 15% as active as in the --B-12 +Met rats. This is comparable with the value of 12% found by Thenen et al. [28] and the value of 15% reported by Brown et al. [5]. Hepatic levels of folate on the --B-12 --Met diet were approx. 40% of those on either the --B-12 +Met or the +B-12 +Met diet. Since the reduction of formiminotransferase activity (measured by [2-~4C]histidine conversion to 14CO2) was greater (15% of normal) than the reduction in liver folate (40% of normal), it appears that there has been a decrease in the functional activity of the remaining folate in the --B-12 --Met animals. Since the liver of the --B-12 --Met animal, containing 40% of normal liver folate level, has a metabolic activity (measured by [2-14C]histidine conversion to ~4CO2) equal to 15% of normal, it would appear that the relative metabolic activity of the folate present in --B-12 --Met animals is about one-third that of the folate in normal animals. This is consistent with the reduced proportion of non-methyltetrahydrofolates as first reported by Noronha and Silverman [29] and later by others [28,30,31]. The increased F I G l u excretion levels and the decreased histidine conversion to CO2 by the --B-12 --Met rats approaches that of the rats on the low folate casein diet where the liver folate dropped to 0.8 pg/g. metabolic activity of the liver folate (5.7 pg/g) in the --B-12 --Met animals seems to approach that of the liver folate level (0.8 pg/g) in the animals on the low folate casein diet. On the basis of these comparisons, the relative activity of folates in the --B-12 --Met animals would appear to be about 15% of normal. The data provide a measure of the magnitude of the relevant amounts of formate which may be oxidized by the two proposed metabolic schemes for formate oxidation. One scheme involves the conversion of formate to 10-formyltetrahydrofolate which is then dehydrogenated to CO2 and tetrahydrofolate [9]. The other involves a catalase coupled system [12--14]. The proportion of formate metabolized by the non-folate-dependent pathway would seem to be about 25%. This is based on the observation that the oxidation of injected formate in a folate-deficient rat (group 5) is 30% of the control animal receiving folate (group 6). These same folate-deficient animals metabolized 5% as much histidine to CO2 as that metabolized by the folate-supplemented rats. This shows that the functional activity of folate in the liver had been reduced to about 5% of normal while the formate oxidation had been reduced to only 30%. Liver perfusion data also point toward a similar value. The oxidation of formate to CO2 from the two deficient animals was 5.6% of the injected dose, while in similar animals with methionine added to the perfusate 16.3% of the formate was converted to CO2. Under similar conditions, Buehring et al. [22] found that 2 pmol of [2-~4C]histidine was oxidized to ~4CO2 while in similar livers supplemented with methionine in the perfusate 40 pmol was oxidized. Thus, in the absence of methionine, histidine metabolism is reduced to 5% of normal and formate oxidation is reduced to only 30% of normal. This suggests that about 25% (30% minus 5%) of the normal formate oxidation proceeds through a non-folate-dependent pathway. Recently, Krebs et al. [32] have reported that the perfused liver or hepatocytes from rats on stock diets containing vitamin B-12 have a reduced ability to

233 oxidize histidine to CO2. This can be increased by adding methionine in vitro, which shows that a functional deficiency of methionine can impair folic acid metabolism in isolated organs or tissues even when the animals had received vitamin B-12 in the diet.

Acknowledgment This research was supported by United States Public Health Service Grant AM-08171 from the National Institutes of Health.

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