反相高效液相色谱法测定红细胞中巯嘌呤甲基转移酶的活性

目的:研究中国健康人群和急性白血病患者红细胞内巯嘌呤甲基转移酶(TPMT)活性。方法:测定TPMT活性的基础是,应用非放射性S-腺苷蛋氨酸(SAM)作为甲基供体,6-MP经TPMT甲基化为6-MeMP。用反相高效液相色谱技术(RP-HPLC),样品经乙酸苯汞加合物(PMA)提取6-MeMP。色谱柱为Dopond C18(10 μm,4.6 mm×250 mm),流动相为甲醇-水(20:80),流速1.5 mL·min～1,检测波长303nm。结果:6-巯基嘌呤(6-MP)和甲基巯嘌呤(6-MeMP)的保留时间分别为3.9 min和5.2 min,日内和日间RSD均小于2％,重复性好。结论:本方法简便、快速、准确,适用于巯嘌呤类药物细胞内药理学研究。     

成人急性淋巴细胞白血病患者巯嘌呤甲基转移酶活性研究

目的应用简易的HPLC方法检测血红细胞中巯嘌呤甲基转移酶（TPMT）的活性,并研究健康汉族成年人群和成人急性淋巴细胞白血病（ALL）患者TPMT活性分布。方法以非放射性S-腺苷-L-甲硫氨酸（SAM）为甲基供体,完成6-巯基嘌呤（6-MP）向6-甲基巯基嘌呤（6-MeMP）的甲基化转变。6-MeMP经乙酸苯汞混和物抽提,采用高效液相色谱法分析。TPMT的活性用37℃时每小时每毫升红细胞生成多少纳摩尔的6-MeMP来表示。结果健康汉族成人和成人ALL患者TPMT活性均呈正态分布,男性和女性相比,TPMT活性没有显著性差异。健康汉族成人与成人ALL患者服用6-MP前TPMT活性相比没有显著性差异。而TPMT＊3C杂合子TPMT平均活性低于野生型TPMT基因（TPMT＊1）纯合子,差异有显著性。结论TPMT活性在健康汉族成人和成人ALL患者均呈正态分布,TPMT基因型和表型存在相关性,TPMT＊3C杂合子平均酶活性低于野生型TPMT基因（TPMT＊1）纯合子。在开始6-MP等药物维持治疗前检测TPMT活性对成人ALL患者有益。     

高效液相色谱法测定急性淋巴细胞白血病患者巯嘌呤甲基转移酶活性

目的:建立一种简易的非放射性方法检测成人急性淋巴细胞白血病患者血红细胞中巯嘌呤甲基转移酶(TPMT)活性。方法:以非放射性S-腺苷-L-甲硫氨酸(SAM)为甲基供体,完成6-巯基嘌呤(6-MP)向6-甲基巯基嘌呤(6-MeMP)的甲基化转变。孵育结束时,6-MeMP经醋酸苯汞混和物抽提。色谱柱为hypersil ODS C18(250mm×4.6mm,5μm),流动相为甲醇-水(加入三乙胺并以磷酸调至pH3.2)(70∶30,v/v),流速为1mL.min-1,检测波长为290nm。结果:线性范围为20~1 280μg.L-1(r=0.999 5,n=7),高、中、低3种浓度的平均回收率为92.3%。结论:该法简便、快速、准确,适用于TPMT药动学研究和临床监测要求。     

硫嘌呤甲基转移酶遗传多态性检测在6-MP个体化治疗中的意义

目的：研究硫嘌呤甲基转移酶活性和基因型检测对6-巯基嘌呤（6-MP）的个体化治疗的意义。方法-94例口服6-MP维持治疗的白血病患者，19例口服6-Mp后出现血液系统危象的为观察组，同时服用6-MP没有出现血液系统损害的75患者为对照组，采用高效液相色谱法测定硫嘌呤甲基转移酶（TPMT）活性，等位基因特异性PCR和限制性片段长度多态性（RFLP）的方法检测TPMT＊2、TPMT＊3A、TPMT＊3B和TPMT＊3C的等位基因频率。通过临床白细胞记数和骨髓象检查及临床表现判定巯嘌呤的疗效和不良反应。结果：观察组TPMT活性是（6．1±2．1）U·mL^-1 pRBCs显著低于对照组（15．3±2．3）U·mL^-1 pRBCs，而观察组的基因突变率10．7％高于对照组1．2％。结论：TPMT活性低下和／或TPMT基因突变型个体，在服用标准剂量的巯嘌呤后，发生不良反应的危险较高，应及时发现此类患者并积极调整剂量实现安全有效的治疗。     

中国维吾尔族和哈萨克族人硫嘌呤甲基转移酶活性的分布

目的研究硫嘌呤甲基转移酶(TPMT)在中国新疆维吾尔族和哈萨克族人中的分布。方法运用改良的RP-HPLC法,测定红细胞中TPMT活性。结果160名维吾尔族成年人TPMT活性呈正态分布,活性均值为(11．57±3．52)U·mL-1 RBC (3．35-29．07 U·mL-1RBC)。327名哈萨克族成年人的TPMT活性呈正态分布,TPMT活性均值(12．27±3．42)U·mL-1 RBC (3．29-24．67 U·mL-1RBC)。在中国新疆维吾尔族和哈萨克族中没有发现酶缺乏者,TPMT活性不存在性别差异。维吾尔族、哈萨克族成年人与汉族TPMT活性相比较,两者差异无统计学意义。结论中国新疆维吾尔族和哈萨克族人TPMT活性呈正态分布。     

高效液相色谱法测定巯嘌呤片含量

巯嘌呤片为常用的抗肿瘤药物制剂 ,主要用于治疗急性白血病 ,收载于美国、英国和中国药典中 ,其含量测定方法均采用紫外分光光度法[1～ 3] ,未见有高效液相色谱法的报道 ,本文介绍用高效液相色谱法测定巯嘌呤片的含量。1　仪器与试药高效液相色谱仪 :日本岛津ＬＣ - 10Ａｖｐ液相色谱仪 ,ＳＰＤ - 10Ａｖｐ紫外检测器 ,Ｃｌａｓｓ -ＶＰ数据处理机 ,ＣＴＯ - 10Ａｖｐ柱温箱。试药 :巯嘌呤、绿原酸对照品均由中国药品生物制品检定所提供 ;甲醇、磷酸二氢钾、磷酸均为分析纯 ;甲酸为色谱纯 ;巯嘌呤片由上海华联制药有限公司提供。2　色谱条件…     

高效液相色谱法测定血浆中硫唑嘌呤和巯嘌呤浓度

本文报道用高效液相色谱法测定人血浆中硫唑嘌呤和巯嘌呤浓度的方法。样品用三氯醋酸沉淀蛋白后，取上清液直接进样。以ＳｐｈｅｒｉｓｏｕｂＣ１８为固定相，甲醇－水－乙二胺（２００：８００：１０Ｖ／Ｖ）为流动相，检测波长为ＵＶ３１３ｎｍ，甲硝唑为内标。硫唑嘌呤和巯嘌呤最低检出浓度分别为０．１μｇ／ｍｌ和０．５μｇ／ｍｌ；平均回收率分别为９６．６±２．７２和１０３．４±２．６５。     

巯基嘌呤甲基转移酶在嘌呤类药物代谢中的作用及其多态性的研究进展

对巯基嘌呤甲基转移酶在巯嘌呤类药物体内代谢过程中的作用、其活性水平与药物活性物质在体内的浓度与药效、药物的毒副作用的关系、酶活性多态性种族差异及基因遗传多态性关系进行综述. 为指导临床合理用药,避免药物不良反应及个体化用药提供参考.     

人血浆中硫唑嘌呤中间代谢物6-巯嘌呤的高效液相色谱测定法

目的:建立人血浆中硫唑嘌呤中间代谢物6-巯嘌呤(6-mercaptopurine,6-MP)的高效液相色谱测定法.方法:取血浆200μL,以6-硫鸟嘌呤(6-TG)为内标,用70%高氯酸沉淀蛋白,6 mol·L-1氢氧化钠调节pH至中性后进样分析.色谱柱:Shimpack CLC-ODS(150 mm×6 mm,5 μm)柱;流动相:乙腈-水-醋酸(10:489:1);检测波长为323 nm.结果:本方法在2～200μg·L-1浓度范围内,线性关系良好(r=0.999 5),RSD为6.79%(n=10);高、中、低3个浓度质控样本的批内及批间RSD在1.3%～6.3%之间,回收率在99.1%～101.5%之间(n=10).结论:本方法简便、准确,能够满足临床研究需要.     

正常中国汉人红细胞中儿茶酚氧位甲基转移酶的活性测定

目的测定正常中国人群红细胞儿茶酚氧位甲基转移酶(COMT)活性浓度值,为研究COMT活性变化在疾病诊断中的应用提供依据。方法采用高效液相色谱法测定279名正常健康人(其中男性134例,女性145例)红细胞中COMT的活性浓度,用SPSS统计软件对性别间测定结果进行检验比较。结果男性和女性红细胞中COMT活性浓度分别为:(16.5±7.4)、(15.5±5.3)nmol.mL RBC-1.h-1。活性频率分布男性在6.0~37.4nmol.mL RBC-1.h-1,女性为5.4~37.2 nmol.mL RBC-1.h-1,男女之间COMT活性浓度的差异无统计学意义(P>0.05)。结论本法灵敏,简便,准确,重复性好,且测定结果与国外一些报道一致,适用于临床应用测定研究。本结果可作为中国健康人红细胞COMT活性正常值及今后研究的重要参考数据。     

HPLC法测定人红细胞中次黄嘌呤鸟嘌呤磷酸核糖转移酶活性

目的:建立一种检测次黄嘌呤鸟嘌呤磷酸核糖转移酶（HGPRT）活性的高效液相色谱法,并检测患者红细胞中HGPRT活性。方法:在5-磷酸核糖-1-焦磷酸（PRPP）条件下,HGPRT催化次黄嘌呤生成次黄嘌呤核苷酸（IMP）,高氯酸沉淀蛋白后进行高效液相色谱法分析。采用Hypersil ODS C₁₈（150 mm×4.6 mm,5μm）柱;流动相为磷酸氢盐缓冲液-乙腈（99.6:0.4）,流速1 ml·min^-1,在262 nm处检测IMP。应用新建立的高效液相色谱法测定68例服用过硫唑嘌呤的肾移植患者红细胞中HGPRT活性。结果:在该条件下,测得的线性范围是20～600μmol·L^-1,检测限是20μmol·L^-1,其日间和日内精密度均小于5%,方法回收率和提取回收率分别在100.81%～101.91%和99.05%～101.40%范围内。65例患者红细胞HGPRT活性范围为44.59～123.86 U,平均（87.83±21.55）U。结论:该法操作简便、灵敏度高、重复性好,适用于临床常规检测。     

Thiopurine S-methyltransferase activity in human erythrocytes: a new HPLC method using 6-thioguanine as substrate

Objectives: To develop a non-radioactive assay to measure thiopurine S-methyltransferase (TPMT) activity. The assay was used to study the distribution of TPMT activity in a healthy German population. Methods: The assay is based on the conversion of 6-thioguanine (6-TG) to 6-methylthioguanine (6-MTG) using non-radiolabelled S-adenosyl-l-methionine (SAM) as the methyl donor. At the end of the incubation period (60 min) 6-MTG is extracted into chloroform/2-propanol and quantitated by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection at Ex 315 nm and Em 390 nm. Results and discussion: The method is rapid, sensitive and reproducible, with an interassay CV of 6.7% (quality control sample with TPMT activity of 43 nmol 6-MTG · g⁻¹ Hb · h⁻¹) and thus suitable for routine monitoring of TPMT activity. The TPMT activity of 219 healthy German blood donors showed the known trimodal distribution with a range from 1.3 to 68.3 nmol 6-MTG · g⁻¹ Hb · h⁻¹ with a median value of 38.8 nmol 6-MTG · g⁻¹ Hb · h⁻¹. When the cut-off value for intermediate to high activity was set at 23.5 nmol 6-MTG · g⁻¹ Hb · h⁻¹, 14.1% belonged to the group with intermediate and 83.6% to the group with high TPMT activity. Five individuals had a very low TPMT activity of <2 nmol 6-MTG · g⁻¹ Hb · h⁻¹. Genetic analysis revealed that these persons were found either homozygote for the variant allele *3A (n=3) or they were compound heterozygotes for the variant alleles *3A/*3C (n=2). With these alleles for low TPMT activity they would run an increased risk of myelosuppression in case of treatment with standard doses of thiopurine drugs. Received: 30 September 1997 / Accepted in revised form: 17 January 1998     

高效液相色谱法测定人血红细胞内硫鸟嘌呤核苷酸浓度

目的:建立以高效液相色谱法测定人血红细胞内硫鸟嘌呤核苷酸浓度的方法。方法:色谱柱为HypersilODS,流动相为甲醇-水(10∶90),检测波长为342nm;硫鸟嘌呤核苷酸经加热水解生成6-硫鸟嘌呤(6-TG),6-TG经提取至0.1mol/L盐酸中进样分析,以外标法定量。结果:6-TG浓度线性范围为30～1200pmol/8×108RBCs,水解时间以1h为宜,提取时pH值以11～12为宜,提取液中醋酸苯汞的量为1.3mmol/L。结论:在优化条件下,本方法测定硫鸟嘌呤核苷酸快速、准确、灵敏度高。     

Thiopurine methyltransferase (TPMT) genotype distribution in azathioprine-tolerant and -intolerant patients with various disorders. The impact of TPMT genotyping in predicting toxicity

Objective To study the distribution of the thiopurine methyltransferase (TPMT) genotype among azathioprine (Aza)-tolerant and -intolerant patients with various disorders, and to investigate a possible relationship with the Aza metabolite levels.Methods Forty-six Aza-tolerant and six Aza-intolerant patients had the TPMT genotype distribution determined using a polymerase chain reaction (PCR) assay and the forty-six Aza-tolerant patients had the Aza metabolite levels determined using a high-pressure liquid chromatography (HPLC) analysis.Results One non-functional TPMT mutant allele was demonstrated in 2 of the 46 Aza-tolerant patients (4.4%) and one or two non-functional mutant alleles in 2 of the 6 Aza-intolerant patients (33.3%). Of the 4 patients, with one or two non-functional mutant alleles 2 (50%) were intolerant to Aza compared with 4 of the 48 patients (8.3%) with no mutations detected. The time to hepatotoxicity did not differ significantly between the 2 patients with one or two non-functional mutant alleles and the remaining 3 patients (P=0.5). The TPMT genotype distribution differed slightly in the three different categories of disorders (P=0.05). The median E-6-TGN level among the 2 TPMT heterozygous patients was 275 pmol/8×10⁸ RBC (range 240–310), whereas the remaining 44 patients had a median E-6-TGN level of 110 pmol/8×10⁸ RBC (range 0–440) (P=0.07).Conclusion Although TPMT genotyping cannot be recommended on behalf of the present study, it is to be expected that half of the patients with one or two non-functional TPMT mutant alleles will develop Aza intolerance leading to withdrawal of therapy. Thus, clinicians may anticipate about 5% of the patients to develop intolerance to Aza therapy solely for that reason.     

Thiopurine S-methyltransferase activity in three major Asian populations: a population-based study in Singapore

Objective The distribution of thiopurine methyltransferase (TPMT) activity in Asian populations has not been well documented. We studied the TPMT phenotype in three major Asian ethnic groups in Singapore, namely the Chinese (Ch), Malays (Mal) and Indians (Ind), with the aim of carrying out a comprehensive survey of the distribution of TPMT activity in Asians. Methods A radiochemical assay was used to measure the enzymatic activity of TPMT in the red blood cells (RBCs) of 479 healthy adults (Ch = 153, Mal = 163 and Ind = 163). Cut-off points for intermediate TPMT activity were validated using a receiver operating curve (ROC) analysis. PCR-based methods were used to screen for the TPMT*3C, TPMT*3A and TPMT*6 variants. Results The histogram of the combined population cohort showed a bimodal distribution of TPMT activity, with no subject having low TPMT activity (<5 units). In total, TPMT variants were detected in 14 subjects (*1/*3C in 13 subjects; *1/*3A in one subject). We observed significant inter-ethnic differences in terms of TPMT activity (p < 0.001), with the Malays showing a higher median activity than the Chinese or Indians (17.8 units vs 16.4 units). The Malays also showed a higher methylation rate – with a cut-off point for intermediate TPMT activity of 11.3 units – than the Chinese (9.9 units) or Indians (9.4 units). A high phenotype–genotype correlation of >97% was observed in all three races. We also genotyped 418 childhood leukaemias. The combined analysis of subjects participating in this and a previous study – 1585 subjects – showed that 4.7% of Chinese (n = 30/644), 4.4% of Malays (n =  24/540) and 2.7% of Indians (n = 11/401) were heterozygous at the TPMT gene locus. Conclusion This is the first comprehensive TPMT phenotype and genotype study in Asian populations, particularly in the Malays and Indians.     

巯嘌呤甲基转移酶基因检测用于指导6-巯基嘌呤在中国儿童急性淋巴细胞白血病中个体化治疗的循证评价

目的　系统评价6-巯基嘌呤(6-mercaptopurine, 6-MP)治疗亚洲儿童急性淋巴细胞白血病(acute lymphoblastic leukemia, ALL)出现骨髓抑制与巯嘌呤甲基转移酶(thiopurine methyltransferase, TPMT)基因多态性的相关性,并分析中国ALL患儿TPMT基因检测的经济性。方法　采用循证医学方法搜集6-MP治疗亚洲儿童ALL相关骨髓抑制与TPMT基因多态性相关的随机对照试验或观察性研究进行Meta分析。借助决策树模型针对中国ALL患儿,对两种6-MP初始给药剂量方案,进行成本效果分析。结果　最终纳入6个观察性研究,共577例亚洲患儿。Meta分析结果显示：TPMT基因多态性与骨髓毒性［OR=5.61,95%CI（2.05,15.34）,P=0.0008］发生有关。在基础数据分析中,针对中国ALL患儿,以上两个方案以严重骨髓抑制发生率为效果指标,增量成本-效果比为10403.83,敏感性分析显示结果稳定。结论　亚洲ALL患儿的TPMT基因多态性与6-MP的骨髓毒性显著相关。决策树模型结果显示,在中国,ALL患儿通过TPMT基因检测调整6-MP初始剂量并不优于标准剂量给药。     

Inhibition of thiopurine S-methyltransferase activity by impurities in commercially available substrates: a factor for differing results of TPMT measurements

Objectives: Thiopurine S-methyltransferase (TPMT) activity, when measured in red blood cells (RBC) with a recently published TPMT activity assay using 6‐thioguanine (6-TG) as substrate, could not be reproduced in another laboratory. We investigated factors which could influence the results of the TPMT activity measurement. Methods: We tested twelve 6-TG and four 6-mercaptopurine (6-MP) compounds from different suppliers as substrates and determined the enzyme kinetic parameters K_m and V_max . Furthermore, we studied the influence of different 6-TG compounds on the affinity of the methyl donor S-adenosyl-l-methionine (SAM) to the TPMT enzyme. Results: All 6-TG products were of equal purity (declared >98% by the supplier); this was ascertained by HPLC. However, the rate of methylation obtained following incubation with 6-TG from different suppliers ranged from 10% to 100% when incubated with the same RBC lysate. The lowest apparent K_m value for a 6-TG was 22.3 μmol · l⁻¹, while the product with the highest methylation rate showed a K_m of 156 μmol · l⁻¹. From these results we assume that there is a contaminant in some 6-TG products, which acts as a strong inhibitor of TPMT activity. Compounds possibly used for the synthesis of 6-TG (guanine, pyridine, 6-chloroguanine) did not affect the methylation rate. Thioxanthine, which is known to be a strong inhibitor of TPMT when added to the assay system to give a 2% contamination, reduced TPMT activity from 100% to 72%. Using 6-MP from different suppliers as substrate resulted in K_m values ranging from 110 to 162 μmol · l⁻¹ and V_max values ranging from 54 to 68 nmol 6‐MMP · g⁻¹Hb · h⁻¹. The K_m value for the methyl donor SAM was similar to and independent from the thiopurine substrates tested (range 4.9–11 μmol · l⁻¹ SAM). In contrast to other investigators, we found non-enzymatic S-methylation, which was negligible under our assay conditions (3% with 128 μmol · l⁻¹ SAM), but could become relevant in experiments using higher SAM concentrations. Conclusions: TPMT enzyme activity determined with 6‐TG as substrate may be strongly inhibited by a contaminant in some of the 6-TG lots distributed. Received: 28 June 1998 / Accepted in revised form: 18 January 1999     

高效液相-荧光检测法测定羟基喜树碱血浓度

目的:建立高效液相-荧光检测法测定人血浆羟基喜树碱的血浓度.方法:色谱柱为DiscoveryC18(15cm×4.6mm,5μm),流动相为枸橼酸缓冲液-乙腈-75nmol·ml-1磷酸二氢钾(70∶23∶7,含0.1%三乙胺),流速为1.0ml*min-1,柱温为50℃,荧光检测波长为λex363nm和λem530nm.结果:该方法的线性范围为19.3～1957.4ng*ml-1(r=0.9995).最低检测限为5.2ng*ml-1,平均加样回收率为91.8%.结论:该方法专属性强,重现性好,操作简便,适用于羟基喜树碱的药代动力学研究和血药浓度监测.     

高效液相色谱荧光检测法测定血浆加巴喷丁含量

目的:建立测定血浆加巴喷丁浓度的高效液相色谱法.方法:待测血浆中加入内标替米沙坦后,用乙腈沉淀蛋白,邻苯二甲醛衍生化,进行高效液相荧光检测.色谱柱为Symmetry C18柱,流动相为0.05mol·L-1磷酸二氢钾溶液-乙腈(57:43),pH 3.6.流速0.8 ml·min-1,激发波长330 nm,发射波长440 nm.结果:线性范围为0.027～16.35 mg·L-,r=0.997 3,日内、日间RSD均小于10%,低、中、高三种浓度的平均回收率分别为91.9%,99.2%,96.0%,最低检测限为0.027 mg·L-1.结论:本法精密、准确,适用于临床上血药浓度监测、人体药动学和生物利用度研究.