Müller Michael, Bünger Jürgen, Voss Michael, Westphal Götz, Ruhnau Peter, Hallier Ernst
Abteilung Arbeits- und Sozialmedizin, Georg-August-Universität Göttingen, Waldweg 37, 37073 Göttingen, Germany.
Arch Toxicol. 2002 Nov;76(11):634-42. doi: 10.1007/s00204-002-0391-1. Epub 2002 Aug 9.
The human glutathione S-transferase hGSTT1-1 is characterized by a polymorphism displaying three phenotypes: the "non-conjugator" (NC) phenotype expresses no or only a residual activity due to a homozygous deletion of the hGSTT1 gene, a medium hGSTT1-1 activity can be demonstrated for the "low conjugator" (LC) phenotype as the heterozygous bearer of one hGSTT1 allele, and a high hGSTT1-1 activity is detected for the "high conjugator" (HC) phenotype as the homozygous bearer of two hGSTT1 alleles. We have developed a routine ex vivo photometric phenotyping procedure based on the determination of bromide release rates from the hGSTT1-1-catalyzed glutathione conjugation of the substrate methyl bromide in EDTA blood samples under standard conditions (1,000 ppm methyl bromide, 10 min incubation). The bromide release rates were standardized to the hemoglobin (Hb) value. Twenty-six individuals were phenotyped following the new procedure. Four individuals were classified as NCs (24-33 pmol Br(-)/mg Hb per min), 21 individuals were regarded as LCs (107-206 pmol Br(-)/mg Hb per min) and one person of the study group was designated HC (236 pmol Br(-)/mg Hb per min). The results were validated by qualitative hGSTT1 genotyping and demonstrated a 100% match for conjugators and non-conjugators. A second HPLC phenotyping routine procedure based on the formation of S-methylglutathione from methyl chloride in erythrocyte lysate incubations (Müller et al. 2001, Arch Toxicol 74:760-767) was established and validated by genotyping. The phenotyping results obtained with both methods were correlated, resulting in a good correlation with R(2)=0.64 (y=0.8997x +51.535). Three distinct phenotype clusters for NCs, LCs and HCs, consistent with the proposed genetics, were demonstrated. Assay-dependent storage experiments revealed an excellent stability of the hGSTT1-1 activity. In conclusion, the evaluated methods provide powerful tools for determination of hGSTT1-1 activity as a clinical parameter.
人类谷胱甘肽S-转移酶hGSTT1-1具有一种多态性，表现为三种表型：“非结合者”（NC）表型由于hGSTT1基因的纯合缺失而不表达或仅表达残余活性；“低结合者”（LC）表型作为一个hGSTT1等位基因的杂合携带者，可表现出中等的hGSTT1-1活性；“高结合者”（HC）表型作为两个hGSTT1等位基因的纯合携带者，可检测到高hGSTT1-1活性。我们开发了一种常规的体外光度法表型分析程序，该程序基于在标准条件下（1000 ppm溴甲烷，孵育10分钟），测定EDTA血样中hGSTT1-1催化底物溴甲烷的谷胱甘肽结合反应中溴离子的释放速率。溴离子释放速率以血红蛋白（Hb）值进行标准化。按照新程序对26名个体进行了表型分析。4名个体被归类为NC（每分钟24 - 33 pmol Br(-)/mg Hb），21名个体被视为LC（每分钟107 - 206 pmol Br(-)/mg Hb），研究组中有1人被指定为HC（每分钟236 pmol Br(-)/mg Hb）。通过定性hGSTT1基因分型对结果进行了验证，结果表明结合者和非结合者的匹配率为100%。建立了第二种基于红细胞裂解物孵育中由氯甲烷形成S-甲基谷胱甘肽的HPLC表型分析常规程序（Müller等人，2001年，《毒理学文献》74:760 - 767），并通过基因分型进行了验证。两种方法获得的表型分析结果具有相关性，相关系数R(2)=0.64（y = 0.8997x + 51.535），呈现出良好的相关性。证明了与所提出的遗传学一致的NC、LC和HC三个不同的表型簇。依赖于检测方法的储存实验显示hGSTT1-1活性具有出色的稳定性。总之，所评估的方法为将hGSTT1-1活性作为临床参数进行测定提供了有力工具。
