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Biochemical and Molecular Action of Nutrients

Intracellular S-Adenosylhomocysteine Concentrations Predict Global DNA Hypomethylation in Tissues of Methyl-Deficient Cystathionine ß-Synthase Heterozygous Mice¹

Marie A. Caudill^*, Jennie C. Wang^*, Stepan Melnyk, Igor P. Pogribny, Stefanie Jernigan, Michael D. Collins, Jesus Santos-Guzman, Marian E. Swendseid, Edward A. Cogger and S. Jill James^,2

Food, Nutrition and Consumer Sciences Department, California State Polytechnic University, Pomona, CA 91768; Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079; and School of Public Health, University of California, Los Angeles, CA 90024 ^*

²To whom correspondence should be addressed. E-mail: jjames{at}nctr.fda.gov

ABSTRACT

Because S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are the substrate and product of essential methyltransferase reactions; the ratio of SAM:SAH is frequently used as an indicator of cellular methylation potential. However, it is not clear from the ratio whether substrate insufficiency, product inhibition or both are required to negatively affect cellular methylation capacity. A combined genetic and dietary approach was used to modulate intracellular concentrations of SAM and SAH. Wild-type (WT) or heterozygous cystathionine ß-synthase (CBS +/-) mice consumed a control or methyl-deficient diet for 24 wk. The independent and combined effect of genotype and diet on SAM, SAH and the SAM:SAH ratio were assessed in liver, kidney, brain and testes and were correlated with relative changes in tissue-specific global DNA methylation. The combined results from the different tissues indicated that a decrease in SAM alone was not sufficient to affect DNA methylation in this model, whereas an increase in SAH, either alone or associated with a decrease in SAM, was most consistently associated with DNA hypomethylation. A decrease in SAM:SAH ratio was predictive of reduced methylation capacity only when associated with an increase in SAH; a decrease in the SAM:SAH ratio due to SAM depletion alone was not sufficient to affect DNA methylation in this model. Plasma homocysteine levels were positively correlated with intracellular SAH levels in all tissues except kidney. These results support the possibility that plasma SAH concentrations may provide a sensitive biomarker for cellular methylation status.

KEY WORDS: • cystathionine ß-synthase • S-adenosylhomocysteine • methylation • homocysteine • mice

S-adenosylmethionine (SAM)³ and S-adenosylhomocysteine (SAH), as components of the methionine cycle, are the substrate and product of essential cellular methyltransferase reactions (Fig. 1 ). SAM is derived from an ATP-dependent transfer of adenosine to methionine via methionine adenosyltransferase (MAT) and serves as the proximal methyl donor for most methylation reactions. Cellular methyl acceptors include phospholipids, proteins, histones, neurotransmitters, DNA and RNA (1 ). After transfer of the methyl group, SAM is converted to SAH, the common product of all SAM-dependent methyltransferase reactions and a potent product inhibitor of the enzyme (2 ). Under normal physiological conditions, SAH is hydrolyzed to homocysteine (Hcy) and adenosine in a reversible reaction catalyzed by SAH hydrolase. However, because the equilibrium constant of SAH hydrolase favors SAH synthesis rather than hydrolysis, SAH hydrolysis depends on the efficient removal of Hcy via remethylation to methionine or degradation to cysteine (Fig. 1) . In most cells, the primary remethylation pathway is catalyzed by folate and vitamin B-12–dependent methionine synthase. However, in liver and kidney cells, betaine-homocysteine methyltransferase can also transfer a methyl group to methionine from betaine, the oxidized form of choline, providing a secondary pathway for Hcy remethylation (3 ). Permanent removal of Hcy from the methionine cycle occurs via the one-way transsulfuration pathway, which involves two vitamin B-6–dependent enzymes, cystathionine ß-synthase (CBS) and cystathionine lyase. CBS condenses Hcy with serine to form cystathionine, and, subsequently, cystathionine lyase converts cystathionine into cysteine and -ketobutyrate. Cysteine is the precursor of several important metabolites, including glutathione. Perturbations of these pathways resulting from nutritional deficiencies or genetic mutations can lead to elevations in Hcy (4 , 5 ) and subsequently SAH (6 ). Hyperhomocysteinemia is an early marker for increased risk of occlusive cardiovascular disease (7 , 8 ) and certain birth defects (9 , 10 ).

View larger version (25K): [in this window] [in a new window]   Figure 1. The methionine cycle. BHMT, betaine-homocysteine methyltransferase; MS, methionine synthase; SAHH, SAH hydrolase; THF, tetrahydrofolate.

It is unclear from the SAM:SAH ratio whether substrate insufficiency, product inhibition or both are required to negatively affect cellular methylation capacity. For example, it is questionable whether alterations in SAM alone are causally related to cellular hypomethylation, because it has been demonstrated that identical decreases in the SAM:SAH ratio are conditionally associated with reduced methylation capacity depending on the absolute value of SAH (21 ). Yi et al. (6 ) found a positive correlation between plasma SAH levels and lymphocyte DNA hypomethylation in healthy young women; however, no association with SAM was observed. SAH is a potent product inhibitor of most SAM-dependent methyltransferase enzymes by binding to the active site with higher affinity than SAM (2 ); thus, increased intracellular concentrations of SAH may be more effective in reducing cellular methylation status than decreased levels of SAM. An evaluation of the tissue-specific alterations in the individual components may provide a more accurate reflection of methylation capacity than the SAM:SAH ratio.

Using a combined genetic and dietary approach, the present study was undertaken to examine the effect of alterations of plasma Hcy and intracellular SAM, SAH and the SAM:SAH ratio on global DNA methylation status in a variety of tissues. CBS heterozygous (22 ) and wild-type mice were fed a control or methyl-deficient diet for 24 wk. A methyl-deficient diet, low in methionine and devoid of folate and choline, was administered to promote accumulation of Hcy (23 ) as well as to increase and decrease intracellular concentrations of SAH and SAM, respectively (Fig. 1) . Heterozygous (+/-) CBS-deficient mice were chosen as the experimental model because CBS is the rate-limiting enzyme in Hcy transsulfuration, and diminished activity of the CBS-dependent transsulfuration pathway will promote accumulation of Hcy and concomitant increases in SAH.

MATERIALS AND METHODS

Mice and experimental diets.

CBS +/- mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred with normal wild-type siblings or wild-type C57BL/6J mice (The Jackson Laboratory) at the School of Public Health, University of California Los Angeles. The male weanling C57BL/6J wild-type and CBS +/- mice entered the study at 4 wk of age, weighing 15.6 ± 2.9 g. The mice were housed four per cage in suspended polycarbonate cages with free access to deionized water in a temperature-controlled (22°C) room with a 12-h light/dark cycle. Wild-type mice (n = 18) and CBS +/- mice (n = 20) were randomly assigned to the methionine-choline-folate–deficient diet (6% casein and 6% gelatin without supplemental methionine, choline or folate) or to the control diet, which was identical to the deficient diet but supplemented with 5 g of L-methionine, 2 mg folic acid and 4.2 g choline bitartrate per kilogram of diet, as previously described (24 ). The diets were stored at 4°C and fed without restriction for 24 wk with replacement every second day. Body weights were recorded at the beginning and end of the 24-wk period. Mice were anesthetized with ethyl ether and killed via cardiac puncture with a heparinized syringe. Blood was collected into vacutainer tubes containing EDTA and immediately centrifuged at 5,000 x g for 8 min. The plasma was subsequently stored at -70°C. Liver, brain, kidneys and testes were excised, immediately put on ice, weighed and stored at -70°C for later analysis. All mice were treated humanely and the experimental protocol was approved by the University of California, Los Angeles’ Animal Care and Use Committees.

Determination of CBS +/- genotype.

Genotyping for the targeted CBS allele was performed by polymerase chain reaction (PCR) as described by Watanabe et al. (22 ). Briefly, DNA was extracted from a tail clip using standard extraction techniques (25 ). DNA aliquots were subjected to PCR amplification using internal standard primers (T-cell receptor) and neomycin cassette primers. Electrophoresis through a 3% agarose gel containing ethidium bromide for 45 min at 175 V was used to fractionate the amplified fragments. The gel was then viewed under ultraviolet light. The neomycin and T-cell receptor primers amplify a 280- and 150-bp fragment, respectively. Only CBS +/- mice should have the neomycin product, while all mice should have the T-cell receptor product.

Plasma total Hcy analyses.

Determination of plasma total Hcy (tHcy) concentrations was based on the methods of Vester and Rasmussen (26 ) and Pfeiffer (27 ). Plasma (50 µL) was mixed with an aqueous solution of internal standard, cystamine (25 µL) and Milli-Q water (25 µL). Tri-N-butylphosphine was added to reduce protein-bound and oxidized thiols (mixed disulfides and disulfides). After a 30-min incubation at 4°C, proteins were precipitated by adding trichloroacetic acid (TCA) (100 µL). An aliquot of the supernatant (80 µL) plus 150 µL potassium borate buffer (pH 10.5) was incubated with ammonium 7-fluoro-benzo-2-oxa-,3-diazole-4-sulfonate (80 µL) for 1 h at 60°C to produce fluorescent derivatives of the reduced thiols. A 100-µL aliquot was then injected onto a C-18 reversed-phase column and eluted with a mobile phase containing 0.1 mol/L acetic acid/acetate (pH 4.0) and 5% (v/v) methanol. Quantitation was performed by peak area ratio (analyte to internal standard) and was based on a standard curve generated by using five different concentrations of external standards (Hcy: 3.12, 6.25, 12.5, 25 and 50 µmol/L). Thiol concentrations of unknown samples were calculated using the intercept and slope of the obtained linear regression line.

Intracellular SAM and SAH analyses.

Measurement of intracellular SAM and SAH was accomplished using coulometric electrochemical detection as previously described in detail (28 ). Briefly, samples of organ tissue (10–15 mg wet wt) were homogenized with 200 µL of phosphate-buffered saline. Ten percent meta-phosphoric acid (50 µL of 100 g/L) was added to cell extract, mixed well and incubated on ice for 20 min. After centrifugation for 15 min at 18,000 x g at 4°C, supernatants were filtered through a 0.2-µm filter, and 20 µL was injected into the high-performance liquid chromatography (HPLC) system. Detection of SAM and SAH was accomplished using a CoulArray electrochemical detector (ESA, Chelmsford, MA). Peak area analysis was performed by HPLC based on calibration curves generated for each compound.

Global DNA methylation using cytosine extension assay.

Assessment of global DNA methylation was accomplished using the cytosine extension assay previously described in detail (29 ). Briefly, genomic DNA (1 µg) was digested with 20 units of HpaII for 16–18 h according to the manufacturer’s protocol (New England Biolabs, Beverly, MA). The single nucleotide extension reaction was performed in a 25-µL reaction mixture containing 0.5 µg of DNA, 1 x PCR buffer II, 1.0 mmol/L MgCl₂, 0.25 units AmpliTaq DNA polymerase and 0.1 µL [³H]dCTP (57.4 Ci/mmol). The reaction mixture was incubated at 56°C for 1 h and subsequently placed on ice. Duplicate aliquots (10 µL) from each reaction were applied onto Whatman DE-81 ion exchange filters and washed three times with 0.5 mol/L sodium phosphate buffer, pH 7.0, at room temperature. The filters were then dried and processed for scintillation counting. The results are expressed as relative [³H]dCTP incorporation/0.5 µg DNA. [³H]dCTP is incorporated at unmethylated sites cleaved by HpaII; thus, an increase in dpm reflects relative DNA hypomethylation.

Statistical analyses.

A two by two factorial study design was used to examine the effects of CBS +/- and methyl deficiency on plasma total Hcy concentrations, intracellular SAM and SAH concentrations and global DNA methylation status in liver, kidney, brain and testes. Data were analyzed using a two-way analysis of variance (ANOVA). Where a significant effect of diet, genotype or their interaction was detected by ANOVA, t tests based on the least-squares means were used to separate the treatment groups. The comparisons of greatest interest included 1) the effect of diet assessed by comparing the reference group (wild-type mice on adequate diet) to the wild-type type mice on the methyl-deficient diet, 2) the effect of genotype assessed by comparing the reference group to the CBS +/- mice on the adequate diet and 3) the combined diet and genotype effect assessed by comparing the reference group to the CBS +/- mice on the deficient diet. A Pearson correlation coefficient (r) was used to measure associations between plasma total Hcy concentrations, intracellular SAM and SAH concentrations and global DNA methylation status. To further explore the relationship between select pairs of variables, linear regression analysis was performed to determine the proportion of variability of global DNA hypomethylation (R²) attributable to SAM and SAH concentrations in the liver. The data are presented as means ± SD. Statistical significance of difference was set at P 0.05.

RESULTS

Biological.

Over the 24-wk experimental period, the mice fed the methyl-deficient diet had approximately 20% less weight gain than the mice fed the control diet (Table 1 ). The liver weights of mice fed the methyl-deficient diet were not different from those of the control mice (Table 1) . Liver (Table 1) and kidney (not shown) protein concentrations were lower in the deficient mice compared with controls (P < 0.05). Liver protein concentration was also lower in the CBS +/- mice compared with wild-type controls, regardless of diet (Table 1) .

Plasma tHcy was 4.2 ± 2.1 µmol/L in the wild-type mice consuming the control diet. Administration of the methyl-deficient diet increased tHcy to 11.23 ± 7.0 µmol/L. In CBS +/- mice, plasma tHcy was 13.32 ± 4.2 µmol/L; administration of the methyl-deficient diet further increased tHcy to 24.22 ± 7.2 µmol/L (Fig. 2 ). Thus, the combination of the heterozygous genotype with the methyl-deficient diet had the greatest impact on plasma tHcy (P 0.05). Regression analysis across groups indicated strong correlations between plasma tHcy and intracellular SAH in liver (r = 0.645; P < 0.0001), brain (r = 0.707; P = 0.0002) and testes (r = 0.654; P < 0.0001), but not kidney (r = 0.237, P = 0.18). Inverse correlations were observed between plasma tHcy and intracellular SAM in liver (r = -0.811; P < 0.0001), kidney (r = -0.765; P < 0.0001) and brain (r = -0.676; P = 0.0005), but not testes (r = -0.324; P = 0.0865).

View larger version (25K): [in this window] [in a new window]   Figure 2. Plasma total Hcy concentrations (mean ± SD; n = 6–13) in wild-type (WT) and CBS +/- mice fed control (C) or methyl-deficient (MD) diet. Bars with different superscript letters are significantly different (P 0.05).

View larger version (16K): [in this window] [in a new window]   Figure 3. A: Relationship between global DNA hypomethylation and hepatocyte intracellular SAH (R2 = 0.224, P = 0.01) in wild-type (WT) and CBS +/- mice fed control (C) or methyl-deficient (MD) diet. B: Relationship between global DNA hypomethylation and SAM within the same groups (R2 = 0.058, P = 0.22).

DISCUSSION

The ratio of SAM to SAH, the substrate and product of most cellular methyltransferase reactions, is generally considered to be predictive of cellular methylation status (11 –13 ). Several studies have emphasized the decrease in SAM as the major effector of the reduced ratio and of methylation status (30 –34 ). While certain liver toxicants or MAT1 inhibitors can severely deplete SAM and lead to DNA hypomethylation (1 , 35 , 36 ), it is doubtful that the more modest reduction in SAM concentrations associated with nutritional deficiencies in methyl donors (folate, B-12 and/or B-6) will approach the K_m of most methyltransferases (2 ). However, the increase in Hcy concentrations associated with methyl donor deficiency will reverse the SAH hydrolase reaction, increase SAH levels, and reduce cellular methylation capacity via product inhibition of methyltransferases. The primary importance of SAH as a mediator of methylation status was recently demonstrated in a human study in which it was found that increased levels of plasma Hcy and intracellular SAH, without concurrent changes in SAM, were associated with lymphocyte DNA hypomethylation (6 ).

In the present study, a dietary and genetic approach was used to modulate SAM and SAH concentrations in a mouse model to determine which intracellular metabolite (substrate or product) has a greater effect on global DNA methylation. The combined results in four different tissues underscore the pivotal importance of tissue-specific metabolic pathways in determining the relative sensitivity to alterations in SAM, SAH and DNA methylation. For example, kidney and liver were least sensitive to alterations in DNA methylation, and both tissues maintain the complete transsulfuration pathway and high levels of SAH hydrolase. In contrast, brain and testes were most sensitive to alterations in SAH and DNA methylation, and both of these tissues lack a complete transsulfuration pathway and both have low SAH hydrolase activity.

In the liver of wild-type and CBS +/- mice, the methyl-deficient diet increased mean levels of SAH, reduced mean levels of SAM and the SAM:SAH ratio and induced modest, nonsignificant increases in mean DNA hypomethylation. The apparent resistance to hepatic DNA hypomethylation, despite an increase in SAH and decrease in SAM, may reflect the established capacity of the methyl-deficient C57Bl/J mouse strain to maintain normal levels of hepatic global DNA methylation (37 ); by contrast, male F344 rats are highly responsive to methyl-deficient diets and hepatic DNA hypomethylation (20 ). Regression analysis across groups was performed to more sensitively explore the relationship between intracellular hepatic concentrations of SAH and SAM with global DNA hypomethylation. The results indicated that intracellular concentrations of hepatic SAH were strongly associated with global DNA hypomethylation (P = 0.01), whereas there was no correlation with SAM levels (P = 0.23).

SAH levels and DNA hypomethylation in the kidney were unaffected by dietary methyl deprivation and/or CBS deficiency (Table 3 ). Significant reductions in SAM levels and in the SAM:SAH ratio were observed in both groups, but these were not independently sufficient to reduce DNA methylation status. These data in the kidney are consistent with our hypothesis that DNA hypomethylation may not be predicted by an independent decrease in SAM or the SAM:SAH ratio. The resistance of the kidney to genetic or dietary modulation of SAH levels is most likely a reflection of tissue-specific metabolic pathways. Schatz et al. (38 ) have shown that the kidney is active in the catabolism of adenosine, which facilitates clearing this tissue of SAH. Furthermore, the kidney can import plasma Hcy (39 ) and SAH (40 ) and excrete these compounds in the urine, thereby preventing intracellular accumulation of toxic levels of SAH. Pertinent to the present results, Perry et al. (41 ) reported increased urinary concentrations of SAH in humans with CBS deficiency. The lack of association between plasma Hcy and renal concentrations of SAH in the present communication lends support to the substantial reserve capacity of the kidney to accommodate elevations in plasma Hcy concentrations and is consistent with the finding of hyperhomocysteinemia in humans with renal failure (42 ).

Brain tissue derived from the methyl-deficient wild-type mice and the control CBS +/- mice exhibited a decrease in SAM and in the SAM:SAH ratio (Table 4) ; however, neither SAH levels nor DNA methylation in brain was significantly affected. In contrast, in brain tissue from the methyl-deficient CBS +/- mice, a significant increase in SAH was associated with DNA hypomethylation. Because brain SAM levels were not affected by methyl deficiency in the CBS +/- mice, the decrease in the SAM:SAH ratio and the increase in DNA hypomethylation were driven by the increase in SAH. The increased sensitivity of brain SAH to deficiencies in the methyl donors folate, methionine and choline may reflect the low levels of SAH hydrolase present in this tissue (43 ). Although CBS is present in brain tissue, it lacks cystathionine lyase and is therefore dependent on plasma-derived cysteine for glutathione synthesis (3 ).

The relationship among SAM, SAH and DNA hypomethylation observed in the testes further confirmed the importance of SAH as the primary determinant of reduced methylation capacity. The independent reduction in the SAM:SAH ratio in wild-type mice fed the methyl/folate–deficient diet was not associated with DNA hypomethylation. This observation in the testes, combined with similar observations in kidney and brain, suggests that the SAM:SAH ratio should be used with caution as a predictor of DNA methylation status. In the CBS +/- mice, no further decrease in SAM was found; however, a significant increase in SAH was associated with DNA hypomethylation, consistent with observations in liver and brain. The greater sensitivity of the testes to increases in SAH and DNA hypomethylation may reflect the low SAH hydrolase activity in this tissue and the lack of the transsulfuration pathway (43 ).

Because severe CBS deficiency in humans is associated with an increase in methionine and SAM levels (44 ), the reduced SAM levels in all tissues from the CBS +/- mice was an unexpected observation; nonetheless, there are several plausible explanations. Elevations in plasma Hcy as well as CBS deficiency with consequent impaired glutathione synthesis have been shown to promote oxidative stress in mammalian liver (45 ). The activities of both methionine synthase (46 ) and MAT1A in the liver (47 ) are reduced under conditions of oxidative stress and may lead to reduced SAM synthesis with moderate CBS deficiency in heterozygous mice. Although methionine synthase is present in all tissues, its activity depends on continued reductive methylation of its cobalamin cofactor and is therefore also sensitive to inactivation under oxidizing conditions (46 ). Finally, the inverse relationship observed between intracellular SAM and SAH in all tissues examined in the present study as well as in human erythrocytes (48 ) is consistent with an inhibitory effect of SAH on methionine synthase and/or MAT. An inhibitory effect of SAH on methionine synthase has been reported in porcine kidney (49 ).

Plasma Hcy concentrations were approximately two times higher in the CBS +/- mice compared with wild-type mice and were highest in the CBS +/- mice fed the methyl/folate–deficient diet. A strong positive correlation was observed between plasma Hcy and intracellular SAH in the liver, brain and testes, but not in kidney. A positive relationship also existed between plasma Hcy and global DNA hypomethylation in the brain and testes, both of which have low SAH hydrolase and transsulfuration activity (43 ). Export of Hcy from tissues is required to maintain intracellular concentrations at a low level to prevent toxic accumulation of SAH. Inefficient removal of Hcy via the remethylation or transsulfuration pathways will promote increases in SAH and product inhibition of SAM-dependent methyltransferases (3 ). Thus, elevated plasma Hcy may be a convenient biomarker for increased SAH and reduced cellular methylation capacity, as suggested by Yi et al. (6 ). Furthermore, because SAH is not readily transported across the plasma membrane, these results support the interesting possibility that Hcy serves as an exportable form of SAH to preserve cellular methylation status.

In summary, these data suggest that in CBS-deficient mice, tissue-specific DNA hypomethylation is more closely related to elevations in intracellular SAH concentrations than to decreases in SAM concentrations or the SAM:SAH ratio. Decreases in SAM alone were not sufficient to induce cellular hypomethylation in this animal model, and decreases in the SAM:SAH ratio were conditionally associated with reduced methylation capacity, depending on the absolute value of SAH. These data also demonstrate that in CBS-deficient mice, elevations of plasma Hcy are associated with increases in intracellular SAH and may serve as a convenient indicator of global DNA methylation status as well as total cellular methylation capacity. It remains to be determined if toxic increases in tissue SAH concentrations, resulting from nutritional or genetic abnormalities that interfere with efficient removal of Hcy, are associated with increased risk for certain cancers, birth defects or chronic diseases via the inhibitory effect of SAH on SAM-dependent methyltransferase reactions.

FOOTNOTES

¹ Supported in part by the California Agricultural Research Initiative, National Cancer Institute (no. P01CA42710) and the FDA Office of Women’s Health. Presented in part at Experimental Biology, April 2, 2001, Orlando, FL [Caudill, M., Wang, J., Collins, M., Swendseid, M., Santos, M., Pogribny, I., Melnyk, S. & James, S.J. Alterations in S-adenosylhomocysteine (SAH) and DNA hypomethylation in tissues from mice heterozygous for the cystathionine beta synthase (CBS) gene. FASEB J. 15:A491.8 (abs)].

³ Abbreviations: ANOVA, analysis of variance; CBS, cystathionine ß-synthase; Hcy, homocysteine; HPLC, high-performance liquid chromatography; MAT, methionine adenosyltransferase; PCR, polymerase chain reaction; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; TCA, trichloroacetic acid.

Manuscript received 3 May 2001. Initial review completed 4 June 2001. Revision accepted 7 August 2001.

LITERATURE CITED

1. Mato, J. M., Alvarez, L., Ortiz, P. & Pajares, M. A. (1997) S-adenosylmethionine synthesis: molecular mechanisms and clinical implications. Pharmacol. Ther. 73:265-280.[Medline]

2. Hoffman, D. R., Cornatzer, W. E. & Duerre, J. A. (1979) Relationship between tissue levels of S-adenosylmethionine, S-adenosylhomocysteine, and transmethylation reactions. Can. J. Biochem. 57:56-65.[Medline]

3. Finkelstein, J. D. (2000) Pathways and regulation of homocysteine metabolism in mammals. Sem. Thromb. Hemostasis 26:219-225.

4. Bailey, L. B. & Gregory, J. F. III. (1999a) Folate metabolism and requirements. J. Nutr. 129:779-782.[Abstract/Free Full Text]

5. Bailey, L. B. & Gregory, J. F. III. (1999b) Polymorphisms of methylenetetrahydrofolate reductase and other enzymes: metabolic significance, risks and impact on folate requirement. J. Nutr. 129:919-922.[Abstract/Free Full Text]

6. Yi, P., Melnyk, S., Pogribna, M., Pogribny, I. P., Hine, R. J. & James, S. J. (2000) Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation. J. Biol. Chem. 275:29318-29323.[Abstract/Free Full Text]

7. Boushey, C. J., Beresford, S. A., Omenn, G. S. & Motulsky, A. G. (1995) A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: probable benefits of increasing folic acid intakes. JAMA 274:1049-1057.[Abstract/Free Full Text]

8. Ueland, P. M., Refsum, H., Beresford, S. A. & Vollset, S. E. (2000) The controversy over homocysteine and cardiovascular risk. Am. J. Clin. Nutr. 72:324-332.[Abstract/Free Full Text]

9. Eskes, T. K. (1998) Neural tube defects, vitamins and homocysteine. Eur. J. Pediatr. 157:S139-S141.

10. James, S. J., Pogribna, M., Pogribny, I. P., Melnyk, S., Hine, R. J., Gibson, J. B., Yi, P., Tafoya, D. L., Swenson, D. H., Wilson, V. L. & Gaylor, D. W. (1999) Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase (MTHFR) gene may be maternal risk factors for Down syndrome. Am. J. Clin. Nutr. 70:495-501.[Abstract/Free Full Text]

11. Cantoni, G. L. (1985) The role of S-adenosylhomocysteine in the biological utilization of S-adenosylmethionine. Prog. Clin. Biol. Res. 198:47-65.[Medline]

12. Chiang, P. K. & Cantoni, G. L. (1979) Perturbation of biochemical transmethylations by 3-deazaadenosine in vivo. Biochem. Pharmacol. 28:1897-1902.[Medline]

13. Hoffman, D. R., Marion, D. W., Cornatzer, W. E. & Duerre, J. A. (1980) S-adenosylmethionine and S-adenosylhomocysteine metabolism in isolated liver. J. Biol. Chem. 255:10822-10827.[Abstract/Free Full Text]

14. Miller, J. W., Nadeau, M. R., Smith, J., Smith, D. & Selhub, J. (1994) Folate deficiency induced homocysteinemia in rats: disruption of S-adenosylmethionine coordinate regulation of homocysteine metabolism. Biochem. J. 298:415-419.

15. Wainfan, E., Dizik, M., Stender, M. & Christman, J. K. (1989) Rapid appearance of hypomethylated DNA in livers of rats fed cancer-promoting, methyl deficient diets. Cancer Res 49:4094-4097.[Abstract/Free Full Text]

16. Jacob, R. A., Gretz, D. M., Taylor, P. C., James, S. J., Pogribny, I. P., Miller, B. J., Henning, S. M. & Swendseid, M. E. (1998) Moderate folate depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women. J. Nutr. 128:1204-1212.[Abstract/Free Full Text]

17. Rampersaud, G. C., Kauwell, G.P.A., Hutson, A. D., Cerda, J. J. & Bailey, L. B. (2000) Genomic DNA methylation decreases in response to moderate folate depletion in elderly women. Am. J. Clin. Nutr. 72:998-1003.[Abstract/Free Full Text]

18. Choi, S. W. & Mason, J. B. (2000) Folate and carcinogenesis: an integrated scheme. J. Nutr. 130:129-132.[Abstract/Free Full Text]

19. Laird, P. W. & Jaenisch, R. (1996) The role of DNA methylation in cancer genetics and epigenetics. Annu. Rev. Genet. 30:441-464.[Medline]

20. Pogribny, I. P., Miller, B. J. & James, S. J. (1997) Alterations in hepatic p53 gene methylation patterns during tumor progression with folate/methyl deficiency in the rat. Cancer Letters 115:31-38.[Medline]

21. Capdevila, A., Decha-Umphai, W., Song, K-H., Borchardt, R. T. & Wagner, C. (1997) Pancreatic expcrine secretion is blocked by inhibitors of methylation. Arch. Biochem. Biophys. 345:47-55.[Medline]

22. Watanabe, M., Osada, J., Aratani, Y., Kluckman, K., Reddick, R., Malinow, M. R. & Maeda, N. (1995) Mice deficient in cystathionine ß -synthase: animal models for mild and severe homocyst(e)inemia. Proc. Natl. Acad. Sci. 92:1585-1589.[Abstract/Free Full Text]

23. Le Leu, R. K., McIntosh, G. H. & Young, G. P. (1998) Ability of endogenous folate from soy protein isolate to maintain plasma homocysteine and hepatic DNA methylation during methyl group depletion in rats. J. Nutr. Sci. Vitaminol. 44:457-464.

24. Henning, S. M., Swendseid, M. E. & Coulson, W. F. (1997) Male rats fed methyl-and folate-deficient diets with or without niacin develop hepatic carcinomas associated with decreased tissue NAD concentrations and altered poly(ADP-ribose) polymerase activity. J. Nutr. 127:30-36.[Abstract/Free Full Text]

25. Miller, S. A., Dykes, D. D. & Polesky, H. F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215.[Free Full Text]

26. Vester, B. & Rasmussen, K. (1991) High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum. Eur. J. Clin. Chem. Clin. Biochem. 29:549-554.[Medline]

27. Pfeiffer, C. M., Huff, D. L. & Gunter, E. W. (1999) Rapid and accurate HPLC assay for plasma total homocysteine and cysteine in a clinical setting. Clin. Chem. 45:290-292.[Free Full Text]

28. Melnyk, S., Pogribna, M., Pogribny, I. P., Ping, Y. & James, S. J. (2000) Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5'-phosphate concentrations. Clin. Chem. 46:265-272.[Abstract/Free Full Text]

29. Pogribny, I. P., Yi, P. & James, S. J. (1999) A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands. Biochem. Biophys. Res. Comm. 262:624-628.[Medline]

30. Lathrop Stern, L., Mason, J. B., Selhub, J. & Choi, S.-W. (2000) Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene. Cancer Epidemiology Biomarkers and Prevention 9:849-853.[Abstract/Free Full Text]

31. Loehrer, F.M.T., Angst, C. P., Haefeli, W. E., Jordon, P. P., Ritz, R. & Fowler, B. (1996) Low whole blood SAM and correlation between 5-methyltetrahydrofolate and homocysteine in coronary heart disease. Arterioscler. Thromb. Vasc. Biol. 16:727-733.[Abstract/Free Full Text]

32. Shivapurkar, N. & Poirier, L. A. (1983) Tissue levels of SAH in rats fed methyl-deficient amino acid-defined diets for one to five wks. Carcionogenesis 4:1051-1057.

33. Simile, M. M., Pascale, R., De Miglio, M. R., Nufris, A., Daino, L., Seddaiu, M. A., Gaspa, L. & Feo, F. (1994) Correlation between S-adenosyl-L-methionine content and production of c-myc, c-Ha-ras, and c-KI-ras mRNA transcripts in the early stages of rat liver carcinogenesis. Cancer Lett 79:9-16.[Medline]

34. Wainfan, E. & Poirier, L. A. (1992) Methyl groups in carcinogenesis: effects on DNA methylation and gene expression. Cancer Res 52:2071s-2077s.[Abstract/Free Full Text]

35. Lu, S. C., Huang, Z-Z., Heping, Y., Mato, J. M., Avila, M. A. & Tsukamoto, H. (2000) Changes in methionine adenosyltransferase and S-adenosylmethionine homeostasis in alcoholic rat liver. Am. J. Physiol. Gastrointest. Liver Physiol. 279:G178-G185.[Abstract/Free Full Text]

36. Varela-Moreiras, G., Alonso-Aperte, E., Rubio, M., Gasso, M., Deulofeu, R., Alvarez, L., Caballeria, J., Rodes, J. & Mato, J. M. (1995) Carbon tetrachloride-induced hepatic injury is associated with global DNA hypomethylation and homocysteinemia: effect of S-adenosylmethionine treatment. Hepatology 22:1310-1315.[Medline]

37. Counts, J. L., McClain, R. M., Harbison, M. L., Sarmiento, J. I. & Goodman, J. I. (1995) Hypomethylation of DNA and heightened sensitivity of the B6C3F1 mouse to liver tumorigenesis. Toxicologist 15:56.

38. Schatz, R. A., Vannam, C. R. & Sellinger, O. Z. (1977) Species and tissue differences in the catabolism of S-adenosyl-L-homocysteine: a quantitative chromatographic study. Life Sci 20:375-384.[Medline]

39. House, J. D., Brosnan, M. E. & Brosnan, J. T. (1998) Renal uptake and excretion of homocysteine in rats with acute hyperhomocysteinemia. Kidney Int 54:1601-1607.[Medline]

40. House, J. D., Brosnan, M. E. & Brosnan, J. T. (1997) Characterization of homocysteine metabolism in the rat kidney. Biochem. J. 328:287-292.

41. Perry, T. L., Hansen, S., MacDougall, L. & Warrington, P. D. (1967) Sulfur-containing amino acids in the plasma and urine of homocytinurics. Clin. Chim. Acta. 15:409-420.[Medline]

42. Hultberg, B., Andersson, A. & Sterner, G. (1993) Plasma homocysteine in renal failure. Clin. Nephrol. 40:230-234.[Medline]

43. Finkelstein, J. D. (1990) Methionine metabolism in mammals. J. Nutr. Biochem. 1:228-237.[Medline]

44. Mudd, S. H. (1997) Cystathionine ß-synthase deficiency: metabolic aspects. Graham, I. Refsum, H. Rosenberg, I.G. Ueland, P.M. eds. Homocysteine Metabolism: From Basic Science to Clinical Medicine 1997:77-82 Kluwer Academic Publishers Norwell, MA. .

45. Huang, R-F.S., Hsu, Y-C., Lin, H- & L. & Yang, F. L. (2001) Folate depletion and elevated homocysteine promote oxidative stress in rat livers. J. Nutr. 131:33-38.[Abstract/Free Full Text]

46. Mosharov, E., Cranford, M. R. & Banerjee, R. (2000) The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes. Biochemistry 39:13005-13011.[Medline]

47. Sanchez-Gongora, E., Ruiz, F., Mingorance, J., An, W., Corrales, F. J. & Mato, J. M. (1997) Interaction of liver methionine adenosyltransferase with hydroxyl radical. FASEB J 11:1013-1019.[Abstract]

48. Loehrer, F. M., Tschopl, M., Angst, C. P., Litynski, P., Jager, K., Fowler, B. & Haefeli, W. E. (2001) Disturbed ratio of erythrocyte and plasma S-adenosylmethionine and S-adenosylhomocysteine in peripheral arterial occlusive disease. Atherosclerosis 154:147-154.[Medline]

49. Finkelstein, J. D., Kyle, W. E. & Harris, B. J. (1974) Methionine metabolism in mammals: regulatory effects of S-adenosylhomocysteine. Arch. Biochem. Biophys. 165:774-779.[Medline]

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