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Nutrient Metabolism

Activities of Hepatic Cytosolic and Mitochondrial Forms of Serine Hydroxymethyltransferase and Hepatic Glycine Concentration Are Affected by Vitamin B-6 Intake in Rats¹

Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611

²To whom correspondence should be addressed. E-mail: jfgy{at}ufl.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate (PLP)–dependent enzyme that exists as cytosolic and mitochondrial isozymes that catalyze the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methyleneTHF. SHMT is a major source of one-carbon units for cellular metabolism, but its sensitivity to various degrees of altered vitamin B-6 nutritional status has not been determined. In this study, cytosolic and mitochondrial SHMT activities were measured in liver from rats fed dietary pyridoxine (PN) ranging from adequate to deficient levels (2, 1, 0.5, 0.1, and 0 mg PN/kg diet; n = 10 per group). Both mitochondrial and cytosolic SHMT activities increased (P < 0.001) with increasing dietary PN over this range, and activities were a linear function of liver PLP concentration. Mitochondrial SHMT comprised 70% of total activity. Assays conducted with and without in vitro addition of PLP indicated that total SHMT (apo- and holoenzyme forms) varied with dietary PN for each isoform, but that the proportion of each present as the apoenzyme was not affected by PN intake. This aspect of SHMT nutritional regulation differs from that of many other PLP-dependent enzymes. Hepatic glycine concentration was inversely related to vitamin B-6 intake (P < 0.05), which suggests a functional effect of altered SHMT activity. Overall these results demonstrate the potential for disruption of SHMT-mediated one-carbon metabolism by inadequate vitamin B-6 intake.

KEY WORDS: • one-carbon metabolism • rat • serine hydroxymethyltransferase • vitamin B-6

Serine hydroxymethyltransferase (SHMT)³ catalyzes the interconversion of serine with glycine and serves to shuttle a one-carbon unit between 5,10-methylenetetrahydrofolate and tetrahydrofolate (THF) (1) for eventual use in cellular methylation reactions and for the synthesis of thymidylate or purines (2,3). In eukaryotes, SHMT is present in both the cytosol and the mitochondria (4). A dissociation constant of 27.5 µmol/L has been reported for the binding of pyridoxal 5'-phosphate (PLP) by apo-SHMT (5), which suggests readily reversible coenzyme binding and the potential for pronounced effects of vitamin B-6 nutritional status on SHMT activity (6).

The 2 isozymes of SHMT exist in cytosolic and mitochondrial compartments (cSHMT and mSHMT, respectively) and may have arisen from a gene duplication event after the divergence of bacterial and eukaryotic proteins (7,8). Although their respective functions have not been fully elucidated, each is thought to have a role in regulating one-carbon metabolism. Communication between the cytosolic and mitochondrial compartments in one-carbon metabolism is achieved using metabolites that can cross the mitochondrial membrane, primarily serine, glycine, and formate (2). Serine is considered the major source of one-carbon units, which are generated through the production of glycine and 5,10-methyleneTHF by both the cytosolic and the mitochondrial forms of SHMT, but primarily by mSHMT (9). In eukaryotic systems, cSHMT generally operates in the direction of serine synthesis, whereas mSHMT primarily works in the opposite direction (10–12). However, in studies of yeast one-carbon metabolism, each SHMT isoform has been reported to be reversible when the supplies of serine and glycine were altered (13). Herbig et al. (14) showed that the level of cSHMT activity in cultured mammalian cells has a role in regulating one-carbon partitioning between methylation reactions and thymidylate synthesis. Girgis et al. (4) examined SHMT expression in human tissues and found a high degree of tissue specificity of cSHMT (highest in liver and kidney) and less tissue-specific variation in mSHMT expression. These investigators proposed that cSHMT is responsible for maintaining a supply of 5,10-methyleneTHF in the cytosol. An alternate hypothesis states that the methyl groups of 5-methylTHF originate from the cytosolic formation of 5,10-methyleneTHF by cSHMT, whereas the one-carbon units for purine biosynthesis originate from the formate produced by the mitochondrial system (12). Evidence that the methyl groups used in the remethylation of homocysteine are generated from both the cytosolic and the mitochondrial pathways in human one-carbon metabolism (15) and in a human breast cancer cell line (14) has been reported.

The objective of this study was to determine the extent to which each of the SHMT isozymes was affected by various degrees of suboptimal vitamin B-6 status. We hypothesized that the mitochondrial enzyme would be shielded from the effects of vitamin B-6 deficiency because of its compartmentalization. We also conjectured that the degree of stimulation of SHMT activity by incubation with PLP, i.e., a stimulation index reflecting the proportion of SHMT in apoenzyme form, would indicate the degree of vitamin B-6 deficiency, as suggested recently by Hansen et al. (16). In addition, we measured the concentration of free hepatic serine, glycine, and methionine as a functional assessment of metabolite pools reflecting changes in one-carbon metabolism.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Materials. [2-³H]Glycine (1.1 TBq/mmol) was obtained from Moravek Biochemicals. Tetrahydrofolate (6RS), PN HCl, PLP, and other chemical reagents were obtained from Sigma Chemical.

    Animals and diets. Weanling male rats (Hsd:Sprague-Dawley SD; Harlan Laboratories), weighing 50 g were housed in hanging wire-mesh stainless-steel cages and maintained at a constant temperature with a 12-h light:dark cycle. This protocol was approved by the University of Florida Institutional Animal Care and Use Committee.

This study was conducted in conjunction with an investigation of the effects of diet composition and vitamin B-6 on intestinal hydrolytic enzymes reported previously (17). Rats (n = 49) were assigned to 10 groups and fed standard rat diet formulations (equally divided between AIN-76A and AIN-93G) modified only with respect to the vitamin B-6 content (18,19). The National Research Council recommendation for vitamin B-6 in purified rat diets for the addition of PN-HCl is 7 mg PN/kg diet (20); however, 2 mg PN/kg diet is nutritionally adequate [e.g., (17)]. The rats in this study were provided free access to diets containing 2, 1, 0.5, 0.1, and 0 mg PN/kg diet for 35 d. Food intake and animal weight were monitored daily. On d 35, the rats were anesthetized by halothane (Alocarbon Laboratories) inhalation and exsanguinated by cardiac puncture. Heparinized needles and syringes were used to collect blood, which was transferred into evacuated tubes containing EDTA. Plasma was obtained by centrifugation at 2000 x g for 20 min and stored at –80°C until analysis. The livers were harvested, cooled on ice, and homogenized immediately in the presence of a general use protease inhibitor cocktail (Sigma). All procedures were performed under gold fluorescent light to minimize the photochemical degradation of vitamin B-6.

    Analytical methods. PLP in liver and plasma was determined using reverse-phase fluorometric HPLC of the semicarbazone derivative by the method of Ubbink et al. (21) with minor modifications.

The concentration of free serine, glycine, and methionine of each liver was determined by reverse-phase HPLC following reaction with DANSYL chloride (22). This method used an octadecylsilyl column (UltraMex 5 C18, 250 x 4.6 mm i.d.; Phenomenex) with a mobile phase consisting of 2.0 mmol/L sodium acetate and 1.5 mL/L phosphoric acid (final pH 3.5) and a nonlinear gradient from 200 to 500 mL/L acetonitrile in 45 min. Peaks were detected using a fluorescence detector (250 nm excitation, 470 nm emission).

    Tissue preparation for enzyme activity assays. Crude liver homogenates containing both cytosolic and dispersed membrane proteins were prepared at 4°C using a glass and Teflon pestle to homogenize 0.5 g liver in 10 vol of buffer containing 0.25 mol/L sucrose, 0.01 mol/L Tris, 0.001 mol/L EDTA, and 10 g/L Triton X-100 at pH 7.4 (23). Homogenization of samples for preparation of cytosolic fractions was conducted in the same manner except with the omission of the detergent Triton X-100. To isolate the cytosolic fraction, the initial homogenate was then centrifuged at 200,000 x g for 45 min and the supernatant was collected. Portions of crude and cytosolic fractions were stored at –80°C until further analysis.

    SHMT activity. SHMT activity assays were conducted at 30°C by measuring the rate of SHMT-catalyzed exchange of the pro-2S proton of purified [³H]glycine in the presence of THF using a minor modification of the method of Kim et al. (24). Assays were performed with reaction mixtures (total volume 110 µL) containing the following final concentrations: 13.6 mmol/L potassium phosphate buffer, pH 7.3, 3.4 mmol/L 2-mercaptoethanol, 0.068 mmol/L ethylenediaminetetraacetate, 4.77 mmol/L THF, and 0.27 mmol/L glycine (providing 68 MBq of [2-³H]glycine). The reactions were terminated by the addition of 10 µL of 100 g/L trichloroacetic acid, and the supernatants were recovered after centrifugation. Each supernatant was applied to a column containing 1 mL (50 mg) of AG 50W-X⁸ ion exchange resin (100–200 mesh, H⁺ form; Bio-Rad Laboratories) which retained residual [³H]glycine and allowed liberated ³H (as ³H₂O) to be eluted into a vial for quantification by liquid scintillation spectrometry. All assays were conducted under conditions that provided linearity in product formation versus time and with rates proportional to enzyme concentration. For each rat, assays were conducted on portions of the liver crude homogenate and cytosol, with mitochondrial SHMT activity assumed to be the difference between the activity in the crude homogenate (designated total activity) and cytosolic activity. Assays for each sample were conducted in parallel reaction mixtures containing either 0 or 0.136 mmol/L added PLP. SHMT activities are presented as pmol of [³H]glycine x min^–1 x g liver^–1.

An inherent assumption in the method of Kim et al. (24) is that the release of the pro-2S proton of glycine is catalyzed only by SHMT and that a background correction for non-SHMT-mediated proton exchange is not necessary. However, such background exchange could complicate the interpretation of SHMT activity measurements (25,26) if not expressly determined. To assess this issue, we conducted an additional experiment in which SHMT assays were conducted on a subset of samples under the standard assay conditions described above (including PLP) except with 50 mmol/L L-serine as a competing substrate. These analyses indicated that the maximum level of background exchange was 8.5%, which was similar to that reported previously for assays of SHMT in human lymphoblasts (26).

    Succinate dehydrogenase activity. Succinate dehydrogenase (SDH) activity in cytosolic and crude homogenate fractions was determined using a modification of the method of Pennington (27) to assess the degree of mitochondrial contamination in cytosolic fractions. A volume of 50 µL of cytosol or crude homogenate was combined in a 2-mL reaction vial with 300 µL of a 0.01 mol/L sodium succinate solution in a 0.05 mol/L phosphate buffer, pH 7.5. After 15 min of incubation at 37°C, 100 µL of 2.5 g/L solution of p-iodonitrotetrazolium violet was added to the reaction vial. The reaction vials were heated in a water bath at 37°C for an additional 10 min. The reaction was stopped by the addition of 1 mL of a 5:5:1 (v:v:w) solution of ethyl acetate:ethanol:trichloroacetic acid. Absorbance was measured at 490 nm. The use of SDH to analyze representative liver preparations as a mitochondrial marker indicated 3.0 ± 9.9% (mean ± SD) contamination of cytosolic fractions (n = 26) by mitochondrial SDH, relative to the total SDH content of crude homogenates.

    Statistical analysis. Differences in plasma and liver PLP and SHMT enzyme activities among the rats fed the diets containing different concentrations of PN were analyzed using one-way ANOVA (28) with the Student-Newman-Keuls pairwise comparison test and SigmaStat software (Jandel). A P value of <0.05 was considered significant. Data are means ± SEM.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Indices of vitamin B-6 status. Mackey et al. (17) reported previously that both liver PLP and plasma PLP concentrations and rat weight gain increased (P < 0.001) with increasing dietary PN concentration in the same rats as evaluated in the current study (Table 1). Rats receiving 0 mg PN/kg diet showed poor weight gain and exhibited classic signs of vitamin B-6 deficiency, including impaired gait and scaling dermatitis on the tail, paws, face, and ears (29,30). These findings demonstrate that this range of dietary PN had the intended effect on vitamin B-6 nutritional status and that the groups demonstrated varying degrees of vitamin B-6 status, ranging from adequate to deficient.

Total SHMT activity in the crude rat liver homogenate increased (P < 0.001) in the absence of exogenous PLP (unstimulated activity) with increasing dietary PN (Fig. 1). The group fed 2 mg PN/kg diet had almost 100% greater activity than the 0 mg PN/kg diet group (2934 ± 157 pmol glycine x min^–1 x g liver^–1 versus 1665 ± 145 pmol glycine x min^–1 x g liver^–1, respectively). All 5 PN treatment groups increased in SHMT activity beyond the unstimulated activity when the assay was performed with exogenous PLP (stimulated activity). The PLP-stimulated activity of the 2 mg PN/kg diet group was 150% of the stimulated activity measured from the deficient group (5097 ± 181 pmol glycine x min^–1 x g liver^–1 and 3290 ± 225 pmol glycine x min^–1 x g liver^–1). The 2 mg/kg and 1 mg/kg groups did not differ in either the basal (P = 0.46) or the stimulated (P = 0.28) states. This is consistent with the minor differences between them in the liver PLP concentration.

View larger version (32K): [in this window] [in a new window]   FIGURE 1 Total serine hydroxymethyltransferase activity in crude liver homogenate of rats receiving graded levels of dietary PN. Values are means ± SEM, n = 10, except 1 mg/kg, n = 9. Bars without a common letter differ, P < 0.05.

View larger version (35K): [in this window] [in a new window]   FIGURE 2 Contribution of liver serine hydroxymethyltransferase isozymes to total measured activity in basal and stimulated states in rats receiving graded levels of dietary PN. Values are means ± SEM, n = 10, except 1 mg/kg, n = 9. Bars without a common letter differ, P < 0.05.

    Coenzyme saturation of SHMT. Measurement of a stimulation index is a commonly used technique for assessing the degree of coenzyme saturation of vitamin-dependent enzymes because the degree of stimulation of activity by the added coenzyme (PLP) reflects the fraction of the total SHMT pool that is initially present as the inactive apoenzyme. The stimulation index for total SHMT activity did not differ (P = 0.38) among the 5 dietary PN groups (Fig. 3). In addition, dietary PN concentration did not influence the stimulation index for either cSHMT (P = 0.58) or mSHMT (P = 0.63). These data indicate that cSHMT and mSHMT exist with 65 and 50% of their respective SHMT pools in apoenzyme form (P < 0.001) and that the fraction in apoenzyme form is not affected by vitamin B-6 status over the broad range examined.

View larger version (29K): [in this window] [in a new window]   FIGURE 3 Stimulation index of liver serine hydroxymethyltransferase activity as a function of dietary PN concentration in rats receiving graded levels of dietary PN. The stimulation index is the ratio of SHMT activity assayed with added PLP/activity without added PLP. Values are means ± SEM, n = 10, except 1 mg/kg, n = 9.

View larger version (12K): [in this window] [in a new window]   FIGURE 4 Total basal serine hydroxymethyltransferase activity as a function of plasma PLP in rats receiving graded levels of dietary PN (n = 49, r2 = 0.466, P < 0.001).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

As expected, the activity of total SHMT (i.e., mitochondrial + cytosolic) in the basal, unstimulated state declined with decreasing dietary PN concentration. Rats consuming diets containing deficient to suboptimal PN (0–0.5 mg/kg) had less hepatic PLP, which yielded a smaller pool of active holoenzyme. The addition of exogenous PLP revealed that the apoenzyme pool, as a percentage of total SHMT, remained approximately constant along the continuum of vitamin B-6 status. However, the finding that the total enzyme pool decreased with decreasing dietary PN was unexpected. The mechanism responsible for this phenomenon is unknown, but it may reflect increased degradation or reduced synthesis of the enzyme. The active form of SHMT is a tetramer and described as a "dimer of dimers" (34). In the absence of bound PLP, dimer formation increases (35). The dimers of SHMT may be preferentially sequestered for lysosomal degradation in a manner similar to the apo-form of cystathionase (36). Sato et al. (36) postulated that the conformational change in a ligand-free enzyme may expose certain domains that exhibit greater recognition by lysosomes; however, increased turnover of dissociated SHMT has not been reported. The decreased enzyme pool may also illustrate a role of vitamin B-6 in regulation of enzyme expression, which has been suggested with other enzymes and genes (37–40). Differences among tissues in the proportion of cSHMT relative to mSHMT (4) provide evidence that these genes exhibit differences in transcriptional regulation.

This variable total enzyme pool with an approximately constant proportion of apoenzyme pool distinguishes cSHMT and mSHMT from other PLP-requiring enzymes such as alanine aminotransferase and aspartate aminotransferase (41). If the SHMT activity in accessible tissues, such as blood cellular fractions, showed behavior similar to that observed in the liver, this would make SHMT a poor choice for determination of a stimulation index for nutritional assessment purposes, as has been proposed (16).

An interesting observation was made when the stimulation index of each isozyme was examined individually (Fig. 3). Although cSHMT and mSHMT exhibited different proportions of apoenzyme, there were no differences in stimulation index between adequate and deficient treatment groups for either SHMT isozyme. Furthermore, our results indicate that cSHMT and mSHMT are not saturated with PLP at PN intakes that are adequate in terms of supporting rat growth (1–2 mg PN/kg).

A direct correlation between PN intake and SHMT activity in vitro is demonstrated, yet these results do not necessarily predict in vivo flux. This laboratory previously examined the in vivo flux of homocysteine remethylation and transsulfuration in vitamin B-6 adequate (2 mg/kg PN) and deficient (0.1 mg/kg PN) rats in addition to hepatic total SHMT activity (6). The deficient group demonstrated a 40% reduction in total SHMT activity and 80% reduction in flux of one-carbon units from serine to methionine via SHMT. Although the previous protocol may not be totally relevant to the present study due to its acute administration of tracers and perturbation of metabolic pools inherent in the flooding dose technique, these results demonstrate the potential for effects of severe vitamin B-6 deficiency on the function of SHMT. Two human vitamin B-6 depletion studies conducted by this laboratory have shown no discernible effects of a moderate vitamin B-6 deficiency state on the overall in vivo rate of homocysteine remethylation in the absence of simultaneous methionine intake during steady-state tracer infusions (42,43). These data concur with a large body of data that indicate little effect of vitamin B-6 status on fasting plasma homocysteine concentration (44). Rather, vitamin B-6 deficiency is more likely to affect homocysteine metabolism when the pathways are challenged as experienced following a methionine load test (45) or after a meal high in protein.

Complete interpretation of the in vitro data requires an evaluation of the functional in vivo consequences of changes in SHMT activity. The large increases observed in hepatic free glycine and in the glycine/serine ratio as a function of dietary PN concentration provide evidence that SHMT activity is altered in vivo in both marginal and more pronounced states of vitamin B-6 deficiency. Similar effects of vitamin B-6 depletion on plasma amino acids have been reported previously (31,46,47). In total, these findings indicate that SHMT in vivo is highly sensitive to vitamin B-6 nutritional status. The observation that hepatic methionine concentration was not influenced by vitamin B-6 intake, despite a functional alteration in SHMT activity, suggests no deficit in one-carbon units for use in overall remethylation processes under the condition of this study. The activity of other PLP-dependent enzymes involved in glycine synthesis and catabolism, including the glycine cleavage system and alanine:glyoxylate aminotransferase, also could be affected by reduced vitamin B-6 intake and could be involved in this elevation in plasma and liver glycine concentration. Further studies will assess the in vivo effects of these vitamin B-6 dependent changes in the activity of SHMT and these other enzymes under conditions requiring additional supply of one-carbon units.

	FOOTNOTES

 
¹ Supported by United States Department of Agriculture National Research Initiative Grant 00–35200-9113 and National Institutes of Health Grants DK37481 and T32 DK07667. This paper is Florida Agricultural Experiment Station Journal Series No. R-10401.

³ Abbreviations used: PLP, pyridoxal 5'-phosphate; PN, pyridoxine; SDH, succinate dehydrogenase; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate.

Manuscript received 18 August 2004. Initial review completed 14 September 2004. Revision accepted 19 November 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Schirch, V. (1982) Serine hydroxymethyltransferase. Adv. Enzymol. Relat. Areas Mol. Biol. 53:83-112.[Medline]

2. Appling, D. (1991) Compartmentation of folate-mediated one-carbon metabolism in eukaryotes. FASEB J. 5:2645-2651.[Abstract]

3. Schirch, V. & Strong, W. B. (1989) Interaction of folylpolyglutamates with enzymes in one-carbon metabolism. Arch. Biochem. Biophys. 269:371-380.[Medline]

4. Girgis, S., Nasrallah, I. M., Suh, J. R., Oppenheim, E., Zanetti, K. A., Mastri, M. G. & Stover, P. (1998) Molecular cloning characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene. Gene 210:315-324.[Medline]

5. Jones, C. & Priest, D. (1978) Interaction of pyridoxal 5'-phosphate with apo-serine hydroxymethyltransferase. Biochem. Biophys. Acta 526:369-374.[Medline]

6. Martinez, M., Cuskelly, G. J., Williamson, J., Toth, J. P. & Gregory, J. F. (2000) Vitamin B-6 deficiency in rats reduces hepatic serine hydroxymethyltransferase and cystathionine beta-synthase activities and rate of in vivo protein turnover, homocysteine remethylation, and transsulfuration. J. Nutr. 130:1115-1123.[Abstract/Free Full Text]

7. Garrow, T., Brenner, A. A., Whitehead, M., Cen, X., Duncan, R., Korenberg, J. & Shane, B. (1993) Cloning of human cDNAs encoding mitochondrial and cytosolic serine hydroxymethyltransferases and chromosomal localization. J. Biol. Chem. 268:11910-11916.[Abstract/Free Full Text]

8. Usha, R., Savithri, H. S. & Rao, N. A. (1994) The primary structure of sheep liver cytosolic serine hydroxymethyltransferase and analysis of the evolutionary relationships among SHMTs. Biochim. Biophys. Acta 1204:75-83.[Medline]

9. Stover, P. & Schirch, V. (1991) 5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase. J. Biol. Chem. 266:1543-1550.[Abstract/Free Full Text]

10. Narkewicz, M., Sauls, S. D., Tjoa, S. S., Teng, C. & Fennessey, P. V. (1996) Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells. Biochem. J. 313:991-996.

11. Jeong, S. S. & Schirch, V. (1996) Role of cytosolic serine hydroxymethyltransferase in one-carbon metabolism in Neurospora crassa. Arch. Biochem. Biophys. 335:333-341.[Medline]

12. Piper, M., Hong, S. P., Ball, E. & Dawes, I. W. (2000) Regulation of the balance of one-carbon metabolism in Saccharomyces cerevisiae. J. Biol. Chem. 275:30987-30995.[Abstract/Free Full Text]

13. Kastanos, E., Woldman, Y. & Appling, D. (1997) Role of mitochondrial and cytoplasmic serine hydroxymethyltransferase isozymes in do novo purine synthesis in Saccharomyces cerevisiae. Biochemistry 36:14956-14964.[Medline]

14. Herbig, K., Chiang, E., Lee, L., Hills, J., Shane, B. & Stover, P. (2002) Cytoplasmic serine hydroxymethyltransferase mediates competition between folate-dependent deoxyribonucleotide and S-adenosylmethionine biosyntheses. J. Biol. Chem. 277:38381-38389.[Abstract/Free Full Text]

15. Gregory, J. F., Cuskelly, G. J., Shane, B., Toth, J. P., Baumgartner, T. G. & Stacpoole, P. W. (2000) Primed, constant infusion with [²H₃]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation and transsulfuration and processes. Am. J. Clin. Nutr. 72:1535-1541.[Abstract/Free Full Text]

16. Hansen, C., Shultz, T., Hunt, K., Hardin, K., Leklem, J., Huang, A. & Ames, B. (2003) Vitamin B-6 intake and smoking status influence lymphocyte serine hydroxymethyltransferase (SHMT) activity in healthy adults. FASEB J. :A277.

17. Mackey, A., Lieu, S., Carmen, C. & Gregory, J. F. (2003) Hydrolytic activity toward pyridoxine-5'-ß-D-glucoside in rat intestinal mucosa is not increased by vitamin B-6 deficiency: effect of basal diet composition and pyridoxine intake. J. Nutr. 133:1362-1367.[Abstract/Free Full Text]

18. American Institute of Nutrition (1977) Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107:1340-1348.

19. Reeves, P., Rossow, K. & Lindlauf, J. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1923-1931.

20. Subcomittee on Laboratory Animal Nutrition Committee on Animal Nutrition Board on Agriculture National Research Council (1995) Nutrient Requirements of Laboratory Animals 4th revised ed. 1995 National Academy Press Washington, DC.

21. Ubbink, J., Serfontein, W. J. & de Villiers, L. S. (1985) Stability of pyridoxal 5'-phosphate semicarbazone: application in plasma vitamin B-6 analysis and population surveys of vitamin B-6 nutritional status. J. Chromatogr. 342:277-284.[Medline]

22. Tapuhi, Y., Schmidt, D. E., Linder, W. & Karger, B. L. (1981) Dansylation of amino acids for high-performance liquid chromatography analysis. Anal. Biochem. 115:123-129.[Medline]

23. Graham, J. M. (1997) Graham, J. M. Rickwood, D. eds. Subcellular Fractionation—A Practical Approach 1997:1-29 Oxford University Press Oxford, UK .

24. Kim, D., Fratte, S. D., Jeong, S. S. & Schirch, V. (1997) Determination of serine hydroxymethyltransferase and reduced folate pools in tissue extracts. Anal. Biochem. 253:201-209.[Medline]

25. Stover, P., Chen, L. H., Suh, J. R., Stover, D. M., Keyomarsi, K. & Shane, B. (1997) Molecular cloning, characterization, and regulation of the human mitochondrial serine hydroxymethyltransferase gene. J. Biol. Chem. 272:1842-1848.[Abstract/Free Full Text]

26. Elsea, S. H., Juya, R. C., Jiralerspong, S., Finucane, B. M., Pandolfo, M., Greenberg, F., Baldini, A., Stover, P. & Patel, P. I. (1995) Haploinsufficiency of cytosolic serine hydroxymethyltransferase in the Smith-Magenis Syndrome. Am. J. Hum. Genet. 57:1342-1350.[Medline]

27. Pennington, R. (1961) Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem. J. 80:649-654.[Medline]

28. Neter, J. & Wasserman, W. (1974) Applied Linear Statistical Models 1974 Irwin Homewood, IL.

29. Sherman, H. (1954) Pyridoxine and related compounds. Sebrell, W. Harris, R. eds. Vitamins 1954:265-276 Academic Press New York, NY. .

30. Schaeffer, M., Cochary, E. & Sadowski, J. (1990) Subtle abnormalities of gait detected early in vitamin B-6 deficiency in aged and weanling rats with hind gait analysis. J. Am. Coll. Nutr. 9:120-127.[Abstract]

31. Schaeffer, M. C., Gretz, D., Mahuren, J. D. & Coburn, S. P. (1995) Tissue B-6 vitamer concentrations in rats fed excess vitamin B-6. J. Nutr. 125:2370-2378.

32. Trumbo, P. R. & Gregory, J. F. (1988) Metabolic utilization of pyridoxine-ß-glucoside in rats: influence of vitamin B-6 status and route of administration. J. Nutr. 118:1336-1342.

33. Stabler, S. P., Sampson, D. A., Wang, L.-P. & Allen, R. H. (1997) Elevations of serum cystathionine and total homocysteine in pyridoxine-, folate-, and cobalamin-deficient rats. J. Nutr. Biochem. 8:279-289.

34. Snell, K., Baumann, U., Byrne, P., Chave, K. J., Renwick, S., Sanders, P. & Whithouse, S. K. (2000) The genetic organization and protein crystallographic structure of human serine hydroxymethyltransferase. Adv. Enzyme Regul. 40:353-403.[Medline]

35. Jagath, J. R., Sharma, B., Rao, N. A. & Savithri, H. S. (1997) The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase. J. Biol. Chem. 272:24355-24362.[Abstract/Free Full Text]

36. Sato, A., Nishioka, M., Awata, S., Nakayama, K., Okada, M., Horiuchi, S., Okabe, N., Sassa, T., Oka, T. & Natori, Y. (1996) Vitamin B-6 deficiency accelerates metabolic turnover of cystathionase in rat liver. Arch. Biochem. Biophys. 330:409-413.[Medline]

37. Allgood, V., Powell-Oliver, F. & Cidlowski, J. (1990) Vitamin B-6 influences glucocorticoid receptor-dependent gene expression. J. Biol. Chem. 265:12424-12433.[Abstract/Free Full Text]

38. Oka, T., Komori, N., Kuwahata, M., Sassa, T., Suzuki, I., Okada, M. & Natori, Y. (1993) Vitamin B-6 deficiency causes activation of RNA polymerase and general enhancement of gene expression in rat liver. FEBS Lett. 331:162-164.[Medline]

39. Oka, T., Komori, N., Kuwahata, M., Okada, M. & Natori, Y. (1995) Vitamin B-6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding protein in rat liver. Biochem. J. 309:243-248.

40. Oka, T., Komori, N., Kuwahata, M., Suzuki, I., Okada, M. & Natori, Y. (1994) Effect of vitamin B-6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle. Experientia 50:127-129.[Medline]

41. Leklem, J. (2001) Vitamin B-6. Rucker, R. Suttie, J. McCormick, D. Machlin, L. eds. Handbook of Vitamins 3rd ed. 2001:339-396 Dekker New York, NY. .

42. Cuskelly, G. J., Stacpoole, P. W., Williamson, J., Baumgartner, T. G. & Gregory, J. F. (2001) Deficiencies of folate and vitamin B-6 exert distinct effects on homocysteine, serine, and methionine kinetics. Am. J. Physiol. Endocrinol. Metab. 281:E1182-E1190.[Abstract/Free Full Text]

43. Davis, S. R., Scheer, J. B., Quinlivan, E. P., Coats, B. S., Stacpoole, P. W. & Gregory, J. F. (2004) Dietary vitamin B6 restriction does not alter rates of homocysteine remethylation or synthesis in healthy young women and men. Am. J. Clin. Nutr. (in press).

44. McKinley, M. (2000) Nutritional aspects and possible pathological mechanisms of hyperhomocysteinaemia: an independent risk factor for vascular disease. Proc. Nutr. Soc. 59:221-237.[Medline]

45. Selhub, J. (1999) Homocysteine metabolism. Annu. Rev. Nutr. 19:217-246.[Medline]

46. Swendseid, M., Villalobos, J. & Friedrich, B. (1964) Free amino acids in plasma and tissues of rats fed a vitamin B-6 deficient diet. J. Nutr. 82:206-208.[Medline]

47. Wolfson, M., Laidlaw, S., Flugel-Link, R., Strong, C., Salusky, E. & Kopple, J. (1986) Effect of vitamin B-6 deficiency on plasma amino acid levels in chronically azotemic rats. J. Nutr. 116:1865-1872.

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