Measurement of Homocysteine and Other Aminothiols in Plasma: Advantages of Using Tris(2-carboxyethyl)phosphine as Reductant Compared with Tri-n-butylphosphine

Abstract

Abstract Background: Aminothiols have been implicated in the pathogenesis of arteriosclerosis, and reliable methods are needed to determine their concentrations in body fluids. We present a comparison of two analytical methods and focus on the reduction of low-molecular weight and protein-mixed disulfides of homocysteine, cysteine, cysteinyl-glycine, and glutathione. Methods: The plasma total aminothiol profile was determined by HPLC with fluorescence detection after derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate. Disulfides and protein-bound aminothiols were reduced by either tri-n-butylphosphine (the TBP method) or tris(2-carboxyethyl)phosphine (the TCEP method); the effects of temperature, time of reduction, and concentration of reductants were evaluated. Results: The intraassay imprecision (CV) was <3% for all aminothiols using both methods. The interassay CVs for total cysteine (tCys), total cysteinyl-glycine (tCys-Gly), and total homocysteine (tHcy) were <4% and <8% for the TCEP and TBP methods, respectively, whereas for total glutathione (tGSH) the interassay CV was >12% for both methods. Deming regression and Bland–Altman difference plots showed positive biases for total aminothiol concentrations determined by the TCEP method relative to the TBP method. The mean proportional biases were 65%, 27%, 6%, and 60% for tCys, tCys-Gly, tHcy, and tGSH, respectively. The calculated concentrations of total aminothiols by the TCEP method were less influenced by changes in temperature and concentration of reducing agent or by calibrator matrix. Conclusions: The agreement between the TCEP and TBP methods was considerably lower for the determination of tCys, tCys-Gly, and tGSH than for tHcy. For total-aminothiol determination, the TCEP method yields better reproducibility and is more robust than the TBP method.