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

Background: The relative changes in plasma and intracellular concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAM) may be impo rtant predictors of cellular methylation potential and metabolic alteration s associated with specific genetic polymorphisms and/or nutritional deficie ncies. Because these metabolites are present in nanomolar concentrations in plasma, methods of detection generally require time-consuming precolumn pr ocessing or metabolite derivatization. Methods: We used HPLC with coulometric electrochemical detection for the si multaneous measurement of SAM and SAH in 200 mu L of plasma, 10(6) lymphocy tes, or 10 mg of tissue. Filtered trichloroacetic acid extracts were inject ed directly into the HPLC system without additional processing and were elu ted isocratically. Results: The limits of detection were 200 fmol/L for SAM and 40 fmol/L SAH. In plasma extracts, the interassay CV was 3.4-5.5% and the intraassay CV w as 2.8-5.6%. The analytical recoveries were 96.8% and 97.3%. for SAM and SA H, respectively. In a cohort of healthy adult women with mean total homocys teine concentrations of 7.3 mu mol/L, the mean plasma value was 156 nmol/L for SAM and 20 nmol/L for SAH. In women with increased homocysteine concent rations (mean, 12.1 mu mol/L), plasma SAH, but not SAM, was increased (P <0 .001), and plasma pyridoxal 5'-phosphate concentrations were reduced (P <0. 001). Plasma SAM/SAH ratios were inversely correlated with homocysteine con centrations (r = 0.73; P < 0.01), and the SAM/SAH ratio in plasma was direc tly correlated with the:intracellular SAM/SAH ratio in lymphocytes (r 0.70; P <0.01). Conclusions: Increased homocysteine in serum is associated with an increase in SAH and a decrease in the SAM/SAH ratio that could negatively affect ce llular methylation potential. Accurate and sensitive detection of these ess ential metabolites in plasma and in specific tissues should provide new ins ights into the regulation of:one-carbon metabolism under different nutritio nal and pathologic conditions. (C) 2000 American Association for Clinical C hemistry.