Volume 9, number 2
MOLECULAR •
CELLULARBIOCHEMISTRY
November 30, 1975
T H E R E L A T I O N S H I P S B E T W E E N V I T A M I N B12 A N D FOLIC A C I D A N D T H E E F F E C T OF M E T H I O N I N E O N F O L A T E M E T A B O L I S M * Yoon Sook SHIN,~ Karl Ulrich B U E H R I N G , + and E. L. R. S T O K S T A D §
Department of Nutritional Sciences, University of California, Berkeley, C A 94720 (Received May 6, 1975)
Summary The relationship between vitamin B,2 and folate and the effect of methionine on folate metabolism during B~2 deficiency in rats is best explained by the prevention of the accumulation of 5-methyl-H4PteGlu by vitamin B12 and/or methionine. Although several points remain to be clarified, the 'methyl trap' hypothesis provides the most satisfactory explanation for the relation between vitamin B,2, methionine and folic acid. This concept is extended by the hypothesis that HePteGlu is the most active substrate for pteroylpolyglutamate synthetase, and thus accounts for the effect of methionine or vitamin B~2 in increasing liver folate levels.
Introduction The biochemical interrelationship between vitamin B~2 and folic acid is illustrated by c o m m o n symptoms which are caused by a deficiency of either vitamin. These include elevated urinary excretion of formiminoglutam.ic acid (F1GLU) '-6, aminoimidazolecarboxamide 7'* and formate 2'9. Methionine is also involved, since its addition to a vitamin B,2 deficient diet can restore to * An invited article. ? Present address: Kinder Klinik der Universit~it Mfinchen, 8 Miinchen 2, Lindwurm str. 4. F.R. Germany $ Present address: c/o E. Merck, 8018 Grating Mtinchen Im Feld 32 § To whom reprint requests should be addressed.
normal in experimental animals the high urinary excretion of F I G L U 3"4'~°, formate 9 and aminoimidazolecarboxamide 7, and the elevated F I G L U excretion in clinical vitamin B12 deficiency ~. The only known metabolic pathway c o m m o n to folic acid, vitamin B12 and methionine is the 5methyltetrahydrofolate :L-homocysteine methyltransferase reaction. The relation of this reaction to other folate dependent reactions is shown in the scheme on the following page. NORONHA and SILVERMAN ~2 observed an increase in methyltetrahydrofolate in vitamin B12 deficient rat liver which was restored to normal by administration of methionine. Based on those observations, they advanced the "methyl trap" hypothesis, which holds that in vitamin B12 deficiency a large amount of folate is trapped as methyl derivatives. These cannot function in most folate-dependent reactions until they have been converted into nonmethylated reduced derivatives by reaction with homocysteine to give methionine via the vitamin B~2 dependent methyltransferase. Methyltetrahydrofolic acid has been identified as the major folate component in human plasma '3 and increased amounts of this have been reported in some cases of pernicious anemia 14~5'~6. Increases in serum folate levels have also been observed in vitamin B~2 deficient rats ~7,~. Work in our laboratory has shown that this is accompanied by decreased heptatic uptake of injected folic acid (GAWTHORNEet al.) ~9 and reduced liver folate levels (KuTZBACH et al.) 2°.
Dr. W. Junk b.v: Publishers - The Hague, The Netherlands
97
5,10-methylene-H4PteGlu
FADH2 ' Reductase
![ 5-methyl-H4PteG1
homocysteine methyltransferase~1~, SAM "* methionine
5_formimino_H4Pteal u ] ~,orn~i~t~f.....
[ H4PteOlu ]
glutamic FIGLU ~cid
Chromatographic separation showed this reduction to be mainly at the expense of the folic acid polyglutamates (THENEN)21. Although the effect of vitamin Blz deficiency in producing a functional folic acid deficiency can easily be understood by a blockage of the reaction of 5-methyltetrahydrofolate with homocysteine to give methionine and tetrahydrofolate, the mode of action of methionine in reversing this effect is more difficult to visualize. In fact, increased levels of tissue methionine might be expected to potentiate vitamin B12 deficiency effects as a result of product inhibition of the transmethylase reaction. The studies to be reported here deal primarily with efforts to elucidate the mechanism of methionine action in modifying folic acid metabolism in vitamin B~2 deficiency. Previous reports from this laboratory have suggested two possible mechanisms: (a) an inhibition of the reduction of methylene-tetrahydrofolate to methyltetrahydrofolate by S-adenosylmethionine22'23; (b) increased uptake of folic acid by the liver and increased hepatic folate levels caused by methionine'9: The observations reported here deal with the effect of methionine on folate metabolism in perfused livers and the effects of methionine on the distribution of folates in the liver and on the levels of certain folate-dependent enzymes.
Materials and Methods
Diets Basal diets deficient in vitamin B12 and marginally deficient in methionine (2 g/kg diet) had the following composition (g/kg): soy assay protein, 200; cerelose, 714; corn oil with vitamins A (15,000 IU), D (2000 IU) and E (~tocopherol acetate, 50 mg), 40; salt mixture 2°, 35; choline chloride, 1; water soluble vitamin 98
pre-mix in cerelose, 102°. Supplemented diets also contained D,L-methionine (15 g/kg), vitamin B12 (10,0/xg), or both. D,L-ethionine (12.5 mg) was given intraperitoneally 10 minutes before the radioactive dose of folic acid.
Animals Male, weanling Sprague-Dawley rats weighing 50-60 g were housed singly in stainless steel metabolic cages in a constant temperature, light-controlled room during the course of the experiment. Food and water were given ad libitum. (3H)folic acid uptake studies 20/xCi (3H)folic acid (10-40 Ci/mmol depending on the batch from the supplier, Amersham Searle Co.), purified by chromatography on DEAE-cellutose or QAE-Sephades 24, was injected intraperitoneally. The animals were given water and food. ad libitum, and 24 hr urine samples were collected in a flask containing 0.1 ml 2-mercaptoethanol. Peffusion studies The perfusion medium was prepared according to the method of HENS et al. 2s. 2.5 g bovine serum albumin powder (Fraction V) and 100 mg glucose were dissolved in 80 ml Krebs-Henseleit buffer and the pH was adjusted to 7.4 with 1 M Na2CO3. 1 ml heparin solution (1000 U.S.P. units/ml) and enough washed human blood cells were added to give a final hemoglobin concentration of 2.5 g per 100 ml. The final volume was adjusted to 100 ml with Krebs-Henseleit buffer. All substrates used for perfusion experiments were dissolved in Krebs-Henseleit buffer and were adjusted to pH 7.4. The perfusion apparatus was a modification of the design of MILLER et al. 26. The apparatus was kept in a plexiglass box with the inside temperature maintained at 35 °C. The temperature in
the oxygenation tube was 39 °C. Perfusion fluid was pumped from the reservoir to the oxygenation tube with a peristaltic pump. The operative technique was based on the procedure of HENS et al. 2~. In the studies with (2-~4C)histidine, ~4CO2 was collected for 30 min periods using 30 ml ethanolamine:ethylene glycol monomethyl ether (1 : 1, v/v) as trapping solution and then counted for radioactivity in a scintillation counter 27. For the identification of the (2-14C)histidine metabolites, perfused livers were homogenized with ice-cold distilled water (total volume liver+water, 30 ml), and protein was precipitated by the addition of 10 ml 20% sulfosalicylate and removed by centrifugation. 5-10 ml of liver extracts were applied to Dowex 50W-X8 columns (0.9 cm × 35 cm), and elution carried out with 100 ml citrate buffer (pH 3.25), 100 ml citrate buffer (pH 4.25) and 300 ml citrate buffer (pH 5.28) at room temperature. A final elution was made with 100 ml dilute N a O H to remove any radioactivity remaining on the columns.
Column chromatography Sephadex G-25 and DEAE-cellulose column chromatography of folate derivatives were done according to the procedures of SHIN et al. 28. Enzyme assays Livers were homogenized in 3 volumes of cold 0.05 M phosphate buffer p H 7.2 with a PotterElvehjem homogenizer and centrifuged at 30,000 x g for 30 minutes. The supernatants were dialyzed for 2 hrs against 2 liters of 0.05 M phosphate buffer p H 7.2 at 4 °C. Methyltetrahydrofolate-homocysteine transmethylase was assayed by the method of D1CKERMAN et a l . 29 a s modified by KUTZBACH et al. 2°. Methylenetetrahydrofolate reductase was assayed in the reverse direction with menadione as the artificial electron acceptor 3°. 14C-formaldehyde formed from (a4C)CH3--HePteGlu was determined as the dimedone adduct which was extracted with toluene ~1. Autopsy and liver folate extraction 24 hrs after injection of tritiated folate, each animal was killed by decapitation and bled for 30 seconds. Livers were perfused with 30 ml ice-cold saline to flush out remaining blood and then quickly excised and weighed. Liver
homogenates and liver extracts in 1.1% ascorbate, p H 6.0 (1:4 wt/vol) were prepared according to the method in our laboratory 24. For the extraction of folates in the perfusion medium, 40 ml of perfusate were poured into 200 ml boiling 0.1% ascorbate solution (pH 7.0), the mixture heated in a water bath at 95 °C, cooled in ice, and the precipitate removed by centrifugation. All extracts were stored at - 2 0 °C until used.
Measurement of [ormiminoglutamic acid and methylmalonic acid in urine Urinary formiminoglutamic acid was determined by the method of TABOR and WYYOARDEN32. The extent of vitamin B12 deficiency was assessed by measuring urinary methylmalonic acid using the method of GIORGIO and PLAUT33 as adapted to automated analysis 34. Urine samples of 1 to 2 ml were passed through 3 x 1.3 cm columns of Bio-Rex 5 anion exchange resin (obtained from Bio-Rad Lab., Richmond, Calif.), 100-200 mesh. The methylmalonic acid was eluted with 25 ml of 0.2 N HC1 and assayed directly with an autoanalyzer using diazotized p-nitroaniline reagent for the color reaction. S-adenosylmethionine assay The determination of S-adenosylmethionine was carried out by the procedure of SALVATOREet al. 35. The efficiency of recovery was measured by the isotope dilution of a sample of ('4C)CH3S-adenosylmethionine added at the beginning of the extraction procedure. Folate analysis The microbiological assay procedures using Lactobacillus casei (ATCC 7469) were essentially those of WATERS and MOLLIN 36 with modifications introduced in this laboratory as described by TAMURA et M. 37.
Results
The first experiments directed toward examining the effect of vitamin B12 and methionine consisted of studying their effect on the levels of certain folate dependent enzymes. The results (Table 1) show that neither vitamin B12 nor methionine had any effect on the levels of 99
E n z y m e levels in rat liver homogenates" E n z y m e activities (nmol min -~ m g ' ± S.D.) ~ Diet
Reductase
Formiminotransferase
Transmethylase
-g~2 Met
0.29±0.06 0.30±0.11 0.31±0.09 0.23±0.04
132± 10 172±53 146±10 139±50
0 . 0 6 ± 0.02 0.27±0.12 0.05±0.01 0.13±0.05
+B,2~-Met d -B12+Met +B~2+Met
and methionine on liver and serum folate levels and on the uptake of a pulse dose of folic acid by the liver is also shown in Table 2. The increased methylmalonic acid excretion in the -B12 groups shows that a vitamin BI2 deficiency had developed in the animals, which was also accompanied by an elevated F I G L U excretion in rats which were deficient in both vitamin B12 and methionine. A double deficiency of vitamin B~2 and methionine decreased hepatic folate uptake and liver folate levels and increased plasma folate. Long-term supplementation with vitamin B12, or the addition of dietary methionine for 24 hours prior to injection of folate, gave normal tritiated folate uptake. This is similar to data reported by GAWTHORNE and STOKS+An19. Attempts were then made to study the effect of methionine on folate metabolism using either tissue homogenates or liver slices. However, neither of these tissue preparations duplicated the whole animal in their response toward methionine. BATRA et a l . 3s found that liver homogenates from vitamin g 1 2 deficient rats degraded F I G L U slower than that from supplemented animals. However, methionine added in vitro slightly depressed the breakdown of F I G L U and therefore had the opposite effect that it had in the whole animal 3'4'1°. In view of the lack of suitability of liver homogenates as a model system for studying the effect of methionine on folate metabolism, the perfused B12
Table 1
a Data courtesy of KUTZBACH et al. z°. Average of four to nine animals in each group. Vitamin B,~ and methionine supplements given for 11 weeks. 100 ~xg/kg diet. d 1 5 g/kg d i e t .
methylenetetrahydrofolate reductase and formiminotransferase. The level of transmethylase, which is vitamin g 1 2 dependent, is reduced in vitamin B~2 deficiency. It thus became apparent that the effect of vitamin g 1 2 deficiency in increasing F I G L U excretion is not due to a reduction in the level of formiminotransferase or methylenetetrahydrofolic acid reductase. The same study ( K u T Z B A C H et al.) 2° showed that liver folate levels were reduced in vitamin g12 deficiency and that they could be restored by addition of methionine. The effect of dietary supplements of vitamin
Table 2 Effect of dietary supplements of vitamin B,2 and methionine on liver and plasma folate levels, the excretion of formiminoglutamic acid (FIGLU) and methylmalonic acid (MMA), and the uptake of tritiated folic acid a
Folate levels Diet h
-B,a-Met Bl~+Met c +B,2-Met +Bi2+Met
Liver
Plasma
p~g/g 3.1+1.1 ° 6.6+2.4 9.5±2.8 9.2+0.7
NG/ML 74±10 55±29 49±6 52±15
Urinary excretion FIGLU
MMA
txmol/100 g body wt/day 33±22 105±80 3.9±3.8 172+116 0.08±0.02 7.5t1.1 0.05±0.01 7.4±1.1
Uptake of Tritiated folate by liver d
7.9±1.7 31.0±7.7 25.5±5.7 25.4±3.8
" D a t a courtesy of S. THENEN41. Diets fed for 18 weeks; 6 animals per group. Methionine given 24 hours before injection. d Uptake by liver in 24 hours following intravenous dose of 5 ~ c or 0.06 txg tritiated folate per 100 g body wt. ± Standard Deviation. b
100
liver was employed by BUEHRING et a l . 39 and was found to duplicate the whole animal in its response to methionine. The metabolism of /(2-14C)histidine added to perfused livers from different dietary groups was studied by measuring its conversion to respiratory 14CO2 and to F I G L U . These results (Table 3) show that the addition of methionine to the perfusion media (Group 2) increased the respired 14CO2 derived from histidine by ten-fold in livers from - B 1 2 Met animals. It also decreased the F I G L U contents of the liver and the perfusion fluid. Homocysteine had no effect. The 14CO2 formation by the perfused liver from an animal receiving methionine in the diet (Group 4) was ca. the same as that from an animal on the - B 1 2 - M e t diet (Group 1). Methionine added to the perfusate of a liver from an animal receiving methionine markedly increased 14CO2 production. This shows that the level of free methionine in the livers of animals from the dietary -B~2 + M e t group was small and rapidly exhausted. Additional methionine must be supplied to the perfusate to maintain the rapid breakdown of F I G L U to CO2. The rate of histidine CO2 respiration (40-80 Ixmol per liver per hr) by the perfused liver in presence of methionine would correspond to 1000 to 2000 txmol per day per animal, which is in large
excess of the dietary intake of histidine (ca. 500 ~mol per day) by these animals. The effect of methionine added to the perfusate thus resembles the effect of methionine in the whole animal in promoting the oxidation of l(214C)histidine to respiratory CO2. A study was then made of the effect of methionine and related compounds on the metabolism of folic acid added to the perfusate. The results, presented in Table 4, show that methionine also has a marked effect on the distribution of the various reduced metabolic forms of folic acid. Addition of methionine decreased the proportion of 5-methyltetrahydrofolate and elevated the level of 10-formyltetrahydrofolate, tetrahydrofolate and polyglutamate forms of folic acid. However, it did not influence the uptake of folic acid by the liver and the rate of reduction to the tetrahydrofolate level. The small amount of unchanged folic acid in the liver compared to the level of reduced folic acid derivatives indicates that folic acid is rapidly reduced to tetrahydrofolic acid. Large amounts of reduced folic acid derivatives are excreted into the perfusion medium. The concentration of reduced folate monoglutamate derivatives in liver is similar to that in the perfusate. A considerably smaller proportion of folic acid potyglutamate forms could be found in
Table 3 Effect of methionine on the metabolism of (2-'~C)histidine in the perfused rat
liver
Group no. 1 2 3 4 5
Diet
Supplement in the perfusion medium
Respired '4CO2 (~xmol)
-B,2-Met -B,2 - Met B~2-Met -B,2+Met -B~2+Met
Hist Hist + Met Hist + Homo Hist Hist+Met
7.5 (2) 80.0 (2) 10.0 (2) 10.0(1) 110.0 (1)
FIGLU in liver (txmol) 220 90 215 170 25
(2) (2) (1) (1) (1)
FIGLU in perfusate (txmol) 65 (2) 55 (2) -60(1) 10(1)
The perfusion time was two hours. The basal perfusion medium (100 ml) contained 500 txmol L-histidine and 1.7 p, Ci L-(2-14C)histidine (sp. act.: 58.3 mCi/mmol). When indicated, 200 ~xmol L-methionine or 400 txmol D,Lhomocysteine thiolactone-HC1 were added in the beginning of the perfusion and 100 p, mol methionine or 200 txmol homocysteine at 30, 60, 90 min. The values are expressed as the average of the number of animals shown in the parentheses. Abbreviations: Hist, Histidine; Met, Methionine; and Homo, Homocysteine.
101
Table 4 The effect of various components on the metabolism of folic acid in perfused rat liver"
Supplement to the perfusion m e d i u m
Diet - B ; : - Met
None
-BI~
Methionine
Met
FIGLU in liver
Folate in liver (perfusate) % of total
124
34 (66) 48 (52) 32 (68) 50 (50) 34 (66) 34 (64) 29
6
-B~+Met
None
140
-B~+Met
Methionine
- e l 2 + Met
Ethionine
-Bx2+Met
Homocysteine
115
-B12+Met
Choline
125
20 8
% Distribution of liver (perfusate) rotates Fb
CH3--FH/
FH4 d C H O - - F H 4 e Poly f
6 (33) 9 (55) 13 (53) 8 (44)
70 (55) 3 (5) 65 (36) 4 (3) 23
10 -46 -6 -36 -40
9 -27 -6 -21 -19
78
6
4
78
4
4
74
6
4
19
27
32
3 8 4 25
(71) -B~2+Met
Betaine
127
-B~2+Met
Homocysteine + Betaine
29 (71) 33 (67)
12
The livers were perfused for 2 hrs with 6 ixCi (3H)folic acid (sp. act.: 28 Ci/mmol), 500 ~xg folic acid and when indicated, 2 0 0 t x m o l L-methionine, 2 0 0 t x m o l betaine, 4 0 0 t x m o l D,L-homocysteine-thiolactone HCI and 400 txmol D,L-ethionine. - - indicates values not determined. b PteGlu. c 5-methyl-H4PteGlu. Sum of H4PteGlu and 5,10-methylene-H~PteGlu. ° Sum of 5,10-methenyl-HaPteGlu, 10-formyl-H4PteGlu and 5-formyl-H4PteGlu. Pteroylpolyglutamates. Table 5 Effect of vitamin B12, methionine and ethionine on hepatic folic acid uptake and on urinary excretion of folic acid derivatives °
Folate distribution in urine
Diet b
-B,2-Met +B12-Met -Bl:+Met +B12 + Met +B12 + E t h ~
Liver uptake % of dose
Radioact. in urine % of dose
PteGlu
4.9 11.3 15.3 14.7 16.0
61 26 45 30 27
1.2 -2.6 1.2 0.8
Methyl" folate
Non-methyV folate
% of Original dose 40 12 13 10 18 14 12 11 11 10
a Data courtesy of VIDAL and STOKSTAD4°. b Diets, B12 and methionine supplements given for 8 weeks. 0.4% ethionine given in diet for 24 hrs plus 12.5 m g ethionine given I.P. just before tritiated folate injection. d CH3--H4PteGlu. 5 - C H O - - H a P t e G l u and 1 0 - C H O - - H 4 P t e G l u . °
102
the perfusion medium than in the liver. Only D,L-ethionine and D,L-homocysteine in combination with betaine could simulate the effect of methionine, i.e., suppress F I G L U and 5-methyltetrahydrofolate accumulation. Neither D,Lhomocysteine, betaine nor choline were effective. Previous experiments in this laboratory 19 showed that the uptake of injected tritiated PteGlu is affected by dietary vitamin B12 and methionine. The experiments shown in Table 5 confirm this effect and also show that a deficiency of vitamin B12 and methionine increases both the excretion of radioactive folates and the 75
relative proportion of 5-methyl-H4PteGlu in urine. In the doubly deficient diet, 40% of the original dose was excreted as 5-methylH4PteGlu, while in those groups receiving either vitamin g12, methionine or ethionine, only 11 to 18% was excreted in this form. The absolute amount of non-methyl folates (5-formylI-LPteGlu+ 10-formyl-HgPteGlu) excreted was about the same in all groups. However, the relative amounts of non-methyl folates to methyltetrahydrofolates was greatly increased by addition of vitamin BI: or methionine. This is shown in the chromatographic curves for these groups which are represented in Figure 1.
CH3 H4 Pte Glu
A
B
~ o
60
J
_ BI2
~- 45
x
IO-CHO-H4PteGIu
-BI2
4.
+ ME THIONINE
5-CHO-H4PteGIu
- METHIONINE
,&
CH3H4 Pie Glu
I0- CHO-H4Pte Glu
IV
30
+
5-CHO-H4PteGlu 3o
g PteGlu
Iv
PteGlu
4'o
20 FRACTION
NUMBER
do
i
4()
20
6o
FRACTION NUMBER(Sml)
(5 m[)
CHzIH4 PteGlu
36
V
30
4- BI2
z THIONINE
D
o
I-
CH3H4PteGlu
2O
v re,
IV
b.
L/
Q.
t, iB I.
=
-
Bi2
--
METHIONINE
+
ETHIONINE
b I
12
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I0
o.
I
FRACTION
25 NUMBER
I
(2.5
50 ML )
\
F"
I
25 FRACTION
50 NUMBER
(5.Oral)
Fig. 1. DEAE-cellulose chromatogram of urinary folate 24 hr after intraperitoneal injection of tritiated PteGlu (10 p~Ci/100 g body weight, sp. act.: 35 Ci/mmol) in rats fed the basal ( - B j 2 - M e t ) diet or diet supplemented with B12, methionine and/or ethionine.
103
The amount of PteGlu excreted was small (0.8-2.6%) in all dietary groups and shows that when tritiated folate is given intraperitoneally, it is almost completely converted to reduced forms before being excreted. A comparison of the effect of ethionine and methionine on folate distribution patterns in the liver is shown in Figure 2. In the - B 1 2 - M e t group, the proportion of methyl to non-methyl folates was high. The administration of methionine (given continuously in the diet) or ethionine (given in diet for 24 hours plus 12.5 mg given by injection) greatly increases the proportion of the non-methyl folate forms. The principal methyl derivatives are Peak V (5-
z9
methyl-H4PteGlu) and Peak IX (5-methylH4PteGlus). The ratio of methyl to non-methyl derivative (Peaks V+IX) divided by non-methyl derivatives (Total-Peaks V+IX) is 0.82 for the - B 1 2 - M e t group, 0.48 for the -B~2 + Met group, and 0.38 for the -B12 +E t h group. Thus, ethionine is as effective as methionine in reducing methyl forms of folic acid. In a separate experiment, it was found that 12.5 mg of ethionine given intraperitoneally was as effective as the combination of dietary and injected ethionine in promoting folate uptake by the liver. Ethionine thus appears to be more effective than methionine since GAWTHORNEe t a l . 22 reported that 40 mg of methionine I.P. was not
z
Io
B
o
,,=®
CH3H4PteGlu 5
==
ix l _ BI2
% 04 X
=E
--
o ®
CH3H4 PteGlu
u.
-BI2 + METHIONINE
ro
-- METHIONINE
~
4O
b
~ H4PteGlu5 Xl
CH3H4PteGlu~ i
20
a
v v, vii
,
50 FRACTION
,~0
I00 NUMBER
Ill IV
(2.5
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ii
¥111
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MOLECULAR •
CELLULARBIOCHEMISTRY
November 30, 1975
T H E R E L A T I O N S H I P S B E T W E E N V I T A M I N B12 A N D FOLIC A C I D A N D T H E E F F E C T OF M E T H I O N I N E O N F O L A T E M E T A B O L I S M * Yoon Sook SHIN,~ Karl Ulrich B U E H R I N G , + and E. L. R. S T O K S T A D §
Department of Nutritional Sciences, University of California, Berkeley, C A 94720 (Received May 6, 1975)
Summary The relationship between vitamin B,2 and folate and the effect of methionine on folate metabolism during B~2 deficiency in rats is best explained by the prevention of the accumulation of 5-methyl-H4PteGlu by vitamin B12 and/or methionine. Although several points remain to be clarified, the 'methyl trap' hypothesis provides the most satisfactory explanation for the relation between vitamin B,2, methionine and folic acid. This concept is extended by the hypothesis that HePteGlu is the most active substrate for pteroylpolyglutamate synthetase, and thus accounts for the effect of methionine or vitamin B~2 in increasing liver folate levels.
Introduction The biochemical interrelationship between vitamin B~2 and folic acid is illustrated by c o m m o n symptoms which are caused by a deficiency of either vitamin. These include elevated urinary excretion of formiminoglutam.ic acid (F1GLU) '-6, aminoimidazolecarboxamide 7'* and formate 2'9. Methionine is also involved, since its addition to a vitamin B,2 deficient diet can restore to * An invited article. ? Present address: Kinder Klinik der Universit~it Mfinchen, 8 Miinchen 2, Lindwurm str. 4. F.R. Germany $ Present address: c/o E. Merck, 8018 Grating Mtinchen Im Feld 32 § To whom reprint requests should be addressed.
normal in experimental animals the high urinary excretion of F I G L U 3"4'~°, formate 9 and aminoimidazolecarboxamide 7, and the elevated F I G L U excretion in clinical vitamin B12 deficiency ~. The only known metabolic pathway c o m m o n to folic acid, vitamin B12 and methionine is the 5methyltetrahydrofolate :L-homocysteine methyltransferase reaction. The relation of this reaction to other folate dependent reactions is shown in the scheme on the following page. NORONHA and SILVERMAN ~2 observed an increase in methyltetrahydrofolate in vitamin B12 deficient rat liver which was restored to normal by administration of methionine. Based on those observations, they advanced the "methyl trap" hypothesis, which holds that in vitamin B12 deficiency a large amount of folate is trapped as methyl derivatives. These cannot function in most folate-dependent reactions until they have been converted into nonmethylated reduced derivatives by reaction with homocysteine to give methionine via the vitamin B~2 dependent methyltransferase. Methyltetrahydrofolic acid has been identified as the major folate component in human plasma '3 and increased amounts of this have been reported in some cases of pernicious anemia 14~5'~6. Increases in serum folate levels have also been observed in vitamin B~2 deficient rats ~7,~. Work in our laboratory has shown that this is accompanied by decreased heptatic uptake of injected folic acid (GAWTHORNEet al.) ~9 and reduced liver folate levels (KuTZBACH et al.) 2°.
Dr. W. Junk b.v: Publishers - The Hague, The Netherlands
97
5,10-methylene-H4PteGlu
FADH2 ' Reductase
![ 5-methyl-H4PteG1
homocysteine methyltransferase~1~, SAM "* methionine
5_formimino_H4Pteal u ] ~,orn~i~t~f.....
[ H4PteOlu ]
glutamic FIGLU ~cid
Chromatographic separation showed this reduction to be mainly at the expense of the folic acid polyglutamates (THENEN)21. Although the effect of vitamin Blz deficiency in producing a functional folic acid deficiency can easily be understood by a blockage of the reaction of 5-methyltetrahydrofolate with homocysteine to give methionine and tetrahydrofolate, the mode of action of methionine in reversing this effect is more difficult to visualize. In fact, increased levels of tissue methionine might be expected to potentiate vitamin B12 deficiency effects as a result of product inhibition of the transmethylase reaction. The studies to be reported here deal primarily with efforts to elucidate the mechanism of methionine action in modifying folic acid metabolism in vitamin B~2 deficiency. Previous reports from this laboratory have suggested two possible mechanisms: (a) an inhibition of the reduction of methylene-tetrahydrofolate to methyltetrahydrofolate by S-adenosylmethionine22'23; (b) increased uptake of folic acid by the liver and increased hepatic folate levels caused by methionine'9: The observations reported here deal with the effect of methionine on folate metabolism in perfused livers and the effects of methionine on the distribution of folates in the liver and on the levels of certain folate-dependent enzymes.
Materials and Methods
Diets Basal diets deficient in vitamin B12 and marginally deficient in methionine (2 g/kg diet) had the following composition (g/kg): soy assay protein, 200; cerelose, 714; corn oil with vitamins A (15,000 IU), D (2000 IU) and E (~tocopherol acetate, 50 mg), 40; salt mixture 2°, 35; choline chloride, 1; water soluble vitamin 98
pre-mix in cerelose, 102°. Supplemented diets also contained D,L-methionine (15 g/kg), vitamin B12 (10,0/xg), or both. D,L-ethionine (12.5 mg) was given intraperitoneally 10 minutes before the radioactive dose of folic acid.
Animals Male, weanling Sprague-Dawley rats weighing 50-60 g were housed singly in stainless steel metabolic cages in a constant temperature, light-controlled room during the course of the experiment. Food and water were given ad libitum. (3H)folic acid uptake studies 20/xCi (3H)folic acid (10-40 Ci/mmol depending on the batch from the supplier, Amersham Searle Co.), purified by chromatography on DEAE-cellutose or QAE-Sephades 24, was injected intraperitoneally. The animals were given water and food. ad libitum, and 24 hr urine samples were collected in a flask containing 0.1 ml 2-mercaptoethanol. Peffusion studies The perfusion medium was prepared according to the method of HENS et al. 2s. 2.5 g bovine serum albumin powder (Fraction V) and 100 mg glucose were dissolved in 80 ml Krebs-Henseleit buffer and the pH was adjusted to 7.4 with 1 M Na2CO3. 1 ml heparin solution (1000 U.S.P. units/ml) and enough washed human blood cells were added to give a final hemoglobin concentration of 2.5 g per 100 ml. The final volume was adjusted to 100 ml with Krebs-Henseleit buffer. All substrates used for perfusion experiments were dissolved in Krebs-Henseleit buffer and were adjusted to pH 7.4. The perfusion apparatus was a modification of the design of MILLER et al. 26. The apparatus was kept in a plexiglass box with the inside temperature maintained at 35 °C. The temperature in
the oxygenation tube was 39 °C. Perfusion fluid was pumped from the reservoir to the oxygenation tube with a peristaltic pump. The operative technique was based on the procedure of HENS et al. 2~. In the studies with (2-~4C)histidine, ~4CO2 was collected for 30 min periods using 30 ml ethanolamine:ethylene glycol monomethyl ether (1 : 1, v/v) as trapping solution and then counted for radioactivity in a scintillation counter 27. For the identification of the (2-14C)histidine metabolites, perfused livers were homogenized with ice-cold distilled water (total volume liver+water, 30 ml), and protein was precipitated by the addition of 10 ml 20% sulfosalicylate and removed by centrifugation. 5-10 ml of liver extracts were applied to Dowex 50W-X8 columns (0.9 cm × 35 cm), and elution carried out with 100 ml citrate buffer (pH 3.25), 100 ml citrate buffer (pH 4.25) and 300 ml citrate buffer (pH 5.28) at room temperature. A final elution was made with 100 ml dilute N a O H to remove any radioactivity remaining on the columns.
Column chromatography Sephadex G-25 and DEAE-cellulose column chromatography of folate derivatives were done according to the procedures of SHIN et al. 28. Enzyme assays Livers were homogenized in 3 volumes of cold 0.05 M phosphate buffer p H 7.2 with a PotterElvehjem homogenizer and centrifuged at 30,000 x g for 30 minutes. The supernatants were dialyzed for 2 hrs against 2 liters of 0.05 M phosphate buffer p H 7.2 at 4 °C. Methyltetrahydrofolate-homocysteine transmethylase was assayed by the method of D1CKERMAN et a l . 29 a s modified by KUTZBACH et al. 2°. Methylenetetrahydrofolate reductase was assayed in the reverse direction with menadione as the artificial electron acceptor 3°. 14C-formaldehyde formed from (a4C)CH3--HePteGlu was determined as the dimedone adduct which was extracted with toluene ~1. Autopsy and liver folate extraction 24 hrs after injection of tritiated folate, each animal was killed by decapitation and bled for 30 seconds. Livers were perfused with 30 ml ice-cold saline to flush out remaining blood and then quickly excised and weighed. Liver
homogenates and liver extracts in 1.1% ascorbate, p H 6.0 (1:4 wt/vol) were prepared according to the method in our laboratory 24. For the extraction of folates in the perfusion medium, 40 ml of perfusate were poured into 200 ml boiling 0.1% ascorbate solution (pH 7.0), the mixture heated in a water bath at 95 °C, cooled in ice, and the precipitate removed by centrifugation. All extracts were stored at - 2 0 °C until used.
Measurement of [ormiminoglutamic acid and methylmalonic acid in urine Urinary formiminoglutamic acid was determined by the method of TABOR and WYYOARDEN32. The extent of vitamin B12 deficiency was assessed by measuring urinary methylmalonic acid using the method of GIORGIO and PLAUT33 as adapted to automated analysis 34. Urine samples of 1 to 2 ml were passed through 3 x 1.3 cm columns of Bio-Rex 5 anion exchange resin (obtained from Bio-Rad Lab., Richmond, Calif.), 100-200 mesh. The methylmalonic acid was eluted with 25 ml of 0.2 N HC1 and assayed directly with an autoanalyzer using diazotized p-nitroaniline reagent for the color reaction. S-adenosylmethionine assay The determination of S-adenosylmethionine was carried out by the procedure of SALVATOREet al. 35. The efficiency of recovery was measured by the isotope dilution of a sample of ('4C)CH3S-adenosylmethionine added at the beginning of the extraction procedure. Folate analysis The microbiological assay procedures using Lactobacillus casei (ATCC 7469) were essentially those of WATERS and MOLLIN 36 with modifications introduced in this laboratory as described by TAMURA et M. 37.
Results
The first experiments directed toward examining the effect of vitamin B12 and methionine consisted of studying their effect on the levels of certain folate dependent enzymes. The results (Table 1) show that neither vitamin B12 nor methionine had any effect on the levels of 99
E n z y m e levels in rat liver homogenates" E n z y m e activities (nmol min -~ m g ' ± S.D.) ~ Diet
Reductase
Formiminotransferase
Transmethylase
-g~2 Met
0.29±0.06 0.30±0.11 0.31±0.09 0.23±0.04
132± 10 172±53 146±10 139±50
0 . 0 6 ± 0.02 0.27±0.12 0.05±0.01 0.13±0.05
+B,2~-Met d -B12+Met +B~2+Met
and methionine on liver and serum folate levels and on the uptake of a pulse dose of folic acid by the liver is also shown in Table 2. The increased methylmalonic acid excretion in the -B12 groups shows that a vitamin BI2 deficiency had developed in the animals, which was also accompanied by an elevated F I G L U excretion in rats which were deficient in both vitamin B12 and methionine. A double deficiency of vitamin B~2 and methionine decreased hepatic folate uptake and liver folate levels and increased plasma folate. Long-term supplementation with vitamin B12, or the addition of dietary methionine for 24 hours prior to injection of folate, gave normal tritiated folate uptake. This is similar to data reported by GAWTHORNE and STOKS+An19. Attempts were then made to study the effect of methionine on folate metabolism using either tissue homogenates or liver slices. However, neither of these tissue preparations duplicated the whole animal in their response toward methionine. BATRA et a l . 3s found that liver homogenates from vitamin g 1 2 deficient rats degraded F I G L U slower than that from supplemented animals. However, methionine added in vitro slightly depressed the breakdown of F I G L U and therefore had the opposite effect that it had in the whole animal 3'4'1°. In view of the lack of suitability of liver homogenates as a model system for studying the effect of methionine on folate metabolism, the perfused B12
Table 1
a Data courtesy of KUTZBACH et al. z°. Average of four to nine animals in each group. Vitamin B,~ and methionine supplements given for 11 weeks. 100 ~xg/kg diet. d 1 5 g/kg d i e t .
methylenetetrahydrofolate reductase and formiminotransferase. The level of transmethylase, which is vitamin g 1 2 dependent, is reduced in vitamin B~2 deficiency. It thus became apparent that the effect of vitamin g 1 2 deficiency in increasing F I G L U excretion is not due to a reduction in the level of formiminotransferase or methylenetetrahydrofolic acid reductase. The same study ( K u T Z B A C H et al.) 2° showed that liver folate levels were reduced in vitamin g12 deficiency and that they could be restored by addition of methionine. The effect of dietary supplements of vitamin
Table 2 Effect of dietary supplements of vitamin B,2 and methionine on liver and plasma folate levels, the excretion of formiminoglutamic acid (FIGLU) and methylmalonic acid (MMA), and the uptake of tritiated folic acid a
Folate levels Diet h
-B,a-Met Bl~+Met c +B,2-Met +Bi2+Met
Liver
Plasma
p~g/g 3.1+1.1 ° 6.6+2.4 9.5±2.8 9.2+0.7
NG/ML 74±10 55±29 49±6 52±15
Urinary excretion FIGLU
MMA
txmol/100 g body wt/day 33±22 105±80 3.9±3.8 172+116 0.08±0.02 7.5t1.1 0.05±0.01 7.4±1.1
Uptake of Tritiated folate by liver d
7.9±1.7 31.0±7.7 25.5±5.7 25.4±3.8
" D a t a courtesy of S. THENEN41. Diets fed for 18 weeks; 6 animals per group. Methionine given 24 hours before injection. d Uptake by liver in 24 hours following intravenous dose of 5 ~ c or 0.06 txg tritiated folate per 100 g body wt. ± Standard Deviation. b
100
liver was employed by BUEHRING et a l . 39 and was found to duplicate the whole animal in its response to methionine. The metabolism of /(2-14C)histidine added to perfused livers from different dietary groups was studied by measuring its conversion to respiratory 14CO2 and to F I G L U . These results (Table 3) show that the addition of methionine to the perfusion media (Group 2) increased the respired 14CO2 derived from histidine by ten-fold in livers from - B 1 2 Met animals. It also decreased the F I G L U contents of the liver and the perfusion fluid. Homocysteine had no effect. The 14CO2 formation by the perfused liver from an animal receiving methionine in the diet (Group 4) was ca. the same as that from an animal on the - B 1 2 - M e t diet (Group 1). Methionine added to the perfusate of a liver from an animal receiving methionine markedly increased 14CO2 production. This shows that the level of free methionine in the livers of animals from the dietary -B~2 + M e t group was small and rapidly exhausted. Additional methionine must be supplied to the perfusate to maintain the rapid breakdown of F I G L U to CO2. The rate of histidine CO2 respiration (40-80 Ixmol per liver per hr) by the perfused liver in presence of methionine would correspond to 1000 to 2000 txmol per day per animal, which is in large
excess of the dietary intake of histidine (ca. 500 ~mol per day) by these animals. The effect of methionine added to the perfusate thus resembles the effect of methionine in the whole animal in promoting the oxidation of l(214C)histidine to respiratory CO2. A study was then made of the effect of methionine and related compounds on the metabolism of folic acid added to the perfusate. The results, presented in Table 4, show that methionine also has a marked effect on the distribution of the various reduced metabolic forms of folic acid. Addition of methionine decreased the proportion of 5-methyltetrahydrofolate and elevated the level of 10-formyltetrahydrofolate, tetrahydrofolate and polyglutamate forms of folic acid. However, it did not influence the uptake of folic acid by the liver and the rate of reduction to the tetrahydrofolate level. The small amount of unchanged folic acid in the liver compared to the level of reduced folic acid derivatives indicates that folic acid is rapidly reduced to tetrahydrofolic acid. Large amounts of reduced folic acid derivatives are excreted into the perfusion medium. The concentration of reduced folate monoglutamate derivatives in liver is similar to that in the perfusate. A considerably smaller proportion of folic acid potyglutamate forms could be found in
Table 3 Effect of methionine on the metabolism of (2-'~C)histidine in the perfused rat
liver
Group no. 1 2 3 4 5
Diet
Supplement in the perfusion medium
Respired '4CO2 (~xmol)
-B,2-Met -B,2 - Met B~2-Met -B,2+Met -B~2+Met
Hist Hist + Met Hist + Homo Hist Hist+Met
7.5 (2) 80.0 (2) 10.0 (2) 10.0(1) 110.0 (1)
FIGLU in liver (txmol) 220 90 215 170 25
(2) (2) (1) (1) (1)
FIGLU in perfusate (txmol) 65 (2) 55 (2) -60(1) 10(1)
The perfusion time was two hours. The basal perfusion medium (100 ml) contained 500 txmol L-histidine and 1.7 p, Ci L-(2-14C)histidine (sp. act.: 58.3 mCi/mmol). When indicated, 200 ~xmol L-methionine or 400 txmol D,Lhomocysteine thiolactone-HC1 were added in the beginning of the perfusion and 100 p, mol methionine or 200 txmol homocysteine at 30, 60, 90 min. The values are expressed as the average of the number of animals shown in the parentheses. Abbreviations: Hist, Histidine; Met, Methionine; and Homo, Homocysteine.
101
Table 4 The effect of various components on the metabolism of folic acid in perfused rat liver"
Supplement to the perfusion m e d i u m
Diet - B ; : - Met
None
-BI~
Methionine
Met
FIGLU in liver
Folate in liver (perfusate) % of total
124
34 (66) 48 (52) 32 (68) 50 (50) 34 (66) 34 (64) 29
6
-B~+Met
None
140
-B~+Met
Methionine
- e l 2 + Met
Ethionine
-Bx2+Met
Homocysteine
115
-B12+Met
Choline
125
20 8
% Distribution of liver (perfusate) rotates Fb
CH3--FH/
FH4 d C H O - - F H 4 e Poly f
6 (33) 9 (55) 13 (53) 8 (44)
70 (55) 3 (5) 65 (36) 4 (3) 23
10 -46 -6 -36 -40
9 -27 -6 -21 -19
78
6
4
78
4
4
74
6
4
19
27
32
3 8 4 25
(71) -B~2+Met
Betaine
127
-B~2+Met
Homocysteine + Betaine
29 (71) 33 (67)
12
The livers were perfused for 2 hrs with 6 ixCi (3H)folic acid (sp. act.: 28 Ci/mmol), 500 ~xg folic acid and when indicated, 2 0 0 t x m o l L-methionine, 2 0 0 t x m o l betaine, 4 0 0 t x m o l D,L-homocysteine-thiolactone HCI and 400 txmol D,L-ethionine. - - indicates values not determined. b PteGlu. c 5-methyl-H4PteGlu. Sum of H4PteGlu and 5,10-methylene-H~PteGlu. ° Sum of 5,10-methenyl-HaPteGlu, 10-formyl-H4PteGlu and 5-formyl-H4PteGlu. Pteroylpolyglutamates. Table 5 Effect of vitamin B12, methionine and ethionine on hepatic folic acid uptake and on urinary excretion of folic acid derivatives °
Folate distribution in urine
Diet b
-B,2-Met +B12-Met -Bl:+Met +B12 + Met +B12 + E t h ~
Liver uptake % of dose
Radioact. in urine % of dose
PteGlu
4.9 11.3 15.3 14.7 16.0
61 26 45 30 27
1.2 -2.6 1.2 0.8
Methyl" folate
Non-methyV folate
% of Original dose 40 12 13 10 18 14 12 11 11 10
a Data courtesy of VIDAL and STOKSTAD4°. b Diets, B12 and methionine supplements given for 8 weeks. 0.4% ethionine given in diet for 24 hrs plus 12.5 m g ethionine given I.P. just before tritiated folate injection. d CH3--H4PteGlu. 5 - C H O - - H a P t e G l u and 1 0 - C H O - - H 4 P t e G l u . °
102
the perfusion medium than in the liver. Only D,L-ethionine and D,L-homocysteine in combination with betaine could simulate the effect of methionine, i.e., suppress F I G L U and 5-methyltetrahydrofolate accumulation. Neither D,Lhomocysteine, betaine nor choline were effective. Previous experiments in this laboratory 19 showed that the uptake of injected tritiated PteGlu is affected by dietary vitamin B12 and methionine. The experiments shown in Table 5 confirm this effect and also show that a deficiency of vitamin B12 and methionine increases both the excretion of radioactive folates and the 75
relative proportion of 5-methyl-H4PteGlu in urine. In the doubly deficient diet, 40% of the original dose was excreted as 5-methylH4PteGlu, while in those groups receiving either vitamin g12, methionine or ethionine, only 11 to 18% was excreted in this form. The absolute amount of non-methyl folates (5-formylI-LPteGlu+ 10-formyl-HgPteGlu) excreted was about the same in all groups. However, the relative amounts of non-methyl folates to methyltetrahydrofolates was greatly increased by addition of vitamin BI: or methionine. This is shown in the chromatographic curves for these groups which are represented in Figure 1.
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Fig. 1. DEAE-cellulose chromatogram of urinary folate 24 hr after intraperitoneal injection of tritiated PteGlu (10 p~Ci/100 g body weight, sp. act.: 35 Ci/mmol) in rats fed the basal ( - B j 2 - M e t ) diet or diet supplemented with B12, methionine and/or ethionine.
103
The amount of PteGlu excreted was small (0.8-2.6%) in all dietary groups and shows that when tritiated folate is given intraperitoneally, it is almost completely converted to reduced forms before being excreted. A comparison of the effect of ethionine and methionine on folate distribution patterns in the liver is shown in Figure 2. In the - B 1 2 - M e t group, the proportion of methyl to non-methyl folates was high. The administration of methionine (given continuously in the diet) or ethionine (given in diet for 24 hours plus 12.5 mg given by injection) greatly increases the proportion of the non-methyl folate forms. The principal methyl derivatives are Peak V (5-
z9
methyl-H4PteGlu) and Peak IX (5-methylH4PteGlus). The ratio of methyl to non-methyl derivative (Peaks V+IX) divided by non-methyl derivatives (Total-Peaks V+IX) is 0.82 for the - B 1 2 - M e t group, 0.48 for the -B~2 + Met group, and 0.38 for the -B12 +E t h group. Thus, ethionine is as effective as methionine in reducing methyl forms of folic acid. In a separate experiment, it was found that 12.5 mg of ethionine given intraperitoneally was as effective as the combination of dietary and injected ethionine in promoting folate uptake by the liver. Ethionine thus appears to be more effective than methionine since GAWTHORNEe t a l . 22 reported that 40 mg of methionine I.P. was not
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