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 (SAH) may be important predictors of cellular methylation potential and metabolic alterations associated with specific genetic polymorphisms and/or nutritional deficiencies. Because these metabolites are present in nanomolar concentrations in plasma, methods of detection generally require time-consuming precolumn processing or metabolite derivatization.


METHODS
We used HPLC with coulometric electrochemical detection for the simultaneous measurement of SAM and SAH in 200 microL of plasma, 10(6) lymphocytes, or 10 mg of tissue. Filtered trichloroacetic acid extracts were injected directly into the HPLC system without additional processing and were eluted 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 was 2.8-5.6%. The analytical recoveries were 96.8% and 97.3% for SAM and SAH, respectively. In a cohort of healthy adult women with mean total homocysteine concentrations of 7.3 micromol/L, the mean plasma value was 156 nmol/L for SAM and 20 nmol/L for SAH. In women with increased homocysteine concentrations (mean, 12.1 micromol/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 concentrations (r = 0.73; P <0.01), and the SAM/SAH ratio in plasma was directly 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 cellular methylation potential. Accurate and sensitive detection of these essential metabolites in plasma and in specific tissues should provide new insights into the regulation of one-carbon metabolism under different nutritional and pathologic conditions.
Melnyk
S
S
Division of Biochemical Toxicology, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079. Author for correspondence. Fax 870-543-7720, USA.
Pogribna
M
M
Pogribny
I P
IP
Yi
P
P
James
S J
SJ
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004563:Electrochemistry; D005260:Female; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry; D008214:Lymphocytes / Q000737:chemistry; D008875:Middle Aged; D011732:Pyridoxal Phosphate / Q000097:blood*; D015203:Reproducibility of Results; D012435:S-Adenosylhomocysteine / Q000032:analysis* / Q000097:blood; D012436:S-Adenosylmethionine / Q000032:analysis* / Q000097:blood; D012680:Sensitivity and Specificity; D014238:Trichloroacetic Acid
D007202:Indicators and Reagents; D006710:Homocysteine; D014238:Trichloroacetic Acid; D011732:Pyridoxal Phosphate; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine