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Department of Veterans Affairs Medical Center, Nashville, TN 37212 and Department of Biochemistry, Vanderbilt University Medical School, Nashville, TN 37232
2To whom correspondence should be addressed. E-mail: donald.w.horne{at}vanderbilt.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • folic acid • one-carbon metabolism • 5,10-methylenetetrahydrofolate • methionine administration • nitrous oxide
Folate deficiency is associated with an increased plasma concentration of homocysteine and increased risk of cardiovascular disease, cancer and neural tube defects (1
–3
). Elevated homocysteine also is associated with polymorphisms in the 5,10-methylenetetrahydrofolate reductase (MTHFR)3 gene and results in a variety of vascular and neurological symptoms (4
).
A key intermediate in folate coenzyme metabolism is 5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlu). It can be synthesized from 5,6,7,8-tetrahydrofolic acid (H4PteGlu) and formate via the trifunctional enzyme, C1-tetrahydrofolate synthetase, and by the reaction of serine with H4PteGlu via serine hydroxymethyltransferase. 5,10-CH2-H4PteGlu may be converted to 10-formyltetrahydrofolic acid (10-HCO-H4PteGlu) which provides one-carbon units for purine synthesis. 5,10-CH2-H4PteGlu also provides the methyl group and reducing equivalents for the synthesis of dTMP from dUMP. 5,10-CH2-H4PteGlu may be reduced by MTHFR to 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu), which is utilized by the vitamin B-12–dependent enzyme, methionine synthase, to methylate homocysteine and regenerate methionine and H4PteGlu.
Administration of methionine results in a redistribution of folate coenzymes such that the level of 5-CH3-H4PteGlu decreases and the level of H4PteGlu increases (5
–7
). This is due to the inhibition of MTHFR by S-adenosylmethionine (AdoMet) whose level increases with methionine administration (8
). In addition, inactivation of the B-12–dependent enzyme, methionine synthase, by nitrous oxide (N2O) results in trapping of folates as 5-CH3-H4PteGlu (9
). This is because only two enzymes use 5-CH3-H4PteGlu, i.e., methionine synthase, which is inactivated, and MTHFR, which is irreversible in vivo (10
).
Because of the key role 5,10-CH2-H4PteGlu plays in the metabolism of folate coenzymes, we devised a HPLC method for measuring this derivative in liver (11
). We have used this procedure to determine the effects of methionine administration and inactivation of methionine synthase on the levels of 5,10-CH2-H4PteGlu in rat liver
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Sodium L-ascorbate, 6-(RS)-5-formyltetrahydrofolate, L-methionine, Hepes and 2-(cyclohexylamino)ethanesulfonic acid (Ches) buffers were purchased from Sigma Chemical (St. Louis, MO), 2-mercaptoethanol was from Acros Organics USA (Fairlawn, NJ), tetrabutylammonium hydroxide and folic acid casei medium were from Fisher Chemical (Atlanta, GA). Lyophilized cultures of Lactobacillus rhamnosus (ATCC 7469) were from the American Type Culture Collection (Rockville, MD). The concentration of 5-formyltetrahydrofolate used as standard for microbiological assay was determined by UV spectroscopy (12
).
Animals.
Male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) weighing 
250 g were used in the studies. They were fed Harlan Teklad (Madison, WI) rodent diet (# 8604), which contains 24% crude protein, 4% fat and 4.5% fiber (13
). In the first experiment, rats were given 1.3 mmol/kg L-methionine intraperitoneally in sterile saline, and liver folate coenzymes were determined at times 0, 30, 60 and 120 min. For exposure to N2O, rats were placed in a chamber with a flow of N2O/O2 (80:20) for 18 h. Liver was removed and folate coenzymes measured. The use of rats in this study was approved by the Institutional Animal Care and Use Committee at Vanderbilt University and the VA Medical Center.
Preparation of liver extracts and analysis of liver folates.
Rats were anesthetized with isofluorane and liver was removed. All procedures were performed at 4°C, unless otherwise stated. Extracts for determination of folates were made by adding 3 volumes of the appropriate ice-cold extract buffer to minced liver; they were processed for HPLC determination of 5,10-CH2-H4PteGlu and other folate coenzymes as described previously (11
). Briefly, for determination of 5,10-CH2-H4PteGlu, liver was homogenized in 0.1 mol/L Ches buffer pH 10, centrifuged (32,000 x g for 30 min), and the 5,10-CH2-H4PteGlu was reduced to 5-CH3-H4PteGlu with sodium borohydride. An equal volume of buffer (0.1 mol/L Hepes, 40 g/L sodium ascorbate, 0.4 mol/L 2-mercaptoethanol, pH 7.85) was added, the pH adjusted to 7.5–7.8 with 1 mol/L HCl and the mixture heated in a boiling water bath for 5 min, cooled, microfuged and the supernatant stored at -20°C until needed for HPLC analysis. For determination of the other folate coenzymes, minced liver was extracted with buffer (20 g/L sodium ascorbate, 0.05 mol/L Hepes, 0.05 mol/L Ches, pH 7.85) in a boiling water bath for 10 min, homogenized, microfuged and the supernatant stored at -20°C.
Folate polyglutamates in both extracts were hydrolyzed using 0.25 volume rat plasma as a source of folylpolyglutamate hydrolase (14
). Folate coenzymes were separated using a Beckman Ultrasphere-IP column (4.6 x 150 mm) and the levels of 5,10-CH2-H4PteGlu and other folate coenzymes determined by Lactobacillus rhamnosus microbiological assays of eluted fractions as described previously (11
).
Statistical treatment of data.
Results are expressed as means ± SEM. Control and N2O-treated rats were compared using Student’s t test with P 
0.05 considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Methionine was administered to the rats and hepatic folate coenzymes were determined at various times. At 30 and 60 min after administration, the level of 5-CH3-H4PteGlu appeared to be greatly decreased (Fig. 1
). This decrease was reflected by increases in the levels of 10-HCO- and H4PteGlu. After 120 min, the level of 5-CH3- and 5-formyltetrahydrofolic acid (5-HCO-H4PteGlu) had returned to normal and the level of H4PteGlu continued to decrease. The amount of 5,10-CH2-H4PteGlu was apparently unchanged at all time intervals.
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In air-breathing control rats, H4PteGlu represented 24% of total hepatic folates; 5,10-CH2-H4PteGlu, 30%; 5-CH3-H4PteGlu, 15%; and the 5- and 10-HCO-H4PteGlu, 30% (Table 1
). As expected, after exposure for 18 h to an atmosphere containing N2O, 5-CH3-H4PteGlu levels increased at the expense of H4PteGlu; no H4PteGlu was detected in the N2O-treated rats. The levels of 5- and 10-HCO-H4PteGlu were not changed by exposure to N2O. The level of 5,10-CH2-H4PteGlu also was not affected (P > 0.18).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,7
,15
,16
). This increased AdoMet concentration leads to decreased levels of 5-CH3-H4PteGlu and increased levels of the other reduced folate coenzymes (6
,7
) because the enzyme that reduces 5,10-CH2-H4PteGlu to 5-CH3-H4PteGlu, MTHFR, is inhibited by AdoMet (8
). Thus, although 5-CH3-H4PteGlu is being utilized to methylate homocysteine, thereby reducing the 5-CH3-H4PteGlu pool, its synthesis is inhibited by the high level of AdoMet. It might be suspected that the inhibition of MTHFR would result in increased levels of 5,10-CH2-H4PteGlu. Unlike MTHFR, which is irreversible in vivo, the trifunctional enzyme, C1-H4PteGlu synthetase, is reversible and may convert 5,10-CH2-H4PteGlu to 5,10-methenyl-H4PteGlu and then to 10-HCO-H4PteGlu, which may be converted to H4PteGlu and CO2 via 10-HCO-H4PteGlu dehydrogenase (17
); however, conversion of 5,10-CH2-H4PteGlu to 5,10-methenyl-H4PteGlu requires NADP+ and the ratio of NADPH/NADP+ of 4 in normal liver (18
) would argue against C1-H4PteGlu synthetase playing a major role in regulating the amount of 5,10-CH2-H4PteGlu. Alternatively, 5,10-CH2-H4PteGlu may be converted to H4PteGlu via cytosolic serine hydroxymethyltransferase, which utilizes 5,10-CH2-H4PteGlu and glycine to synthesize serine (19
). The fact that we found a large increase in the amount of H4PteGlu and a rather small increase in the amount of 10-HCO-H4PteGlu (Fig. 1)
after administration of methionine would support the role of cytosolic serine hydroxymethyltransferase in maintaining the concentration of 5,10-CH2-H4PteGlu in the liver under these conditions.
Earlier studies showed that inactivation of methionine synthase or severe vitamin B-12 deficiency leads to an accumulation of folates as 5-CH3-H4PteGlu. This is called the methyl trap hypothesis and was first described by Noronha and Silverman (20
) and Herbert (21
). 5-CH3-H4PteGlu accumulates because in vitamin B-12 deficiency (either dietary or induced by N2O), the B12-dependent enzyme methionine synthase is inactive and the enzyme that catalyzes the synthesis of 5-CH3-H4PteGlu, MTHFR, is irreversible in vivo (10
). Many studies have confirmed this hypothesis (5
,6
,22
,23
). However, no information is available concerning the effects of B-12 deficiency on the level of 5,10-CH2-H4PteGlu. Therefore, we exposed rats to N2O and measured the distribution of hepatic folate coenzymes. As expected, N2O exposure resulted in an increase in the amount of hepatic 5-CH3-H4PteGlu compared with air-breathing control rats. The amounts of 5- and 10-HCO-H4PteGlu were unchanged. The amount of 5,10-CH2-H4PteGlu was not affected. Surprisingly, we detected no H4PteGlu in livers from the N2O-exposed rats. All previous studies, including ours, have shown that H4PteGlu levels decreased in the N2O group (5
,6
,22
,23
). However, all of these studies employed tissue extraction procedures performed at pH <9.5 and/or with 2-mercaptoethanol, which would lead to dissociation of 5,10-CH2-H4PteGlu to H4PteGlu and formaldehyde (11
,24
). Thus, it appears that there is little H4PteGlu present in livers of rats exposed to N2O.
In conclusion, we investigated whether two conditions known to perturb folate coenzyme metabolism altered the concentration of 5,10-CH2-H4PteGlu in liver. 1) Administration of methionine might be expected to increase the concentration of 5,10-CH2-H4PteGlu because methionine raises the concentration of AdoMet and inhibits the utilization of 5,10-CH2-H4PteGlu for the biosynthesis of 5-CH3-H4PteGlu. 2) Inactivation of methionine synthase, which results in the trapping of folates as 5-CH3-H4PteGlu, might be expected to result in a decrease in the concentration of 5,10-CH2-H4PteGlu because it is being reduced to 5-CH3-H4PteGlu by MTHFR and the resulting 5-CH3-H4PteGlu is trapped. However, neither of these conditions resulted in changes in the concentration of 5,10-CH2-H4PteGlu. These results may be explained because 5,10-CH2-H4PteGlu may be redistributed by the reversible activities of the C1-H4PteGlu synthetase to 5,10-methenyl-H4PteGlu and 10-HCO-H4PteGlu and/or by the activity of cytosolic serine hydroxymethyltransferase, thus maintaining a relatively constant concentration of this key folate metabolite.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AdoMet, S-adenosyl-L-methionine; Ches, 2-(cyclohexylamino)ethanesulfonic acid; 5,10-CH2-H4PteGlu, 5,10-methylenetetrahydrofolic acid; 5-CH3-H4PteGlu, 5-methyltetrahydrofolic acid; 5-HCO-H4PteGlu, 5-formyltetrahydrofolic acid; 10-HCO-H4PteGlu, 10-formyltetrahydrofolic acid; H4PteGlu, 5,6,7,8-tetrahydrofolic acid; MTHFR, 5,10-methylenetetrahydrofolate reductase.
Manuscript received 18 September 2002. Initial review completed 21 October 2002. Revision accepted 15 November 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Selhub, J. (1999) Homocysteine metabolism. Annu. Rev. Nutr. 19:217-246.[Medline]
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5. Makar, A. B. & Tephly, T. R. (1987) The role of formate and S-adenosylmethionine in the reversal of nitrous oxide inhibition of formate oxidation in the rat. Mol. Pharmacol. 32:309-314.[Abstract]
6. Horne, D. W. (1989) Effects of nitrous oxide inactivation of vitamin B12 and of methionine on folate coenzyme metabolism in rat liver, kidney, brain, small intestine and bone marrow. BioFactors 2:65-68.[Medline]
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9. Shane, B. & Stokstad, E. L. R. (1985) Vitamin B12-folate interrelationships. Annu. Rev. Nutr. 5:115-141.[Medline]
10. Green, J. M., Ballou, D. P. & Matthews, R. G. (1988) Examination of the role of methylenetetrahydrofolate reductase in incorporation of methyltetrahydrofolate into cellular metabolism. FASEB J. 2:42-47.[Abstract]
11. Horne, D. W. (2001) High-performance liquid chromatographic measurement of 5,10-methylenetetrahydrofolate in liver. Anal. Biochem. 297:154-159.[Medline]
12. Blakley, R. L. (1969) The Biochemistry of Folic Acid and Related Pteridines 1969 North-Holland Amsterdam, The Netherlands.
13. Harlan Teklad Standard Diets Available at: http://www.teklad.com/standard/index.htm (accessed October 23, 2002).
14. Wilson, S. D. & Horne, D. W. (1984) High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver. Anal. Biochem. 142:529-535.[Medline]
15. Eloranta, T. O. (1977) Tissue distribution of S-adenosylmethionine and S-adenosylhomocysteine in the rat. Effect of age, sex and methionine administration on the metabolism of S-adenosylmethionine, S-adenosylhomocysteine and polyamines. Biochem. J. 166:521-529.[Medline]
16. Makar, A. B. & Tephly, T. R. (1983) Effect of nitrous oxide and methionine treatments on hepatic S-adenosylmethionine and methylation reactions in the rat. Mol. Pharmacol. 24:124-128.[Abstract]
17. Cook, R. J. (2001) Folate metabolism. Carmel, R. Jacobsen, D. W. eds. Homocysteine in Health and Disease 2001:113-134 Cambridge University Press New York, NY. .
18. Shin, M., Iwamoto, N., Yamashita, M., Sano, K. & Umezawa, C. (1998) Pyridine nucleotide levels in liver of rats fed clofibrate- or pyrazinamide-containing diets. Biochem. Pharmacol. 55:367-371.[Medline]
19. Herbig, K., Chiang, E. P., Lee, L. R., Hills, J., Shane, B. & Stover, P. J. (2002) Cytoplasmic serine hydroxymethyltransferase mediates competition between folate-dependent deoxyribonucleotide and S-adenosylmethionine biosyntheses. J. Biol. Chem. 277:38381-38389.
20. Noronha, J. M. & Silverman, M. (1962) On folic acid, vitamin B12, methionine, and formiminoglutamate metabolism. Heinrich, H. C. eds. Vitamin B12 and Intrinsic Factor, 2nd European Symposium 1962:728-736 Ferdinand Enke Stuttgart, Germany. .
21. Herbert, V. & Zalusky, R. (1962) Interrelations of vitamin B12 and folic acid metabolism: folic acid clearance studies. J. Clin. Investig. 41:1263-1276.
22. Horne, D. W., Patterson, D. & Cook, R. J. (1989) Effect of nitrous oxide inactivation of vitamin B12-dependent methionine synthetase on the subcellular distribution of folate coenzymes in rat liver. Arch. Biochem. Biophys. 270:729-733.[Medline]
23. Wilson, S. D. & Horne, D. W. (1986) Effect of nitrous oxide inactivation of vitamin B12 on the levels of folate coenzymes in rat bone marrow, kidney, brain, and liver. Arch. Biochem. Biophys. 244:248-253.[Medline]
24. Osborn, M. J., Talbert, P. T. & Huennekens, F. M. (1960) The structure of "active formaldehyde" (N5,N10-methylene tetrahydrofolic acid). J. Am. Chem. Soc. 82:4921-4927.
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