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Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5'-phosphate concentrations.

Chronic imbalance in the integrated pathways of one-carbon metabolism as a consequence of genetic aberrations and/or nutritional insufficiencies underlies the pathogenesis of many chronic human disease states (1-5). Heterozygous or homozygous mutations in genes coding for enzymes that participate directly or indirectly in one-carbon metabolism, such as methylenetetrahydrofolate reductase (MTHFR), [1] methionine synthase reductase, or cystathionine [beta]-synthase (CBS), can interact with inadequate dietary intake to exacerbate metabolic pathology (3, 6, 7). Gene-nutrient interactions that compromise normal one-carbon metabolism are associated with increased risk of cardiovascular disease (2), certain cancers (8), birth defects (9, 10), recurrent early pregnancy loss (11), central nervous system demyelinization (12), and neuropsychiatric disease (13, 14). In many cases, appropriate nutritional intervention can normalize metabolic imbalance and retard the progression of pathology. Early diagnosis and preventive intervention strategies depend on the development of simple, sensitive, and reproducible methodologies to monitor plasma concentrations of homocysteine and the multiple thiols involved in homocysteine and methionine metabolism. For example, a new method utilizing HPLC with coulometric electrochemical (EC) detection for the simultaneous measurement of plasma methionine, homocysteine, cysteine, cystathionine, cysteinylglycine, and glutathione could aid in differential diagnoses and in the design of intervention strategies (15). Rapid coulometric EC detection of homocysteine has also been reported recently (16).

An abbreviated overview of one-carbon metabolism with emphasis on essential cellular methylation reactions is presented in Fig. 1. Methionine is converted to S-adenosylmethionine (SAM), the major intracellular methyl donor, by methionine adenosyltransferase (EC 2.5.1.6) and subsequently to S-adenosylhomocysteine (SAH) by a variety of cellular methyltransferases present in all cells. This one-way reaction is subject to competitive product inhibition by SAH because SAH has a higher affinity for the methyltransferase active site than does its precursor, SAM (17). The pathologic accumulation of SAH can lead to a decrease in the SAM/SAH ratio and inhibition of most cellular methyltransferases (17-19). SAH is hydrolyzed to homocysteine and adenosine by SAH hydrolase (EC 3.3.1.1), a reversible reaction with thermodynamics that actually favor SAH synthesis (20, 21). Accumulation of SAH and the associated inhibition of cellular methyltransferases will therefore occur under metabolic conditions that interfere with product removal of homocysteine or adenosine (17,22-25).

[FIGURE 1 OMITTED]

Although the regulatory role of SAM on the enzyme activities of MTHFR, CBS, and methionine synthase has been emphasized (21, 22, 26), the regulatory importance of SAH in the maintenance of balanced one-carbon metabolism may be underestimated. Under physiologic conditions, SAH concentrations generally are several-fold lower than SAM concentrations, and SAH concentrations in plasma have only recently become detectable with newer methods (27, 28). Alterations in cytosolic SAH have tissue-specific bioregulatory functions and have been reported to up-regulate CBS activity (18), decrease betaine-homocysteine methyltransferase activity (18), and decrease MTHFR activity (29). Inhibition of the several methyltransferase reactions by SAH would be expected to spare the substrate, SAM. Consistent with this notion, an increase in SAM with increased intracellular SAH has been observed in end stage renal patients (30) and in cultured mouse lymphoma cells (31). Homozygous mutations in the CBS gene (32) or the adenosine deaminase gene (33) lead to substantial increases in homocysteine and SAH without affecting methionine synthase activity. By contrast, drugs or nutritional deficiencies that negatively affect the methionine synthase reaction are generally associated with a decrease in SAM and an increase in homocysteine and SAH (34, 35). The extent of reduction in SAM concentrations with folate or [B.sub.12] deficiencies, however, does not approach the [K.sub.m] of most methyltransferases (17) and therefore may be less likely to reduce methyltransferase activity than an increase in SAH. An effect of SAH on cellular methylation is most likely in nonhepatic tissues in which the concentration of SAH is more variable than SAM (21).

Although several HPLC methods exist for the simultaneous measurement of SAM and SAH, most of these methods rely on precolumn derivatization and ultraviolet detection, which are more time-consuming and less sensitive than direct injection using coulometric EC detection (36-39). In addition, these methods often require the use of internal standards to correct for sample losses during preparative procedures (27). In this report, we present a new method in which neutralized trichloroacetic acid (TCA) extracts are injected directly into the HPLC system without any further manipulation. SAM and SAH are separated by isocratic elution and coulometric EC detection to provide femtomolar detection limits with exceptional reproducibility and recovery.

Materials and Methods

REAGENTS

SAM, SAH, TCA, sodium phosphate monobasic, monohydrate, and 1-heptanesulfonic acid were obtained from Sigma. HPLC grade methanol was purchased from J.T. Baker. Deionized HPLC-grade water was prepared by passage through a Syrbon/Barusted NANOpure II filtration system and subsequent passage through a [C.sub.18] SepPak cartridges (Millipore).

SUBJECTS AND SAMPLE PREPARATION

Subjects were 58 healthy adult females with a mean age of 37.2 years (range, 19-53 years) who had participated in a previous clinical trial (40). Fasted blood samples were collected into EDTA Vacutainer Tubes, chilled immediately in an ice-water bath, and centrifuged at 4100g for 15 min at 4[degrees]C. Aliquots of the plasma layer were transferred into multiple cryostat tubes and stored at -20[degrees]C until analysis. Separate aliquots were thawed for determination of plasma homocysteine, pyridoxal 5'-phosphate (PLP), SAM, and SAH. In a subset of women, mononuclear cells were isolated by carefully layering whole blood onto an equal volume of Histopaque[R]-1077 (Sigma Diagnostics) and centrifuged at 400g for 30 min. Mononuclear cells were recovered from the interface and washed several times as described by the manufacturer. Samples of liver tissue (10-15 mg wet weight) or isolated lymphocytes ([10.sup.6] cells) were homogenized with 200 [micro]L of phosphate-buffered saline. To precipitate protein, 40 [micro]L of 400 g/L TCA was added to 200 [micro]L of plasma or cell extract, mixed well, and incubated on ice for 30 min. After centrifugation for 15 min at 18 000g at 4[degrees]C, supernatants were filtered through a 0.2 [micro]m filter, and 20 [micro]L was injected into the HPLC system.

CHROMATOGRAPHY

Separation of SAM and SAH in plasma and cell extracts was accomplished by HPLC with a Shimadzu solvent delivery system (ESA model 580) and a reversed-phase [C.sub.18] column (5 [micro]m bead size; 4.6 x 150 mm; MCM) obtained from ESA. Isocratic elution with a mobile phase consisting of 50 mmol/L sodium phosphate monobasic, monohydrate; 10 mmol/L heptanesulfonic acid; and 75 mL/L methanol adjusted to pH 3.4 with concentrated phosphoric acid, was performed at ambient temperature at a flow rate of 1.0 mL/min and a pressure of 100-110 kfg/[cm.sup.2] (1500-1800 psi). Extracts were injected directly onto the column using a Beckman autosampler (model 507E). To assure standardization between sample runs, calibration and reference plasma samples were interspersed at intervals during each run. Total homocysteine (tHcy) and cysteine concentrations were quantified using HPLC and coulometric EC detection as described previously in detail (15).

COULOMETRIC EC DETECTION

After HPLC separation, detection of SAM and SAH was accomplished using a model 5200A Coulochem II electrochemical detector (ESA) equipped with a dual analytical cell (model 5010) and a guard cell (model 5020). A guard cell was placed in line before the injector to remove oxidizable impurities present in the mobile phase that might compromise baseline stability. The dual analytical cell contained two porous graphite electrodes in series. The first electrode (E1) is used as an oxidative screen and was set at a lower voltage than the second electrode (E2) to remove interfering compounds that oxidize at lower potentials than the compounds of interest. For selectivity, E2 was set at or above the established oxidation potential of the compounds of interest. For optimum detection of SAM and SAH, the electrode potentials for the guard cell, electrode E1, and electrode E2 were set at +1000 mV, +400 mV, and +920 mV, respectively. The current generated at E2 represents the oxidation of the active species between +400 mV and +920 mV, which encompasses the peak oxidative range for both SAM and SAH. These potentials provide peak area response with minimum background and are the basis for quantification. Peak area analysis was provided by GOLD Nouveau software (Beckman Instruments) based on calibration curves generated for each compound. Each day, electrode sensitivity and baseline stability were confirmed by the application of -400 mV at each electrode for 30 min with the mobile phase at 1.5 mL/min, followed by a 30-min water rinse at 1 mL/min with the electrodes turned off, and a final 30-min rinse with 500 mL/L methanol (electrodes off). Before injection of the first sample, the potential at the electrodes was increased in a stepwise fashion to the final working potential and the HPLC-EC system was equilibrated for ~1 h with the mobile phase at 1 mL/min. The use of an autosampler is highly recommended and allows continuous sampling overnight. When not running samples, the mobile phase was set at a rate of 0.2 mL/min with lower voltages of +50 mV, +100 mV, and +200 mV at E1, E2, and the guard cell, respectively.

[FIGURE 2 OMITTED]

CURRENT/VOLTAGE CURVES, CALIBRATION CURVES, AND LIMITS OF DETECTION

Curves reflecting the current generated by the oxidation of SAM and SAH with increasing voltage at E2 were used to estimate the respective voltage requirements for peak sensitivity and reproducibility. Linear calibration curves for SAM and SAH in 100 [micro]mol/L HCl were generated in the following physiologic ranges for each compound: 1-600 nmol/L for SAM, and 0.2-100 nmol/L for SAH. Before each analysis, calibration curves were generated from aliquots of frozen calibrators and examined for reproducibility.

[FIGURE 3 OMITTED]

RECOVERY, PRECISION, AND STATISTICAL ANALYSIS

To determine analytical recovery, known concentrations of SAM and SAH within the physiologic range were added to plasma and mouse liver tissue extracts. The concentrations of SAM and SAH in the supplemented samples were determined in five independent samples, and the mean quantitative recoveries were calculated. To determine the intraassay precision, 10 replicates of the same sample were analyzed in a single analytical run. The intraassay precision was determined by analyzing aliquots from a single sample on 10 different days over a 1-month period. The CV was calculated as the standard deviation expressed as a percentage of the mean values. Statistical differences between means were calculated using the Student t-test and Sigmastat software (Jandel Scientific).

PLP

Concentrations of PLP were determined in fasting plasma by the radioenzymatic assay described previously in detail (41).

Results

CHROMATOGRAPHY

Details of the theory and advantages of coulometric EC detection over amperometric EC detection have been described previously (15). Based on the current/voltage curve in Fig. 2A, the voltage was set at 400 mV and 920 mV for electrodes E1 and E2, respectively, for maximum sensitivity and reproducibility without damage to the electrodes. In Fig. 2, B and C, the linearity of detection within the physiologic range for each compound was confirmed. Typical chromatograms from calibrators, plasma extracts, and mouse liver extracts are presented in Fig. 3, A, B, and C, respectively. The concentration of the ion-pairing reagent, heptanesulfonic acid, was critical for optimum separation. The retention times were strongly affected by the pH and methanol concentration in the mobile phase. Under the conditions selected, the retention times for SAH and SAM were ~10.5 and 20.5 min, respectively. Excellent separation without interfering peaks was achieved in both plasma and cell extracts, as shown in Fig. 3. With EC detection, the selectivity for SAM and SAH was greatly enhanced at E2 by the oxidation and elimination of interfering compounds at E1. Biological samples required ~35 min between injections to eliminate a large unidentified peak that elutes at ~30 min.

LIMITS OF DETECTION, PRECISION, AND RECOVERY

The limit of detection for the calibrators, defined as the concentration that produced a signal-to-noise ratio >5, was 200 fmol/L for SAM and 40 fmol/L for SAH. These limits of detection are several-fold lower than those reported previously with methods using derivatization and ultraviolet detection (27, 28, 38). The interassay/intraassay precision and product recovery are presented in Table 1. In plasma extracts, the mean within-run (intraassay) CV was 3.4% for SAM and 5.5% for SAH, with mean recoveries of 96.8% for SAM and 97.3% for SAH. In mouse liver extracts; the mean intraassay CV was 6.1% for SAM and 8.8% for SAH, with mean recoveries of 95.2% for SAM and 97.1% for SAH. The mean between-run (interassay) CV in plasma extracts was 2.8% for SAM and 5.6% for SAH; for mouse liver extracts, the mean interassay CV was 7.3% for SAM and 7.6% for SAH.

PLASMA SAM, SAH, PLP, AND CYSTEINE CONCENTRATIONS IN WOMEN WITH NORMAL AND INCREASED tHcy

In Table 2, plasma SAM, SAH, PLP, and cysteine are shown as a function of fasting plasma tHcy in the 58 young women. The women were stratified by tHcy based on previously published reference values for adult females (42, 43). In women with normal tHcy values (mean, 7.3 [+ or -] 1.1 [micro]mol/L; range, 5.8-8.7 [micro]mol/L), the mean SAM and SAH concentrations were 155.9 and 20.0 nmol/L, respectively, with a mean SAM/SAH ratio of 8.5. In women with increased tHcy (mean, 12.3 [+ or -] 1.8 [micro]mol/L; range, 9.3-16.5 [micro]mol/L), SAM concentrations were not altered, but SAH concentrations were increased twofold relative to women with normal tHcy, and the SAM/SAH ratio was decreased by one-half. Interestingly, increased tHcy was associated with significantly reduced concentrations of plasma PLP and cysteine in this cohort of women (P <0.001). A significant negative correlation was observed when SAM/SAH ratios were plotted as a function of tHcy concentrations (r = 0.73; P <0.01; Fig. 4).

INTRACELLULAR SAM AND SAH CONCENTRATIONS AND SAM/SAH RATIO IN HUMAN LYMPHOCYTES AND MOUSE LIVER EXTRACTS

The applicability of the assay for measurement of SAM and SAH in cell and tissue extracts is presented in Table 3. Lymphocytes were isolated from whole blood obtained from a subset of women with a mean plasma tHCy of 7.8 [+ or -] 1.1 [micro]mol/L and a mean plasma SAM/SAH ratio of 8.8 [+ or -] 1.6 (n = 7). The mean intracellular SAM/SAH ratio (9.4 [+ or -] 3.1) was not significantly different from the plasma ratio. Fig. 5 is a scatter plot of the SAM/SAH ratios in plasma and in lymphocytes obtained from the same individuals. Regression analysis revealed a significant positive association (r = 0.70; P <0.01), suggesting that the plasma SAM/SAH ratios may be a convenient reflection of the intracellular ratio in lymphocytes. In the mouse liver samples, expression of SAM and SAH concentrations per milligram of protein produced smaller variation than expression per gram wet weight. The values obtained for mouse liver TCA extracts were within previously published ranges obtained with a different methodology (35).

[FIGURE 4 OMITTED]

Discussion

The intracellular concentrations of SAM and SAH, as the substrate and product of essential cellular methyltransferase reactions, are important metabolic indicators of the cellular methylation capacity (17, 30, 44). Although emphasis has been placed on the toxicity of increased tHcy and depressed SAM concentrations in metabolic pathology of various diseases (22, 26, 45), a chronic increase in SAH, secondary to the homocysteine-mediated reversal of the SAH hydrolase reaction, may also have significant, albeit indirect, pathologic consequences. Because SAH can bind to and inhibit multiple cellular methyltransferases, increased SAH concentrations can lead to DNA hypomethylation and altered chromatin configuration, reduced membrane phosphatidylcholine concentrations, reduced protein and RNA methylation, and reduced neurotransmitter synthesis (17,19, 22, 30, 46). These alterations have been associated with functional abnormalities, including inappropriate gene expression, altered signal transduction, immune deficiency, and cytotoxicity (21, 31, 47-49).

The results presented in Table 2 indicate that increased plasma tHcy was associated with a twofold increase in SAH, no change in SAM, and a twofold decrease in the SAM/SAH ratio in this cohort of women. Whether the decrease in SAM/SAH is of sufficient magnitude to affect methyltransferase activity and cellular methylation reactions cannot be determined from the present data. Nonetheless, these observations are consistent with a homocysteine-mediated reversal of the SAH hydrolase reaction. The regression analysis in Fig. 4 indicates that plasma tHcy concentrations are inversely correlated with SAM/ SAH ratios and suggests the possibility that tHcy could have indirect effects on cellular methylation potential. The decreases in plasma PLP concentrations in these women further suggest the possibility that nutritional vitamin [B.sub.6] deficiency may have contributed to the increase in tHcy; the decrease in cysteine concentrations is consistent with this interpretation. Similar increases in SAH concentrations associated with [B.sub.6] deficiency previously have been observed in rat liver (28). In Fig. 5, the significant positive correlation between plasma and lymphocyte SAM/SAH ratios suggests that the plasma SAM/SAH ratio may be a reasonable reflection of the intracellular ratio. In lymphocytes, export of SAH has been well documented and appears to be carrier-mediated and largely unidirectional (50). The kidney appears to be the only route for SAH removal from plasma (51). The origin of SAM in plasma is still an open and interesting question because export from the hepatocytes does not occur despite the fact that more SAM is used for creatine synthesis in the liver than in all extrahepatic tissues combined (52).

[FIGURE 5 OMITTED]

The mean plasma concentration of SAM detected with the present HPLC-EC method in women with normal tHcy was 156 nmol/L, which is considerably higher than previously published values of 26.5 nmol/L (38), 60 nmol/L (30), and 102 nmol/L (27) in plasma from healthy individuals obtained with other methods of detection. The higher values with the present method most likely reflect the direct injection of the extract without the need for precolumn processing and derivatization, which can lead to significant losses. The SAM peak was completely eliminated by boiling the extract for 5 min before injection, suggesting that coelution of a contaminating metabolite is unlikely, although this possibility cannot be definitively ruled out. Adenosine elutes before the SAH peak, at ~8 min. The mean plasma concentration of SAH obtained with the present method was 20 nmol/L and is within the ranges reported previously using other methods (27). The limits of detection using the coulometric EC detector were 200 fmol/L for SAM and 40 fmol/L for SAH. These values far exceed previous ultraviolet detection limits of 10 nmol/L (30),25 pmol (28), and 5 pmol/L (27) for both SAM and SAH. Using the present method, we obtained a SAM/SAH ratio of 8.5 for plasma from healthy individuals, which is considerably higher than previously reported values (38). A disadvantage of the present method is that 40 min is required for complete elution of the accompanying peaks in biological samples; however, the precolumn processing time is minimal, and with the use of an autoinjector, ~20 samples per day can be processed. Because of the relatively long elution times, this method for measurement of SAM and SAH is best suited for research purposes and refinement of diagnoses rather than routine clinical analyses that require high throughput methodology. The ability to sensitively and reproducibly detect these important metabolites in plasma should provide new insights into the differential regulation of one-carbon metabolism with specific nutritional deficiencies and specific genetic polymorphisms.

This work was supported by a grant from the US Food and Drug Administration Office of Women's Health (to S.J.J.) and by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the US Food and Drug Administration (S.M.). We thank Dr. James D. Finkelstein for critical reading of the manuscript and insightful comments.

Received October 25, 1999; accepted December 1, 1999.

References

(1.) Zittoun J, Tonetti C, Bories D, Pignon JM, Tulliez M. Plasma homocysteine levels related to interactions between folate status and methylenetetrahydrofolate reductase: a study in 52 healthy subjects. Metabolism 1998;47:1413-8.

(2.) Christensen B, Frosst P, Lussier-Cacan S, Selhub J, Goyette P, Rosenblatt DS, et al. Correlation of a common mutation in the methylenetetrahydrofolate reductase gene with plasma homocysteine in patients with premature coronary artery disease. Arterioscler Thromb Vasc Biol 1997;17:569-73.

(3.) Wilson A, Platt R, Wu RK, Leclerc D, Christensen B, Yang HT, Rozen R. A common variant in methionine synthase reductase combined with low cobalamin (vitamin 1312) increases risk for spina bifida. Mol Genet Metab 1999;67:317-23.

(4.) Ma J, Stampfer MJ, Giovannucci E, Artigas C, Hunter DJ, Fuchs C, et al. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res 1997;57: 1098-102.

(5.) Dierkes J, Domrose U, Ambrosch A, Schneede J, Guttormsen AB, Neumann KH, Luley C. Supplementation with vitamin 1312 decreases homocysteine and methylmalonic acid but also serum folate in patients with end-stage renal disease. Metabolism 1999;48:631-5.

(6.) Rozen R. Molecular genetic aspects of hyperhomocysteinemia and its relation to folic acid. Clin Investig Med 1996;19:171-8.

(7.) Botto LD, Mastroiacovo P. Exploring gene-gene interactions in the etiology of neural tube defects. Clin Genet 1998;53:456-9.

(8.) Chen J, Giovannucci EL, Hunter DJ. MTHFR polymorphism, methylreplete diets and the risk of colorectal carcinoma and adenoma among US men and women: an example of gene-environment interactions in colorectal tumorigenesis. J Nutr 1999;129:560S-4S.

(9.) Eskes TK. Neural tube defects, vitamins and homocysteine. Eur J Pediatr 1998;157:S139-41.

(10.) James SJ, Pogribna M, Pogribny IP, Melnyk S, Hine RJ, Gibson JB, et al. Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase (MTHFR) gene may be maternal risk factors for Down syndrome. Am J Clin Nutr 1999;70:495-501.

(11.) Grandone E, Margaglione M, Colaizzo D, d'Addedda M, D'Andrea G, Pavone G, Di Minno G. Methylene tetrahydrofolate reductase 677C [right arrow] T mutation and unexplained early pregnancy loss. Thromb Haemost 1998;79:1056-7.

(12.) Surtees R. Demyelination and inborn errors of the single carbon transfer pathway. Eur J Pediatr 1998;157:S118-21.

(13.) Arinami T, Yamada N, Yamakawa-Kobayashi K, Hamaguchi H, Toru M. Methylenetetrahydrofolate reductase variant and schizophrenia/depression. Am J Med Genet 1997;74:526-8.

(14.) Bottiglieri T. Folate, vitamin [B.sub.12], and neuropsychiatric disorders. Nutr Rev 1996;54:382-90.

(15.) Melnyk S, Pogribna M, Pogribny IP, Hine RJ, James SJ. A new HPLC method for the simultaneous determination of oxidized and reduced plasma aminothiols using coulometric electrochemical detection. J Nutr Biochem 1999;10:490-7.

(16.) Martin SC, Tsakas-Ampatzis I, Bartlett WA, Jones AF. Measurement of plasma total homocysteine by HPLC with coulometric detection. Clin Chem 1999;45:150-2.

(17.) Hoffman DR, Cornatzer WE, Duerre JA. Relationship between tissue levels of S-adenosylmethionine, S-adenosylhomocysteine, and transmethylation reactions. Can J Biochem 1979;57:56-65.

(18.) Finkelstein JD, Kyle WE, Harris BJ. Methionine metabolism in mammals: regulatory effects of S-adenosylhomocysteine. Arch Biochem Biophys 1974;165:774-9.

(19.) De Cabo SF, Santos J, Fern andez-Piqueras J. Molecular and cytological evidence of Sadenosyl-L-homocysteine as an innocuous undermethylating agent in vivo. Cytogenet Cell Genet 1995;71:187-92.

(20.) Ueland PM. Pharmacological and biochemical aspects of S-adenosylhomocysteine hydrolase. Pharmacol Rev 1982;34:233-53.

(21.) Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem 1990;1:228-37.

(22.) Chiang PK, Gordon RK, Tal J, Zeng GC, Doctor BP, Pardhasaradhi K, McCann PP. S-adenosylmethionine and methylation. FASEB J 1996:10:471-80.

(23.) Finkelstein JD. The metabolism of homocysteine: pathways and regulation. Eur J Pediatr 1998;157:S40-4.

(24.) Fleischman A, Hershfield MS, Toutain S, Lederman HM, Sullivan KE, Fasano MB, et al. Adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency in common variable immunodeficiency. Clin Diagn Lab Immunol 1998;5:399-400.

(25.) Hershfield MS, Francke U. The human genes for S-adenosylhomocysteine hydrolase and adenosine deaminase are syntenic on chromosome 20. Science 1982;216:739-42.

(26.) Miller JW, Nadeau MR, Smith J, Smith D, Selhub J. Folatedeficiency-induced homocysteinaemia in rats--disruption of S adenosylmethionines coordinate regulation of homocysteine metabolism. Biochem J 1994;298:415-9.

(27.) Capdevila A, Wagner C. Measurement of plasma S-adenosylmethionine and S-adenosylhomocysteine as their fluorescent isoindoles. Anal Biochem 1998;264:180-4.

(28.) She Q-B, Nagao I, Hayakawa T, Tsuge H. A simple HPLC method for the determination of S-adenosylmethionine and S-adenosylhomocysteine in rat tissues: the effect of vitamin [B.sub.6] deficiency on these concentrations in rat liver. Biochem Biophys Res Commun 1994;205:1748-54.

(29.) Jencks DA, Mathews RG. Allosteric inhibition of methylenetetrahydrofolate reductase by S-adenosylmethionine. J Biol Chem 1987;262:2485-93.

(30.) Loehrer FM, Angst CP, Brunner FP, Haefei WE, Fowler B. Evidence for disturbed S-adenosylmethionine:S-adenosylhomocysteine ratio in patients with end-stage renal failure: a cause for disturbed methylation reactions? Nephrol Dial Transplant 1998;13:656-61.

(31.) Kredich NM, Martin DW. Role of S-adenosylhomocysteine in adenosine-mediated toxicity in cultured mouse T lymphoma cells. Cell 1977;12:931-8.

(32.) Kraus JP. Biochemistry and molecular genetics of cystathionine R-synthase deficiency. Eur J Pediatr 1998;157:S50-3.

(33.) Kredich NM, Hershfield MS. S-adenosylhomocysteine toxicity in normal and adenosine kinase deficient lymphoblasts of human origin. Proc Natl Acad Sci U S A 1979;76:2450-4.

(34.) Molloy AM, Weir DG, Kennedy G, Kennedy S, Scott JM. A new high performance liquid chromatographic method for the simultaneous measurement of S-adenosylmethionine and S-adenosylhomocysteine: concentrations in pig tissues after inactivation of methionine synthase by nitrous oxide. Biomed Chromatogr 1990;4:257-60.

(35.) Henning SM, McKee RW, Swendseid ME. Hepatic content of S-adenosylmethionine and S-adenosylhomocysteine and glutathione in rats receiving treatments modulating methyl donor availability. J Nutr 1989;119:1478-82.

(36.) Wise CK, Cooney CA, Ali SF, Poirier LA. Measuring S-adenosylmethionine in whole blood, red blood cells and cultured cells using a fast preparation method and high-performance liquid chromatography. J Chromatogr Biomed Sci Appl 1997;696:145-52.

(37.) Luippold G, Delabar U, Kloor D, Muhlbauer B. Simultaneous determination of adenosine, S-adenosylhomocysteine, and S adenosylmethionine in biological samples using solid phase extraction and high performance liquid chromatography. J Chromatogr 1999;724:231-8.

(38.) Loehrer FM, Haefei WE, Angst CP, Browne G, Frick G, Fowler B. Effect of methionine loading on 5-methyltetrahydrofolate, S-adenosylmethionine, and S-adenosylhomocysteine in plasma of healthy humans. Clin Sci 1996;91:79-86.

(39.) Weir DG, Molloy AM, Keating JN. Correlation of the ratio of S-adenosylmethionine to S-adenosylhomocysteine in the brain and cerebrospinal fluid of the pig: implications for the determination of this methylation ratio in human brain. Clin Sci 1992;82:93-7.

(40.) James SJ, Pogribna M, Pogribny IP, Melnyk S, Hine RJ, Gibson JB, et al. Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome. Am J Clin Nutr 1999;70:495-501.

(41.) Camp VM, Chisari FV, Faraj BA. Radioenzymatic assay for direct measurement of pyridoxal-5-phosphate. Clin Chem 1983;29:642-4.

(42.) Jacobsen DW, Gatautis VJ, Greene R, Robinson K, Savon SR, Secic M, et al. Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects. Clin Chem 1994;40:873-81.

(43.) Pastore A, Massoud R, Motti C, Lo R, Fucci G, Cortese C, Federici G. Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine. Clin Chem 1998;44:825-32.

(44.) Shivapurkar N, Poirier LA. Tissue levels of S-adenosylhomocysteine in rats fed methyl-deficient amino acid-defined diets for one to five weeks. Carcinogenesis 1983;4:1051-7.

(45.) Loehrer FM, Angst CP, Haefei WE, Jordon PP, Ritz R, Fowler B. Low whole blood S-adenosylmethionine and correlation between 5-methyltetrahydrofolate and homocysteine in coronary heart disease. Arterioscler Thromb Vasc Biol 1996;16:727-33.

(46.) Jacob RA, Gretz DM, Taylor PC, James SJ, Pogribny IP, Miller BJ, et al. Moderate folate depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women. J Nutr 1998;128:1204-12.

(47.) Cantoni GL. The role of S-adenosylhomocysteine in the biological utilization of S-adenosylmethionine. Prog Clin Biol Res 1985;198: 47-65.

(48.) Wainfan E, Poirier LA. Methyl groups in carcinogenesis: effects on DNA methylation and gene expression. Cancer Res 1992;52: 2071s-7s.

(49.) Chiang PK, Cantoni GL. Perturbation of biochemical transmethylations by 3-deazaadenosine in vivo. Biochem Pharmacol 1979; 28:1897-902.

(50.) Greenberg ML, Chaffee S, Hershfield MS. Basis for resistance to 3-deazaaristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase, in human lymphocytes. J Biol Chem 1989;264:795-803.

(51.) Duerre JA, Miller CH, Reams GG. Metabolism of S-adenosyl-L-homocysteine in vivo in the rat. J Biol Chem 1969;244:107-11.

(52.) Hoffman DR, Marion DW, Cornatzer WE, Duerre JA. S-Adenosylmethionine and S-adenosylhomocysteine metabolism in isolated liver. J Biol Chem 1980;22:10822-7.

[1] Nonstandard abbreviations: MTHFR, methylenetetrahydrofolate reductase; CBS, cystathionine [beta]-synthase; EC, electrochemical; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; TCA, trichloroacetic acid; PLP, pyridoxal 5'-phosphate; and tHcy, total homocysteine.

Stepan Melnyk, Marta Pogribna, Igor P. Pogribny, Ping Yi, and S. Jill James *

Division of Biochemical Toxicology, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079.

* Author for correspondence. Fax 870-543-7720; e-mail jjames@nctr.fda.gov.
Table 1. Precision and recovery of assays.

A. Precision of the assay

 Intraassay (n = 10)

 Mean SD CV, %

Plasma extract, nmol/L
 SAM 151.5 5.1 3.4
 SAH 22.6 1.3 5.5

Mouse liver extract, pmol/mg protein
 SAM 495.3 29.7 6.1
 SAH 93.7 8.3 8.8

 Interassay (n = 10)

 Mean SD CV, %

Plasma extract, nmol/L
 SAM 147.5 4.1 2.8
 SAH 21.8 1.2 5.6

Mouse liver extract, pmol/mg protein
 SAM 466.2 34.2 7.3
 SAH 87.3 6.6 7.6

B. Recovery of the assay (a)

 Sample Added

Plasma extract, nmol/L
 SAM 150.9 100
 SAH 23.3 20

Mouse liver extract,
 pmol/mg protein
 SAM 467.8 400
 SAH 91.4 80

 Mean
 Mean So recovery, %

Plasma extract, nmol/L
 SAM 243.1 3.8 96.8
 SAH 42.2 1.3 97.3

Mouse liver extract,
 pmol/mg protein
 SAM 824.7 16.4 95.2
 SAH 166.5 6.9 97.1

(a) Mean of five independent experiments.

Table 2. Plasma concentrations of SAM, SAH, cysteine,
and PLP in women with normal and increased tHcy
concentrations (mean [+ or -] SD).

 Normal tHcy Increased tHcy
 (7.3 [+ or -] 1.1 (12.3 [+ or -] 1.8
 [micro]mol/L; [micro]mol/L;
 n = 28) n = 30)

SAM, nmol/L 155.9 [+ or -] 14.1 154.8 [+ or -] 16.9
SAH, nmol/L 20.0 [+ or -] 5.6 40.1 [+ or -] 12.5 (a)
SAM/SAH ratio 8.5 [+ or -] 2.9 4.5 [+ or -] 2.7 (a)
PLP, nmol/L 48.1 [+ or -] 16.2 31.3 [+ or -] 7.2 (a)
Cysteine, mol/L 219.1 [+ or -] 23.4 191.8 [+ or -] 22.0 (a)

(a) P <0.001 relative to control.

Table 3. Intracellular SAM and SAH concentrations, and
SAM/SAH ratio in human lymphocytes and mouse
liver tissue (mean [+ or -] SD).

 Human lymphocytes, Mouse liver,
 nmol/[10.sup.6] cells pmol/mg protein
 (n = 7) (n = 4)

SAM 4.7 [+ or -] 1.4 515.4 [+ or -] 110.3
SAH 0.54 [+ or -] 0.19 96.5 [+ or -] 26.1
SAM/SAH ratio 9.4 [+ or -] 3.1 6.7 [+ or -] 1.3
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Title Annotation:Endocrinology and Metabolism
Author:Melnyk, Stepan; Pogribna, Marta; Pogribny, Igor P.; Yi, Ping; James, S. Jill
Publication:Clinical Chemistry
Date:Feb 1, 2000
Words:5243
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