Protein-bound Homocystamide Measured in Human Plasma by HPLC

Abstract

Homocysteine (Hcy) is a sulfur-containing nonprotein amino acid derived from the metabolism of methionine. Plasma Hcy is an independent risk factor for atherosclerosis (1)(2)(3)(4)(5). The cyclic internal lactone form of Hcy, homocysteine thiolactone, may be the critical molecular form in the pathogenesis of atherosclerosis. In cultured cells, homocysteine thiolactone is synthesized through the reaction of methionyl-tRNA synthase (6)(7). This enzyme reacts with free amino groups of proteins to produce protein-bound homocystamide (7)(8)(9). The toxicity of homocysteinylation to the cell has been demonstrated in both in vitro and in vivo experiments. Lysyl oxidase, which catalyzes the posttranslational modification essential to the pathogenesis of connective tissue matrices, is irreversibly inactivated by homocysteine thiolactone (10). Homocysteinylation increases the internalization of LDL by macrophages (11). Furthermore, homocysteinylated LDL elicits an autoimmune response (12). Homocysteine thiolactone hydrolase is present in human plasma and is identical to paraoxonase (13). Homocysteine thiolactone hydrolase may hydrolyze homocysteine thiolactone to Hcy and thereby prevent the homocysteinylation of proteins. In the absence of a suitable assay system for homocysteine thiolactone, however, no detailed study is available on homocysteine thiolactone or Hcy-related proteins. We have developed and evaluated an assay system for measuring protein-bound homocystamide in plasma and used it in samples from healthy adults and hemodialysis patients. Here we report the results of that study. We obtained plasma samples from 20 healthy volunteers (12 males, 8 females; age range, 29–38 years) and 15 nondiabetic hemodialysis patients [7 males and 8 females; mean (SD) age, 49 (7) years] treated at the Suiyukai Clinic (Nara, Japan) after receiving their informed consent for this study. The patients were on hemodialysis using bicarbonate dialysate with systemic heparinization two or three times week a week. Blood was collected by …