采用 LC-MS/MS 同时定量测定淋巴细胞中 IMPDH 活性和嘌呤碱：评估生物标志物对霉酚酸的反应。
Simultaneous quantification of IMPDH activity and purine bases in lymphocytes using LC-MS/MS: assessment of biomarker responses to mycophenolic acid.
机构信息
*Department of Medical Biochemistry, Oslo University Hospital; †School of Pharmacy, University of Oslo; and Departments of ‡Anaesthesiology, §Pharmacology, and ¶Transplantation, Oslo University Hospital, Oslo, Norway.
出版信息
Ther Drug Monit. 2014 Feb;36(1):108-18. doi: 10.1097/FTD.0b013e3182a13900.
BACKGROUND
The development of biomarkers describing the individual responses to the immunosuppressant mycophenolic acid (MPA) has focused on the target enzyme activity [inosine 5'-monophosphate dehydrogenase (IMPDH)]. An extended strategy is to quantify the metabolic consequences of IMPDH inhibition. The aim of this study was to develop an assay for quantification of IMPDH activity and related purine bases and to provide preliminary data on the behavior of these biomarkers during clinical exposure to MPA.
METHODS
Liquid chromatography-mass spectrometry was used to determine xanthine (IMPDH activity in incubated cell lysate), hypoxanthine, guanine, and adenine derived from free nucleotides in lymphocytes. Analytical performance was assessed, and the biomarkers were examined in CD4⁺ cells from 2 groups: Healthy individuals in a single-dose MPA study (n = 5) and liver transplant recipients on MPA therapy (n = 15).
RESULTS
Coefficients of variation between series were below 10% and 15% for measurement of the purines and IMPDH activity, respectively. Although IMPDH was inhibited, the purine levels increased in response to MPA in 3 of the 5 healthy individuals, and this positive response seemed to be associated with IMPDH1 c.579 + 119 G/G and c.580 - 106 G/G. In the liver transplant study, guanine was not reduced in response to the transient drop in IMPDH activity after MPA dosing. However, there were trends toward decrease in guanine and elevation of hypoxanthine during prolonged MPA therapy. The guanine/hypoxanthine ratio (median) was 37% lower and the adenine level was 21% lower at day 17 compared with day 4 after transplantation.
CONCLUSIONS
The assay allows precise quantification of IMPDH activity, hypoxanthine, guanine, and adenine in lymphocytes. Some individuals may possess a counteracting purine response to the MPA-mediated inhibition of IMPDH. Reduction of the guanine/hypoxanthine ratio may be related to prolonged inhibition of IMPDH and seems as an intriguing pharmacodynamic biomarker for MPA.
背景
描述个体对免疫抑制剂麦考酚酸（MPA）反应的生物标志物的发展侧重于靶酶活性[肌苷 5'-单磷酸脱氢酶（IMPDH）]。一种扩展的策略是量化 IMPDH 抑制的代谢后果。本研究的目的是开发一种用于定量测定 IMPDH 活性和相关嘌呤碱的测定法，并提供这些生物标志物在临床暴露于 MPA 期间行为的初步数据。
方法
使用液相色谱-质谱法测定孵育细胞裂解物中黄嘌呤（IMPDH 活性）、次黄嘌呤、鸟嘌呤和腺嘌呤，这些嘌呤来自淋巴细胞中的游离核苷酸。评估分析性能，并在 2 组 CD4⁺细胞中检查生物标志物：单次 MPA 研究的健康个体（n = 5）和 MPA 治疗的肝移植受者（n = 15）。
结果
嘌呤和 IMPDH 活性测量的系列间变异系数分别低于 10%和 15%。尽管 IMPDH 受到抑制，但在 5 名健康个体中的 3 名中，MPA 会导致嘌呤水平升高，这种阳性反应似乎与 IMPDH1 c.579 + 119 G/G 和 c.580-106 G/G 有关。在肝移植研究中，在 MPA 给药后 IMPDH 活性短暂下降时，鸟嘌呤并未减少。然而，在长期 MPA 治疗期间，鸟嘌呤和次黄嘌呤的水平呈下降趋势。与移植后第 4 天相比，第 17 天的鸟嘌呤/次黄嘌呤比值（中位数）降低了 37%，腺嘌呤水平降低了 21%。
结论
该测定法可精确定量淋巴细胞中的 IMPDH 活性、次黄嘌呤、鸟嘌呤和腺嘌呤。一些个体可能对 MPA 介导的 IMPDH 抑制具有拮抗的嘌呤反应。鸟嘌呤/次黄嘌呤比值的降低可能与 IMPDH 的长期抑制有关，并且似乎是 MPA 的一个有趣的药效动力学生物标志物。
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