基于高效液相色谱法测定人血样本中硫嘌呤S-甲基转移酶活性：儿童红细胞中的参考值

Measurement of thiopurine S-methyltransferase activity in human blood samples based on high-performance liquid chromatography: reference values in erythrocytes from children.

作者信息

Keizer-Garritsen Jenneke J, Brouwer Connie, Lambooy Lambert H J, Ter Riet Patricia, Bökkerink Jos P M, Trijbels Frans J M, De Abreu Ronney A

机构信息

Center for Pediatric Oncology, SE Netherlands, Department of Pediatrics, University Medical Center St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

出版信息

Ann Clin Biochem. 2003 Jan;40(Pt 1):86-93. doi: 10.1258/000456303321016222.

DOI:10.1258/000456303321016222

PMID:12542916

Abstract

BACKGROUND

Monitoring 6-thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) activity is especially important when patients are treated with 6-thiopurine drugs, since severe bone marrow toxicity may be induced if patients have deficient TPMT activity.

METHODS

We have developed a method based on high-performance liquid chromatography (HPLC) for the measurement of TPMT activity in various cell types: erythrocytes (RBC), human peripheral blood mononuclear cells (pMNC) and human malignant lymphoblasts (Molt-F4). The enzymatic activity is measured by the amount of 6-methylmercaptopurine formed, using 6-mercaptopurine (6MP) as substrate and S-adenosylmethionine as co-substrate.

RESULTS

The K(m) values calculated for 6MP were 0.54 (RBC), 0.85 (pMNC) and 0.65 (Molt-F4 cells) mmol/L. The K(m) values for S-adenosylmethionine were 11.9 (RBC), 16.4 (pMNC) and 6.65 (Molt-F4 cells) micro mol/L. The assay variation was 8.2-17%. TPMT activity was determined in a control group of 103 children and young adults (44 female, 59 male). The values observed were (mean +/- standard deviation): female children and young adults, 15.1 +/- 4.8 pmol/10(7) cells per h (n = 44); male children and young adults, 15.8 +/- 6.4 pmol/10(7) cells per h (n = 59). No gender or age differences were found.

CONCLUSION

The HPLC-based method enables the rapid screening of TPMT activities in large groups of patients treated with 6-thiopurines.

摘要

背景

当患者接受6-硫嘌呤类药物治疗时，监测6-硫嘌呤S-甲基转移酶（TPMT；EC 2.1.1.67）活性尤为重要，因为如果患者TPMT活性不足，可能会诱发严重的骨髓毒性。

方法

我们开发了一种基于高效液相色谱（HPLC）的方法，用于测定各种细胞类型中的TPMT活性：红细胞（RBC）、人外周血单核细胞（pMNC）和人恶性淋巴母细胞（Molt-F4）。以6-巯基嘌呤（6MP）为底物、S-腺苷甲硫氨酸为共底物，通过测定生成的6-甲基巯基嘌呤的量来测量酶活性。

结果

计算得出6MP的米氏常数（K(m)）值在红细胞中为0.54 mmol/L，在外周血单核细胞中为0.85 mmol/L，在Molt-F4细胞中为0.65 mmol/L。S-腺苷甲硫氨酸的K(m)值在红细胞中为11.9 μmol/L，在外周血单核细胞中为16.4 μmol/L，在Molt-F4细胞中为6.65 μmol/L。测定的变异系数为8.2%-17%。在103名儿童和青年成人（44名女性，59名男性）的对照组中测定了TPMT活性。观察到的值为（平均值±标准差）：女性儿童和青年成人，15.1±4.8 pmol/10(7)细胞/小时（n = 44）；男性儿童和青年成人，15.8±6.4 pmol/10(7)细胞/小时（n = 59）。未发现性别或年龄差异。