Yang B I, Harris D J
Division of Molecular Biology & Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City 64110-2499.
Anal Biochem. 1991 Nov 15;199(1):18-24. doi: 10.1016/0003-2697(91)90263-s.
Pyridoxamine 5'-phosphate in 18 microliters of human capillary blood plasma is determined by catalytic amplification using the apoenzyme of aspartate aminotransferase. Prior isolation from interfering substances is accomplished by employment of a cation exchange resin in batch operation. The procedure consists of the following stages. Stage I, denaturation of proteins. Trichloroacetic acid is used to precipitate plasma proteins and liberate any bound coenzyme. Dilute NaCl is added to expand the volume thus minimizing coenzyme entrapment in the precipitate. Stage II, isolation of the coenzyme. A sulfonated polystyrene ion exchange resin is used inside a centrifugal filter. Pyridoxamine 5'-phosphate in the supernatant from Stage I adsorbs to the resin. Pyridoxal 5'-phosphate, other organic phosphates, and Pi are removed by centrifugation. Rinsing with dilute NaBH4 destroys traces of pyridoxal 5'-phosphate and washes off residual inhibitors. Pyridoxamine 5'-phosphate is then desorbed with NaOH and Tris buffer and recovered by centrifugation. Stage III, reconstitution and assay. The desorbate from Stage II is incubated with excess apoenzyme. Specific activity of the reconstituted enzyme is measured. Interpolation from a standard curve relating enzyme specific activity and pyridoxamine 5'-phosphate concentration yields the plasma level of the cofactor. Approximately 3 h are required to carry out the procedure. Much of the coenzyme was found not be assayable if plasma was refrigerated overnight or if whole blood was left standing at room temperature for a few hours. The degradation was arrested with freezing at -80 degrees C. In a 13-day experiment involving a healthy subject, sharp rises of plasma pyridoxamine 5'-phosphate were found to occur in response to small doses of oral vitamin B6.(ABSTRACT TRUNCATED AT 250 WORDS)
采用天冬氨酸转氨酶的脱辅基酶，通过催化扩增法测定18微升人毛细血管血浆中的5'-磷酸吡哆胺。在批量操作中，使用阳离子交换树脂从干扰物质中预先分离出该物质。该程序包括以下几个阶段。第一阶段，蛋白质变性。用三氯乙酸沉淀血浆蛋白并释放任何结合的辅酶。加入稀氯化钠以扩大体积，从而将辅酶截留于沉淀中的量降至最低。第二阶段，辅酶的分离。在离心过滤器中使用磺化聚苯乙烯离子交换树脂。第一阶段上清液中的5'-磷酸吡哆胺吸附到树脂上。通过离心去除5'-磷酸吡哆醛、其他有机磷酸盐和无机磷酸盐。用稀硼氢化钠冲洗可破坏痕量的5'-磷酸吡哆醛并洗去残留抑制剂。然后用氢氧化钠和Tris缓冲液解吸5'-磷酸吡哆胺，并通过离心回收。第三阶段，重构与测定。将第二阶段的解吸物与过量的脱辅基酶一起孵育。测定重构酶的比活性。根据酶比活性与5'-磷酸吡哆胺浓度的标准曲线进行插值，得出血浆中该辅因子的水平。完成该程序大约需要3小时。如果血浆冷藏过夜或全血在室温下放置数小时，发现许多辅酶无法测定。在-80℃冷冻可阻止降解。在一项涉及一名健康受试者的为期13天的实验中，发现小剂量口服维生素B6会导致血浆5'-磷酸吡哆胺急剧升高。（摘要截短于250字）
