Sciotti Michel A, Hasan Lara, Scholer Andre, Jermann Thomas M, Weber Jakob M, Gygax Daniel
Hochschule für Life Sciences, Fachhochschule Nordwestschweiz Gründenstrasse 40, CH-4132 Muttenz, Universitätsspital Basel.
Chimia (Aarau). 2010;64(11):793-8. doi: 10.2533/chimia.2010.793.
Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for the development of a simple and rapid screening method as an alternative to the currently available, technically demanding diagnostic methods. We have developed a rapid enzymatic assay based on the GHB dehydrogenase of Ralstonia eutropha. The enzyme is expressed as a recombinant protein in Escherichia coli and characterized in terms of reaction mechanism and kinetic parameters for the catalysis of conversion of GHB into succinic semialdehyde (SSA). The concomitant NADH production enables spectrophotometric monitoring of the reaction and the quantification of GHB in physiological fluids depending on initial velocities. We have tested a panel of twelve serum and urine samples containing GHB concentrations from 0.0 to 2.1 mmol/L. GHB dehydrogenase activity obeys a non classical bi bi ping pong mechanism exhibiting substrate inhibition by NAD+. With an optimal NAD+ concentration of 3.7 mmol/L in the reaction, the enzyme yields a K(M) of 1.0 mmol/L for GHB and a Vmax of 3.37 mmol/min/mg. The assay shows a linear standard curve from 0.1 to at least 1 mmol/L of GHB. Spiking experiments result in mean recoveries of 92% for urine and 114% for serum, respectively. The comparison to an ion chromatographic reference method exhibits a mean difference of 10% divergence from the target values in urine and 9% in serum, respectively.
γ-羟基丁酸（GHB）是一种受管制的治疗药物，它作为γ-氨基丁酸（GABA）神经递质代谢的中间体天然存在于哺乳动物脑组织中。近年来，GHB作为麻醉剂或滥用药物的滥用情况日益增加，这就需要开发一种简单快速的筛查方法，以替代目前可用的、技术要求较高的诊断方法。我们基于真养产碱菌的GHB脱氢酶开发了一种快速酶促测定法。该酶在大肠杆菌中作为重组蛋白表达，并根据反应机制和催化GHB转化为琥珀酸半醛（SSA）的动力学参数进行了表征。伴随产生的NADH能够通过分光光度法监测反应，并根据初始速度对生理流体中的GHB进行定量。我们测试了一组12份血清和尿液样本，其GHB浓度范围为0.0至2.1 mmol/L。GHB脱氢酶活性遵循非经典的双底物双产物乒乓机制，表现出受NAD+的底物抑制作用。在反应中NAD+的最佳浓度为3.7 mmol/L时，该酶对GHB的K(M)为1.0 mmol/L，Vmax为3.37 mmol/min/mg。该测定法显示，GHB在0.1至至少1 mmol/L范围内呈线性标准曲线。加标实验结果表明，尿液和血清的平均回收率分别为92%和114%。与离子色谱参考方法相比，尿液和血清与目标值的平均差异分别为10%和9%。
