Refsum H, Ueland P M, Svardal A M
Department of Pharmacology and Toxicology, University of Bergen, Haukeland Hospital, Norway.
Clin Chem. 1989 Sep;35(9):1921-7.
Homocysteine exists in human plasma as various (mixed) disulfides. Most plasma homocysteine (about 70%) is protein bound, probably via a disulfide bond to albumin, whereas homocysteine-cysteine mixed disulfide is the predominating form in the free fraction. We here present a method for the determination of total homocysteine, which includes both fractions. Plasma was initially treated with sodium borohydride to reduce the disulfide bonds, and the liberated thiols were derivatized with monobromobimane. The derivatized sample, still containing the plasma proteins, was injected onto a strong cation-exchange column, from which the homocysteine derivative was directed by column switching into a cyclohexyl silica (CH) column. The homocysteine derivative was top-concentrated on the CH column, then rapidly eluted with a steep gradient of methanol. Both the derivatization procedure and chromatography were performed with a combined sample processor and sample injector from Gilson (Model 232-401). Within-run and between-run precision (CV) was less than 4%, and the detection limit of 0.2 pmol was sufficiently low for monitoring homocysteine in plasma. We verified the assay against two established manual methods for the determination of total homocysteine in plasma. This, the first fully automated assay for total plasma homocysteine, allows the unattended analysis of 70 samples per 24 h.
同型半胱氨酸在人体血浆中以各种（混合）二硫化物的形式存在。大多数血浆同型半胱氨酸（约70%）与蛋白质结合，可能通过二硫键与白蛋白结合，而游离部分中同型半胱氨酸 - 半胱氨酸混合二硫化物是主要形式。我们在此介绍一种测定总同型半胱氨酸的方法，该方法包括这两种组分。血浆首先用硼氢化钠处理以还原二硫键，释放出的硫醇用单溴代联苯胺进行衍生化。仍含有血浆蛋白的衍生化样品被注入强阳离子交换柱，通过柱切换将同型半胱氨酸衍生物导入环己基硅胶（CH）柱。同型半胱氨酸衍生物在CH柱上进行顶部富集，然后用甲醇的陡梯度快速洗脱。衍生化过程和色谱分析均使用吉尔森公司（型号232 - 401）的组合样品处理器和进样器进行。批内和批间精密度（CV）小于4%，0.2皮摩尔的检测限对于监测血浆中的同型半胱氨酸来说足够低。我们将该检测方法与两种已确立的测定血浆总同型半胱氨酸的手工方法进行了验证。这是首个用于血浆总同型半胱氨酸的全自动检测方法，每24小时可无人值守分析70个样品。
