Abstract
Homocysteine (Hcy) is a sulfur-containing nonprotein amino acid, a metabolite of methionine. Mechanisms by which Hcy is involved in the pathogenesis of vascular diseases remain unclear. One of the potential mechanisms underlying harmful effects of Hcy is the protein N-homocysteinylation induced by Hcythiolactone. Proteolytic degradation of N-homocysteinylated protein yields Nɛ-homocysteinyl-lysine, a novel and important component of Hcy metabolism. Here we describe new high-performance liquid chromatography assay for the determination of Nɛ-homocysteinyl-lysine and γ-glutamylcysteine in plasma, based on a derivatization with 2-chloro-1-methyllepidinium tetrafluoroborate and UV detection. Baseline separation was achieved on an analytical column from Phenomenex (Kinetex C18, 100 × 4.6 mm, 2.6 μm) using gradient elution, with a mobile phase consisting 0.1 M trichloroacetic acid (pH 2.3) — acetonitrile. The quantification limits for Nɛ-homocysteinyl-lysine and γ-glutamylcysteine in plasma were 0.1 and 0.2 μM, respectively. Other main endogenous thiols can also be measured during the same analytical run. The proposed method was applied for the analysis of 15 plasma samples for total form of Nɛ-homocysteinyl-lysine and γ-glutamylcysteine.
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