Jacobsen D W, Gatautis V J, Green R
Department of Laboratory Hematology, Cleveland Clinic Foundation, Ohio 44106.
Anal Biochem. 1989 Apr;178(1):208-14. doi: 10.1016/0003-2697(89)90381-3.
Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.
严重高胱氨酸血症常与血管疾病相关，而血浆同型半胱氨酸中度或轻度升高的病理后果尚不清楚。钴胺素和叶酸缺乏可能导致血浆同型半胱氨酸升高。已经开发出一种用于检测血浆总同型半胱氨酸的灵敏且可重复的检测方法。该检测方法的基本步骤包括：(i) 用硼氢化物还原将同型半胱氨酸二硫化物转化为游离同型半胱氨酸；(ii) 使同型半胱氨酸与一溴双马来酰亚胺结合；(iii) 通过反相高效液相色谱法将同型半胱氨酸-双马来酰亚胺与其他血浆硫醇-双马来酰亚胺加合物分离；(iv) 通过荧光法检测和定量同型半胱氨酸-双马来酰亚胺。该方法对同型半胱氨酸的检测灵敏度为4.4皮摩尔，且具有高度可重复性（批内和批间变异系数分别为4.97%和4.53%）。非空腹成年男性（n = 12）和女性（n = 12）血浆总同型半胱氨酸的平均浓度分别为15.8（范围7.0 - 23.7）和16.5纳摩尔/毫升（范围8.6 - 20.7）。在钴胺素和叶酸缺乏的患者中发现同型半胱氨酸水平明显升高。在正常个体的血浆中，血浆总同型半胱氨酸约占硼氢化物产生的硫醇反应性的4%。
