Fujima James M, Danielson Neil D
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
J Capill Electrophor Microchip Technol. 2002;7(1-2):19-22.
The application of substrate cycling for alpha-ketoglutarate (AKG) through enzymatic amplification has been demonstrated in conjunction with capillary electrophoresis. AKG is determined using an off-line assay, which involves the coupled enzymatic reactions catalyzed by glutamate dehydrogenase and glutamic oxalacetic transaminase. Varying concentrations of the substrate and set concentrations of cofactors and enzyme are incubated together outside the CE instrument. The accumulated product, nicotinamide adenine dinucleotide, reduced form (NADH), was detected at 340 nm using CE. Amplification of a factor of 10 over just the reversed glutamate dehydrogenase reactions permitted an AKG detection limit of 10 microM. Throughput for each assay is about 5 min (including wash and equilibrium steps). After simple dilution as the only sample pretreatment, spiked AKG serum samples were determined with an average recovery of 96% with a 3% RSD.
通过酶促扩增实现的α-酮戊二酸（AKG）底物循环应用已与毛细管电泳相结合得到证实。AKG采用离线测定法进行测定，该方法涉及由谷氨酸脱氢酶和谷氨酸草酰乙酸转氨酶催化的偶联酶促反应。将不同浓度的底物以及设定浓度的辅因子和酶在毛细管电泳仪外部一起孵育。使用毛细管电泳在340nm处检测积累的产物还原型烟酰胺腺嘌呤二核苷酸（NADH）。仅通过反向谷氨酸脱氢酶反应就实现了10倍的放大，使得AKG的检测限达到10微摩尔。每次测定的通量约为5分钟（包括清洗和平衡步骤）。作为唯一的样品预处理，经过简单稀释后，加标AKG血清样品的测定平均回收率为96%，相对标准偏差为3%。
