Lennard L, Singleton H J
Department of Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, UK.
J Chromatogr B Biomed Appl. 1994 Nov 4;661(1):25-33. doi: 10.1016/0378-4347(94)00327-0.
An assay is described for the determination of red blood cell (RBC) thiopurine methyltransferase (TPMT) activity. TPMT S-methylates the antileukaemic drugs 6-mercaptopurine (6-MP) and 6-thioguanine and enzyme activity is inherited as a genetic trait. The assay is based on the TPMT-catalysed conversion of 6-MP to 6-methylmercaptopurine (methyl-MP) with non-radioactive S-adenosyl-L-methionine as the methyl donor. The methyl-MP metabolite is extracted into toluene as a phenyl-mercury adduct and back-extracted into 0.1 M HCl. Methyl-MP is quantitated by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet detection using a C18 Resolve cartridge and a mobile phase of methanol-water (20:80, v/v) with 100 mM triethylamine adjusted to pH 3.2 with orthophosphoric acid. There was a strong correlation between the HPLC assay and the established radiochemical assay (P < 0.0001). TPMT activities were measured by both methods in a population study of 111 children. There was no significant difference between the two frequency distribution histograms (P > 0.6).
本文描述了一种用于测定红细胞（RBC）硫嘌呤甲基转移酶（TPMT）活性的分析方法。TPMT可使抗白血病药物6-巯基嘌呤（6-MP）和6-硫鸟嘌呤发生S-甲基化，且酶活性作为一种遗传性状遗传。该分析方法基于以非放射性S-腺苷-L-甲硫氨酸作为甲基供体，TPMT催化6-MP转化为6-甲基巯基嘌呤（甲基-MP）。甲基-MP代谢产物作为苯基汞加合物萃取到甲苯中，然后反萃取到0.1M盐酸中。使用C18 Resolve柱和以甲醇-水（20:80，v/v）为流动相、含100mM三乙胺并用正磷酸调至pH 3.2的流动相，通过反相高效液相色谱（HPLC）和紫外检测对甲基-MP进行定量。HPLC分析方法与既定的放射化学分析方法之间存在强相关性（P < 0.0001）。在一项对111名儿童的群体研究中，用这两种方法测量了TPMT活性。两个频率分布直方图之间无显著差异（P > 0.6）。
