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T Polymorphism and Folate Status Affect One-Carbon Incorporation into Human DNA Deoxynucleosides1,2
Food Science & Human Nutrition Department, Institute of Food and Agricultural Sciences,
* Division of Endocrinology and Metabolism, College of Medicine,
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32611;
** Vitamin Metabolism Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111; and
Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA
3To whom correspondence and reprint requests should be addressed. E-mail: jfgy{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T polymorphism is thought to influence the partitioning of 1-carbon units between methylation and other components of 1-carbon metabolism and to influence the risk and etiology of several major cancers and cardiovascular disease. Our objective was to determine the effect of the MTHFR 677CT polymorphism and folate status on the relative rate and extent of in vivo synthesis of DNA precursors. Adequately nourished, healthy women (9 CC, 9 TT) were infused with [3-13C]serine and [13C5]methionine for 9 h before and after 7 wk of consumption of a low-folate diet. Blood was drawn over 5 d for monocyte DNA isolation. Isotopic enrichment of the nucleosides in DNA digests was determined by LC-MS/MS. Maximum thymidine enrichment tended to be higher (P = 0.07) in TT than in CC subjects, suggestive of marginally higher mean thymidylate synthesis. However, the subset of TT subjects who exhibited formyltetrahydrofolate in erythrocytes (an indicator of 1-carbon partitioning) had greater (P = 0.036) thymidine enrichment than CC subjects, who had no erythrocyte formyltetrahydrofolate. Purine enrichment was not affected by genotype or folate depletion. However, the deoxyadenosine to deoxyguanosine enrichment ratio was significantly higher in TT subjects, suggesting a greater relative rate of adenine synthesis. The 
40% greater (P = 0.012) labeling of the methyl group of methyldeoxycytidine during folate depletion suggests a change in the origin of this 1-carbon unit. This is the first time that 1-carbon incorporation into human DNA has been measured in vivo after infusion of 13C-labeled 1-carbon precursors. These findings support the feasibility of further assessment of factors affecting deoxynucleotide synthesis and DNA methylation in human 1-carbon metabolism.
KEY WORDS: • thymidine • methyldeoxycytidine • deoxypurine • LC-MS/MS • isotope
Folates, as substituted forms of tetrahydrofolate (THF),4 serve as in vivo carriers of active carbon groups that lead primarily to cellular methylation reactions and the synthesis of nucleotides. Folate-mediated acquisition, processing, and 1-carbon transfer reactions are employed in the metabolism of all 4 DNA bases. Specifically, 10-formyltetrahydrofolate (10-CHO-THF) provides 1-carbon units for the 2 transformylation reactions in the formation of the purine rings of deoxyguanosine (dG) and deoxyadenosine (dA), and thymidine (dT) is formed through the transfer of the 1-carbon unit of 5,10-methylene-THF to deoxyuridine triphosphate (dUTP) to form thymidylate (thymidine triphosphate) (Fig. 1). In addition, S-adenosylmethionine (SAM), which is a component of the folate-mediated methylation cycle, serves as methyl donor for in situ methylation of DNA deoxycytidine (dCMdC). Aberrations in 2 of these reactions, thymidylate synthesis and dC methylation, are thought to contribute to the etiology of cancer by effecting genomic stability and the regulation of gene transcription.
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DNA methylation, through formation of MdC, plays an important role in regulating gene transcription, either by directly inhibiting transcription factor binding (7,8) or by initiating chromatin formation (9,10). DNA hypomethylation may also contribute to an increased incidence of strand breakage (11), which leads to chromosomal instability.
Methylenetetrahydrofolate reductase (MTHFR) irreversibly commits 1-carbon units toward the methylation cycle and away from thymidylate and purine synthesis. The MTHFR 677TT genotype is associated with general hypomethylation of DNA (12,13), whose extent is inversely related to folate nutritional status. Such global DNA hypomethylation was observed in association with localized hypermethylation of tumor suppressor genes (14,15), particularly in subjects with the MTHFR 677TT genotype (15). The TT genotype is associated with an increased risk of having a child with a neural tube defect (16) and is a risk factor for breast (17), cervical (18), and some colon cancers (15); however, it may protect against other forms of colon cancer (19). The TT genotype also may protect against adult acute lymphocytic leukemia (ALL) (20,21), but conflicting data exist regarding the effect of this genotype on the occurrence of childhood ALL (22,23). The modulation in human health risk between MTHFR genotypes can be accentuated by low folate status (17,19). The MTHFR 677TT genotype also affects the efficacy of various chemotherapeutic cancer treatments (24). It has been suggested that the modulation of cancer risk associated with the MTHFR 677 TT genotype is due to differential partitioning of 1-carbon groups between the methylation cycle and thymidine synthesis.
We report here the results of a stable isotopic investigation of the relative synthesis rates of DNA bases and the origin of methyl groups employed in DNA methylation by analysis of human monocyte DNA. Also reported is a newly developed method for analysis of all of the DNA deoxynucleosides by LC-MS/MS. This study provided an opportunity to gather initial data on this aspect of human 1-carbon metabolism and to evaluate the merits of a tracer protocol based mainly on primed, constant infusion of the 1-carbon sources [3-13C]serine and [13C5]methionine, along with preformed substrates to assess nucleoside pool sizes (i.e., [2H4]thymidine and [13C5]hypoxanthine). Additional objectives were to determine the quantitative effects of folate nutritional status and the MTHFR 677 CT polymorphism.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Human subjects.
Subjects were healthy 20- to 26-y-old women, nonsmokers, who did not use oral contraceptives or other medications known to interfere with folate metabolism. The subjects also abstained from alcohol consumption during the study period. Informed consent was obtained from all subjects. MTHFR 677CT genotype was determined by a PCR/restriction fragment length polymorphism procedure (28), and subjects were selected on the basis of their genotype (9 CC, 9 TT). Plasma concentrations of folate (11–32 nmol/L), vitamin B-12 (178–448 pmol/L) (Quantaphase II B12/folate competitive binding assay, Bio-Rad), pyridoxal 5'-phosphate (43–81 nmol/L) (29), and homocysteine (6.3–12.1 µmol/L) (30) were within acceptable limits in all subjects. A medical history questionnaire, physical examination, and clinical blood chemistry screening, including tests of thyroid and renal function, were used to confirm general health. Erythrocyte folate distribution was also measured as previously described (31). The protocol was approved by the University of Florida Institutional Review Board and the General Clinical Research Center (GCRC) Scientific Advisory Committee. Infusions were performed between January 2001 and December 2001.
Infusion protocol.
Subjects were admitted to the GCRC on the evening before each infusion protocol and consumed no food between 2030 h and initiation of the infusion the following morning. On the morning of the infusion, a heparin lock was established in 1 vein of each arm: 1 for blood collection and 1 for the tracer infusion. Infusions were initiated at 0830 h with a 5-min, 20 mL priming dose that delivered 9.26, 1.62, 1.87, 0.187, and 0.187 µmol/kg of [3-13C]serine, [13C5]methionine, [2H3]leucine, [2H4]thymidine, and [13C5]hypoxanthine, respectively. The 9-h constant infusion immediately followed the priming dose and delivered 20 mL of infusion solution/h, providing 9.26, 1.62, 1.87, 0.187, and 0.187 µmol/(kg · h) of [3-13C]serine, [13C5]methionine, [2H3]leucine, [2H4]thymidine, and [13C5]hypoxanthine, respectively. Every hour, during the infusion, subjects consumed a protein-free formulation containing carbohydrate (70% of energy) and fat (30% of energy) that provided one twenty-fourth of the daily energy requirement per serving {1.25 kcal/(kg · h) [5.23 kJ/(kg · h)]}.
Blood samples were taken before the infusion (d 1) to measure plasma concentrations of folate, vitamin B-12, and pyridoxal 5'-phosphate, and homocysteine, and to isolate the monocytes to determine the natural isotopic abundance of the DNA. Monocytes have a half-life of 1.9–2.2 d in the blood (32) and take 1 d to appear in circulation after tracer introduction. Consequently, blood was collected on the mornings of d 3, 4, and 5 for monocyte DNA isolation, around the time of expected maximum DNA enrichment.
After the initial infusion, subjects consumed a 7-wk controlled folate depletion diet [which achieved a 60% decrease in serum folate and a 24% decrease in RBC folate concentrations (26,27)], after which the infusion and blood collections were repeated. Due to scheduling problems, 2 subjects (1 TT, 1 CC) consumed the folate depletion diet for only 5 wk before their 2nd infusion. The [13C5]hypoxanthine tracer was not available early in the study. Consequently, 2 subjects (1 TT, 1 CC) did not receive [13C5]hypoxanthine in either infusion, whereas 2 others (2 CC) did not receive [13C5]hypoxanthine in their first infusion.
Dietary treatments. All meals were prepared by the GCRC. Meals were nutritionally adequate, except for their folate content (115 ± 20 µg dietary folate equivalents/d), as described in detail elsewhere (33).
Monocyte isolation and DNA purification.
Monocytes were partially purified from whole blood using Vacutainer Sodium Heparin CPT tubes (Becton Dickinson). Final purification was achieved using specific magnetic labeling of the monocytes with CD14 MicroBeads (Miltenyi Biotec) followed by retention and elution from a MiniMACS MS Column (Miltenyi Biotec). A pilot study showed that monocytes isolated in this manner were >98% pure, as determined by flow cytometry. DNA was purified from the isolated monocytes using a QIAamp DNA Blood kit (Qiagen). The concentration of DNA in each sample was determined by measuring the absorbance at 
= 260 nm. DNA purity was estimated from the ratio of absorbance at 260 and 280 nm (A260:280 = 1.8–1.9).
Enzymatic hydrolysis of purified DNA extracts. DNA was hydrolyzed to nucleosides by sequential enzymatic digestion using a modification of the method of Crain (34). In brief, 2–3 µg of DNA, in 10 volumes of DNA isolation buffer, was precipitated with 1 volume of 0.1 mol/L ammonium acetate and 70% (final volume) ethanol. The DNA pellet, washed with 70% (v:v) ethanol, was resuspended in 60 µL H2O and denatured in a water bath at 100°C for 3 min. The denatured sample was cooled rapidly on ice before the addition of 6 µL ammonium acetate (0.1 mol/L, pH 5.3). The sample was incubated with 6 U P1 nuclease (2 U/µL; Sigma N-8630) at 45°C for 2 h. Then, 6.5 µL ammonium bicarbonate (1 mol/L, pH 7.75) and 3.3 mU phosphodiesterase (1.1 mU/µL; Sigma P-3243) were added to the tube and incubated at 20°C for 2 h. Finally, 2 U alkaline phosphatase (1 U/µL; Sigma P-4252) was added and the tube incubated at 30°C for 2 h. The digested samples were stored at –20°C.
LC-MS/MS analysis of DNA digests.
We developed a chromatographic method (Fig. 2) for the separation of all 6 DNA nucleosides; (although deoxyuracil was resolved from other nucleosides, it was present in DNA digests at concentrations below detection limits). DNA digests were chromatographed using a 5-µm Discovery HS C18 column (50 x 2.1 mm, Supelco) eluted with 5 mmol/L ammonium acetate:0.1% acetic acid (v:v) using a methanol gradient (Fig. 2) at a flow rate of 0.6 mL/min. The methanol gradient increased linearly from 2 to 16% over an 8-min period and was held at 16% for 1 min before returning to baseline (2%). The column was reequilibrated for 2 min between runs. LC-MS analysis was conducted using a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer with APCI Interface in selected reaction monitoring (SRM) mode. N2 pressure was 80 psi for the sheath and 10 psi auxiliary. The vaporizer was held at 500°C and the capillary was maintained at 200°C. The corona current was set to 3 kA to maintain a voltage of 4 kV. Argon was used as collision cell gas at 2.5 mTorr with a collision energy of 20 V. For the determination of isotopic enrichment of the nucleosides, scanning was performed in SRM mode, using the parameters shown in the inset of Figure 2.
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Statistical analysis. Data were analyzed by 2-way repeated-measures ANOVA using SigmaStat for Windows (Version 3.0, SPSS). Genotype and folate depletion status (baseline vs. postdepletion) were the independent variables. Post hoc analyses were performed using the Holm-Sidak method for multiple comparisons. Differences were considered to be significant at P < 0.05, whereas values of 0.05 < P < 0.1 were considered to be indicative of a trend.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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= 260 nm) to ensure that the deoxynucleoside peaks had not drifted from their allotted detection window, and to verify completeness of enzymatic digestion. Although incomplete digestion would result in an overall decrease in sensitivity, the use of chromatographic separation, coupled with SRM, ensured selectivity. Potentially contaminating RNA nucleosides did not interfere with the analysis for a similar reason. Thymidine 1-carbon enrichment. Maximum thymidine enrichment from [3-13C]serine tended (P = 0.070) to be greater for TT subjects than for CC subjects (Table 1). The data were further analyzed on the basis of the propensity of subjects to accumulate 10-CHO-THF in erythrocytes (27). The 10-CHO-THF distributions were not available for 1 TT subject (who was excluded from the analysis) and 2 CC-subjects (the analysis was conducted with and without their inclusion). Although 75% (6/8) of TT subjects exhibited 10-CHO-THF in erythrocytes (range = 2–32%), none (0/7) of the CC subjects showed such accumulation, a result consistent with previous reports (12). TT subjects who exhibited 10-CHO-THF accumulation in erythrocytes had higher monocyte dT enrichment than CC subjects (P = 0.036). When the 2 CC subjects for whom 10-CHO-THF data were not available were included on the assumption that they, like other CC-individuals, would not have erythrocyte 10-CHO-THF, this analysis yielded P = 0.039. The isotopic enrichment of dT was not affected by folate status.
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Adjustment for precursor enrichment. A difficulty in interpreting the enrichment of cellular DNA constituents is the uncertain enrichment of the 1-carbon donors. Such donors include SAM, 5,10-methylene-THF, and 10-CHO-THF, and their precursors, whose enrichment cannot readily be measured at the site of monocyte synthesis and development. The sources of 13C units in this study include the infused tracers [13C5]methionine and [13C]serine and metabolically generated [13C1]methionine, as well as the various metabolic intermediates (i.e., 1-carbon substituted forms of THF).
The steady-state enrichment of the labeled forms of plasma methionine and serine was measured during the 9-h infusion as part of a larger study (26,27). In an exploratory analysis, plasma serine and methionine plateau enrichments were employed as divisors to adjust monocyte DNA nucleoside enrichment for precursor enrichment (Table 2). We also evaluated the relation between the raw enrichment data (i.e., M + 1 enrichment of dT, dA, and dG) and the precursor-adjusted enrichment values using linear regression correlation analysis. The direct measures of nucleoside enrichment and the enrichment values adjusted for plasma precursor enrichment were highly correlated (Table 3). This suggests that the use of plasma precursor enrichment does not aid in the interpretation of the labeling patterns of monocyte DNA and supports the suitability of the raw enrichment data.
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141), and 6 subjects (2 baseline and 4 postdepletion) for whom [13C5]deoxyguanosine enrichment was not detected (i.e., there was no peak for the m/z transition 273157). These data indicate that the overall purine pool was sufficiently large that the [13C5]hypoxanthine infusion rate used in this study yielded only minimal enrichment of the purine pool. Alternatively, there may not have been sufficient activity of the purine salvage pathway to allow substantial enrichment of dA and dG from the infused [13C5]hypoxanthine. Methyldeoxycytidine 1-carbon enrichment. MdC enrichment (i.e., 13C labeling of the methyl group of MdC) of monocyte DNA increased (P = 0.012) with folate depletion (Table 1). However, MdC 1-carbon enrichment did not differ between genotypes.
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Although the infusion protocol was designed and validated primarily to study other aspects of 1-carbon metabolism (25–27), our findings also support the feasibility of using monocyte DNA to evaluate the cellular synthesis of DNA precursors. Furthermore, the analysis of MdC labeling allows inferences to be made regarding the origin of the 1-carbon unit employed in the DNA methylation. Our finding that nucleoside enrichment tended to peak by d 4, i.e., 3 d postinfusion, is consistent with the observations that monocytes have a half-life of 1.9–2.2 d in the blood (32) and that it takes 1 d for enriched monocytes to appear in circulation after tracer introduction. More frequent sampling should be made to determine nucleoside enrichment kinetics more precisely and to determine maximum peak enrichment more accurately.
Our LC-MS/MS method was not calibrated to allow calculation of the ratio of MdC to dC, and thus overall DNA methylation. However, we employed an adaptation of the method (37) to determine overall methylation of leukocyte DNA in a larger study group (19 TT, 22 CC) and found no effects of genotype at the level of folate deficiency attained by our subjects. However, leukocyte DNA tended (P = 0.07) to exhibit higher methyl-acceptance in the TT samples postdepletion (37), which was indicative of less DNA methylation.
A complexity of this protocol is the difficulty in determining the enrichment of the cellular precursor pool(s) used for nucleotide synthesis and DNA methylation. The benefit of using plateau enrichment in plasma as a surrogate for 1-carbon precursor enrichment is uncertain because it may not accurately reflect intracellular conditions over the several days it takes to produce and release monocytes into the blood. In addition, the measures of plasma enrichment of serine and methionine isotopomers reflect only circulating pools, not the enrichment of tissue pools. Plasma [methyl-13C1]methionine is produced through cellular remethylation of homocysteine, and its enrichment in plasma is a useful tool in assessing whole-body 1-carbon metabolism (25–27). However, such kinetic measures of whole-body metabolic processes do not necessarily reflect those within the developing monocyte.
This study was designed to examine only the folate component of the 1-carbon cycle. Thus, subjects were maintained nutritionally replete for vitamin B-12, pyridoxal 5'-phosphate, and riboflavin. It is therefore not clear what effect deficiency in these vitamins might have on 1-carbon utilization when coupled with the folate depletion achieved in this study.
Thymidine synthesis. Our analysis indicated that the MTHFR 677TT genotype had a significant effect on dT synthesis, mainly when the TT subjects exhibited 10-CHO-THF as a component of their erythrocyte folate pool. This is the first direct evidence to support the hypothesis that the MTHFR 677TT genotype caused the preferential incorporation of 1-carbon groups into dT. In view of the evidence from the [2H4]thymidine tracers that the thymidine pool size was not affected by genotype or folate status, the observed differences in dT enrichment are indicative of a greater rate of thymidylate synthesis in MTHFR 677TT subjects. Our observation is consistent with that of Friso et al. (12) that lymphocyte DNA methylation is inversely proportional to erythrocyte 10-CHO-THF concentrations, i.e., by diverting 1-carbon group toward dT synthesis (as shown in this study), the MTHFR 677TT genotype diverts 1-carbon groups away from dC methylation (12).
We saw no effect of folate depletion on thymidine enrichment. This was unexpected in view of observations that thymidylate concentrations decrease in lymphoblasts in severe folate deficiency (38). However, thymidylate synthase and thymidine kinase activities were shown to increase in folate-deficient lymphoblasts (38). Thus, in the mild folate deficiency achieved in our study, increased thymidylate synthase and thymidine kinase activity may have compensated for the decrease in folate coenzyme concentrations. In more severe folate deficiency, a 2-fold increase (38) in activity of these enzymes is unlikely to be sufficient to maintain thymidylate synthesis rates needed for DNA synthesis. We would expect to see such an effect of folate status on dT synthesis if this technique were employed with truly folate-deficient subjects, rather than those with marginal deficiency examined in this study.
Methyldeoxycytidine enrichment. Interpretation of the MdC results is complicated by the fact that the labeling of the methionine methyl group of SAM, and consequently MdC enrichment, was derived from 2 sources: directly from the infused [13C5]methionine and from [methyl-13C1]methionine methyl group derived from [3-13C]serine via the 1-carbon cycle. Although we observed little effect of folate depletion on the plateau plasma enrichment of infused [13C5]methionine, or synthesized [13C1]methionine, or on the plasma concentration of SAM (26,27), it must be remembered that these are measurements of whole-body metabolism. It is not clear what effect folate depletion would have on tracer enrichment in individual tissues, particularly in rapidly proliferating tissues like monocytes. For instance, in a larger (19 TT, 22 CC) study group (37), of which our subjects were a subgroup, dC methylation tended (P = 0.08) to be lower in leukocytes postdepletion, measured by the methyl-acceptance test, suggesting a decrease in folate-derived 1-carbon groups for DNA methylation. Consequently, because the abundance of the infused [13C5]methionine was several-fold that of [13C1]methionine derived from [13C]serine (25–27), the increased enrichment of MdC in folate deficiency (Tables 2, and 3) suggests that folate-dependent intracellular 1-carbon metabolism was suppressed and the infused [13C5]methionine became a greater source of methyl groups for DNA methylation. An alternative tracer protocol will be required to allow unambiguous interpretation of this phenomenon.
Purine enrichment.
There was no significant effect of genotype or folate depletion on either dA or dG enrichment. However, there was a significant effect of genotype on the dA:dG enrichment ratio. Despite having common precursors, the 2 deoxypurine species fluctuate independently of each other with perturbation of the 1-carbon cycle (38,39). Although dG triphosphate concentrations are particularly prone to depletion under conditions of folate deficiency (38) or antifolate treatment (39), dA triphosphate concentrations are not substantially changed by folate depletion (2,38) and can increase with antifolate treatment (38). The disparate effect of folate depletion on deoxypurine concentrations may be related to the difference in size (38,39), importance (40), and turnover rates (41) of the adenosine and guanosine pools. By using the dA: dG ratio, we may have controlled for precursor enrichment and intersubject variability, making it possible to detect a genotype effect on relative deoxypurine enrichment and apparent synthesis rates. The physiologic and epidemiologic consequences of modulating the purine biosynthetic pathway are not clear; however, our findings suggest a potentially new avenue of investigation of the metabolic consequences of the MTHFR 677CT polymorphism.
Enrichment from [2H4]-thymidine and [13C5]-hypoxanthine. [2H4]thymidine and [13C5]hypoxanthine were infused to provide a source of "premade" forms, i.e., distal to the 1-carbon reactions, of thymidine and purines. The intent was to aid in evaluating the relative pools sizes and for comparison against the rate and extent of M + 1 enrichment that is indicative of de novo synthesis. The [2H4]thymidine infusion rate was based on enrichment data from rats (42), rather than from humans. We used the same rate for the [13C5]hypoxanthine infusion because no data were available concerning hypoxanthine salvage. However, the level of monocyte DNA enrichment from [2H4]thymidine was low, and the resulting imprecision reduced statistical sensitivity. Purine enrichment from [13C5]hypoxanthine was even lower, and was undetectable in some cases. One factor that might have contributed to this low level of enrichment was the high baseline folate status of the subjects, presumably related to the folic acid fortification of grain products in the United States (43). Under the conditions of this study, subjects reached only a mild state of folate deficiency (27); thus, consistent activation of the purine and thymidine salvage pathways may not have occurred. Cost and safety considerations may limit the rate at which labeled thymidine and hypoxanthine can be infused as reference tracers with which to assess relative rates of folate-dependent deoxynucleotide synthesis. Therefore, it may be preferable to use alternative methods of DNA labeling such as with D2O (44) or labeled forms of glucose (32) or glycine (45).
In conclusion, we measured for the first time the in vivo 1-carbon incorporation into DNA bases in human monocytes after the infusion of [13C]-labeled 1-carbon precursors. In doing so, we showed an effect of the MTHFR 677CT genotype on thymidine synthesis and demonstrated asymmetry in the metabolism of the 2 purine bases of DNA. We also showed that moderate folate deficiency affects 1-carbon metabolism and leads to changes in DNA methylation.
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FOOTNOTES |
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T polymorphism and folate status. FASEB J. 17: A276–A277 (abs.)]. 
2 Supported by National Institutes of Health grants DK56274 (J.F.G.), DK42033 (B.S.), and GCRC M01-RR00082, United States Department of Agriculture and National Research Institute grants 00–35200-9113 (J.F.G.) and 00–35200-9102 (L.B.B.). This paper is Florida Agricultural Experiment Station Journal Series No. R-10437.
4 Abbreviations used: ALL, acute lymphocytic leukemia; 10-CHO-THF, 10-formyltetrahydrofolate; 5-CH3-THF, 5-methyltetrahydrofolate; dA, deoxyadenosine; dC, deoxycytidine; dG, deoxyguanosine; dT, thymidine; dUTP, deoxyuridine triphosphate; GCRC, General Clinical Research Center; MdC, methyldeoxycytidine; MTHFR, methylenetetrahydrofolate reductase; SAM, S-adenosylmethionine; SRM, selected reaction monitoring.
Manuscript received 20 August 2004. Initial review completed 12 November 2004. Revision accepted 29 November 2004.
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LITERATURE CITED |
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