Development and validation of an LC-MS/MS assay for the quantification of the trans-methylation pathway intermediates S-adenosylmethionine and S-adenosylhomocysteine in human plasma

Abstract

Background Although increased levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) have been implicated as markers for renal and vascular dysfunction, until now there have been no studies investigating their association with clinical post-transplant events such as organ rejection and immunosuppressant nephrotoxicity. Methods A newly developed and validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the quantification of SAM and SAH in human EDTA plasma was used for a clinical proof-of-concept pilot study. Retrospective analysis was performed using samples from a longitudinal clinical study following de novo kidney transplant patients for the first year (n = 16). Results The ranges of reliable response were 8 to 1024 nmol/l for SAM and 16 to 1024 nmol/l for SAH. The inter-day accuracies were 96.7–103.9% and 97.9–99.3% for SAM and SAH, respectively. Inter-day imprecisions were 8.1–9.1% and 8.4–9.8%. The total assay run time was 5 min. SAM and SAH concentrations were significantly elevated in renal transplant patients preceding documented acute rejection and nephrotoxicity events when compared to healthy controls (n = 8) as well as transplant patients void of allograft dysfunction (n = 8). Conclusion The LC-MS/MS assay will provide the basis for further large-scale clinical studies to explore these thiol metabolites as molecular markers for the management of renal transplant patients.

Endothelial dysfunction and oxidative stress in polycystic kidney disease

“…Endothelial dysfunction markers, including arginine (Arg), ADMA, and symmetric dimethylarginine (SDMA), cysteine (Cys), glutathione, homocysteine (Hcy), methionine (Met), S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM), were quantified using a validated HPLC-MS/MS assay (for more detail, see Ref. 26). In brief, to 100 l serum 50 l of internal standard containing solution (50 M d7-ADMA, d4-cystine, d8-homocystine, d3-methionine and S-methylglutathione, all in HPLC water), and 40 l of 500 mM DTT solution were added.…”

Pravastatin Therapy and Biomarker Changes in Children and Young Adults with Autosomal Dominant Polycystic Kidney Disease

“…Endothelial dysfunction markers, including arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), citrulline, cysteine, homocysteine (HCy), methionine (Met), S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM), were quantified in plasma using an HPLC-tandem mass spectrometry (MS/MS) assay (27). In brief, the API5000 mass spectrometer (AB Sciex, Concord, ON, Canada) was run in the positive electrospray ionization mode (ESI) using multiple reaction monitoring (MRM Urinary PGs.…”

Reversed phase and cation exchange liquid chromatography with spectrophotometric and elemental/molecular mass spectrometric detection for S-adenosyl methionine/S-adenosyl homocysteine ratios as methylation index in cell cultures of ovarian cancer

“…These effects can be minimized by the use of stable isotopes labeled analogs as internal standards. As previously mentioned, SAM and SAH determination by LC-MS/MS have been utilized in human biological fluids such as plasma, [20,21] serum [22] and cerebrospinal fluid, [23,24] and also in animal tissues as mice liver [25] or mouse embryos [26] but relatively little attention has been paid to analysis of SAM and SAH in cultured cells [27,28].…”