Determination of thiazole-4-carboxamide adenine dinucleotide (TAD) levels in mononuclear cells of leukemic patients treated with tiazofurin.
Determination of thiazole-4-carboxamide adenine dinucleotide (TAD) levels in mononuclear cells of leukemic patients treated with tiazofurin.
复制标题
测定接受噻唑呋林治疗的白血病患者单核细胞中噻唑-4-甲酰胺腺嘌呤二核苷酸 (TAD) 水平。
DOI：
10.1016/0006-2952(91)90487-p
复制
发表时间：
1991
影响因子：
5.8
通讯作者：
Weber,G
中科院分区：
文献类型：
--
作者：
Zhen,W;Jayaram,HN;Weber,G
噻唑呋喃是一种溶瘤药物，已在终末期急性非淋巴细胞白血病(ANLL)和慢性粒细胞白血病(CGL-BC)急变期显示出治疗活性。噻唑呋喃被合成为活性代谢物--噻唑-4-甲酰胺腺嘌呤二核苷酸(TAD)，它能抑制IMP脱氢酶活性，导致鸟苷酸库的减少和癌细胞的停止增殖。接受噻唑呋喃治疗的患者肿瘤细胞中TAD的浓度应该是对药物敏感性的一个很好的指标，也可能预示着耐药细胞的出现。因此，在噻唑呋喃类药物治疗过程中，肿瘤细胞中TAD的精确定量是至关重要的。本文报道了一种测定生物样品中TAD的高灵敏方法。利用这项技术，除了TAD之外，还可以准确地分离和定量其他13种与生物相关的腺嘌呤、鸟嘌呤、胞嘧啶和尿苷核苷酸。TAD标准品在Waters Partisil 10-SAX色谱柱上，在RCM-10模块中使用磷酸铵缓冲系统进行分离。TAD洗脱时间为21min，最低检测限为ISpmo1，线性范围达3nmo1。保留时间和TAD浓度的变异系数分别为0.6±0.1%和2±0.3%。TAD的回收率为96%，重现性为98%。为了检验这种方法在临床上的适用性，采集了一名CGL-BC患者的血液样本，在Ficoll-Hypaq梯度上分离白细胞，用三氯乙酸提取，并在高效液相色谱法上分析了等量。通过比较保留时间和标准物质的光谱分析，确定TAD峰。患者接受2200 mg/m2(12.7 mM)的噻唑呋喃治疗后，2、6、24小时单核细胞TAD浓度分别为23.1、13.6、0.8μ，3300 mg/m2(21.1 mM)治疗后2、6、24小时TAD分别为42.8、26.1、1.4μM。
Tiazofurin is an oncolytic agent which has shown therapeutic activity in end-stage acute nonlymphocytic leukemia (ANLL) and blast crisis of chronic granulocytic leukemia (CGL-BC). Tiazofurin is anabolized to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), which inhibits IMP dehydrogenase activity, leading to reduction of guanylate pools and cessation of cancer cell proliferation. The concentration of TAD in neoplastic cells of patients treated with tiazofurin should be a good indicator of sensitivity to the drug and also might herald the emergence of drug-resistant cells. Therefore, the precise quantitation of TAD in cancer cells during tiazofurin treatment is essential. In this paper we report a highly sensitive method for the determination of TAD in biological samples. With this technique, in addition to TAD, thirteen other biologically relevant adenine, guanine, cytosine and uridine nucleotides can be separated and quantitated accurately. TAD standard was separated on a Waters Partisil 10-SAX column in a RCM-10 module using an ammonium phosphate buffer system. TAD eluted at 21 min with a limit of detection of ISpmol and linearity up to 3nmol. The coefficient of variation was 0.6 ± 0.1% for retention time and 2 ± 0.3% for TAD concentration. Recovery of TAD was 96% with reproducibility of 98%. To examine the applicability of this method to a clinical setting, blood samples were obtained from a patient with CGL-BC and leukocytes were separated on a Ficoll-Hypaq gradient, extracted with trichloroacetic acid, and an aliquot was analyzed on HPLC. The TAD peak was identified by comparing the retention time and spectral analysis of the standard. After the patient was treated with a 2200 mg/m2(12.7 mM) dose of tiazofurin, the TAD concentrations in the mononuclear cells at 2, 6, and 24 hr were 23.1, 13.6, and 0.8 μM. TAD levels at 2, 6, and 24 hr after a tiazofurin dose of 3300 mg/m2(21.1 mM) were 42.8, 26.1, and 1.4 μM respectively.