Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699, USA.
Talanta. 2012 Oct 15;100:7-11. doi: 10.1016/j.talanta.2012.08.022. Epub 2012 Aug 24.
Two enzymatic assays were developed for the analysis of α-ketoglutaramate (KGM)-an important biomarker of hepatic encephalopathy and other hyperammonemic diseases. In both procedures, KGM is first converted to α-ketoglutarate (KTG) via a reaction catalyzed by ω-amidase (AMD). In the first procedure, KTG generated in the AMD reaction initiates a biocatalytic cascade in which the concerted action of alanine transaminase and lactate dehydrogenase results in the oxidation of NADH. In the second procedure, KTG generated from KGM is reductively aminated, with the concomitant oxidation of NADH, in a reaction catalyzed by L-glutamic dehydrogenase. In both assays, the decrease in optical absorbance (λ=340 nm) corresponding to NADH oxidation is used to quantify concentrations of KGM. The two analytical procedures were applied to 50% (v/v) human serum diluted with aqueous solutions containing the assay components and spiked with concentrations of KGM estimated to be present in normal human plasma and in plasma from hyperammonemic patients. Since KTG is the product of AMD-catalyzed hydrolysis of KGM, in a separate study, this compound was used as a surrogate for KGM. Statistical analyses of samples mimicking the concentration of KGM assumed to be present in normal and pathological concentration ranges were performed. Both enzymatic assays for KGM were confirmed to discriminate between the predicted normal and pathophysiological concentrations of the analyte. The present study is the first step toward the development of a clinically useful probe for KGM analysis in biological fluids.
两种酶促分析方法被开发出来用于分析α-酮戊二酸（KGM）——肝性脑病和其他高氨血症疾病的一个重要生物标志物。在这两种程序中，KGM 首先通过ω-酰胺酶（AMD）催化的反应转化为α-酮戊二酸（KTG）。在第一个程序中，AMD 反应中生成的 KTG 启动了一个生物催化级联反应，其中丙氨酸转氨酶和乳酸脱氢酶的协同作用导致 NADH 的氧化。在第二个程序中，从 KGM 生成的 KTG 在 L-谷氨酸脱氢酶催化的反应中被还原胺化，同时 NADH 被氧化。在这两种测定中，与 NADH 氧化相对应的光吸收（λ=340nm）的减少用于定量 KGM 的浓度。这两种分析程序被应用于 50%（v/v）的人血清，用含有测定成分的水溶液稀释，并加入 KGM 的浓度，这些浓度估计存在于正常人血浆和高氨血症患者的血浆中。由于 KTG 是 AMD 催化水解 KGM 的产物，在一项单独的研究中，该化合物被用作 KGM 的替代物。对模拟正常和病理浓度范围内 KGM 浓度的样品进行了统计学分析。两种用于 KGM 的酶促分析方法均被证实可以区分预测的正常和病理生理浓度的分析物。本研究是开发用于生物体液中 KGM 分析的临床有用探针的第一步。
