The Use of N-Ethylmaleimide for Mass Spectrometric Detection of Homocysteine Fractions in Blood Plasma

Abstract

Fraction analysis of homocysteine in biological fluids is important for the diagnosis and studies of cardiovascular, nervous, urological, and other diseases. Measurements of total, free, and reduced homocysteine by mass spectrometry with HPLC by means of its modification with N-ethylmaleimide are proposed. The linearity and detection threshold were 0.025-10 and 0.001 μM, respectively, for reduced homocysteine fraction and 0.2-100 and 0.1 μM, respectively, for the rest fractions. The accuracy and reproducibility of the method were within 12%.

HPLC Analysis of Homocysteine and Related Compounds

“…These S-linked forms are converted to the reduced form and analyzed as the total Hcy. The disulfide can be reduced using sodium borohydride [25,26], tributylphosphine (TBP) [27,28], tris(2-carboxyethyl)phosphine (TCEP) [6,[29][30][31][32][33][34][35][36][37][38][39][40], dithiothreitol (DTT) [41][42][43][44], 1,4-dithioerythritol (DTE) [45][46][47], and mercaptoethanol [48]. Bai et al developed a unique online reduction quartz column packed with a Zn(II)-TCEP complex [33,49].…”

A more accurate indicator to evaluate oxidative stress in rat plasma with osteoporosis

“…[29][30][31][32] The total plasma concentration is mostly measured as an important element in clinical diagnostic evaluations. [33][34][35] However, total thiols are the sum of free and protein-bound thiols and disuldes (derived from autooxidation of thiols). [32][33][34][35][36] In fact, there is a state of dynamic equilibrium between their own oxidized and reduced thiols in the aminothiols.…”

“…[33][34][35] However, total thiols are the sum of free and protein-bound thiols and disuldes (derived from autooxidation of thiols). [32][33][34][35][36] In fact, there is a state of dynamic equilibrium between their own oxidized and reduced thiols in the aminothiols. The ratio of the reduced to the oxidized thiols can characterize the redox status.…”

“…The ratio of the reduced to the oxidized thiols can characterize the redox status. [33][34][35][36][37][38][39] Perturbation of plasma redox status and tissue aminothiol levels is an important indicator of chronic oxidative stress. 36 It has been reported that aminothiol concentration disturbances can also correspond to metabolic disorders, and the ratio of reduced and oxidized thiols indicates the redox state in the internal environment.…”

“…However, these methods suffer from several problems. 13,34 The relatively low concentration in plasma (especially the very low concentration of oxidized thiols), as well as the poor traditional assay method sensitivity, has presented a major challenge in accurate and precise physiological concentration quantitation in clinical research. [36][37][38][39][40] A formula for quantifying disuldes is based on thiols aer and before reduction 41 or indirectly calculated from the difference between the measurements obtained in the two separate steps.…”