Casal Enriqueta, Palomo Laura, Cabrera Diana, Falcon-Perez Juan M
Metabolomics Platform, CIC bioGUNE, CIBERehd, Bizkaia Technology Park Bizkaia, Spain.
Exosomes Laboratory, Metabolomics Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park Bizkaia, Spain.
Front Pharmacol. 2016 Dec 23;7:501. doi: 10.3389/fphar.2016.00501. eCollection 2016.
There is a clear need for drug treatments to be selected according to the characteristics of an individual patient, in order to improve efficacy and reduce the number and severity of adverse drug reactions. One of the main enzymes to take into account in pharmacogenomics is catechol O-methyltransferase (COMT), which catalyzes the transfer of a methyl group from -adenosylmethionine to catechols and catecholamines, like the neurotransmitters dopamine, epinephrine, and norepinephrine. Although, most of this enzyme is associated to intracellular vesicles, recently it has also been detected in extracellular vesicles secreted by hepatocytes and in serum circulating vesicles. COMT has implications in many neurological and psychiatric disorders like Parkinson's disease, chronic fatigue, pain response, schizophrenia, and bipolar disorders. Remarkably, genetic variations of COMT affect its activity and are associated to various human disorders from psychiatric diseases to estrogen-induced cancers. Consequently, the establishment of new methods to evaluate COMT activity is an important aspect to investigate the biology of this drug-metabolizing enzyme. Herein, we have developed a sensitive and selective method to determine COMT activity. We first optimized the activity in rat liver incubated with two different substrates; norepinephrine and dopamine. The enzymatically formed products (normetanephrine and 3-methoxytyramine, respectively) were extracted by solid-phase extraction using weak cation exchange cartridges, chromatographically separated, and detected and quantified using a mass spectrometer. The range of quantitation for both products was from 0.005 to 25 μg/mL. This methodology offers acceptable recovery for both enzymatic products (≥75%) and good accuracy and precision (≤15%). The lower limit of quantifications were 0.01 and 0.005 μM for 3-methoxytyramine and normetanephrine, respectively. Importantly, this sensitive assay was able to detect the presence of COMT activity in extracellular vesicles secreted by hepatocytes supporting a potential role of these vesicles in catecholamines and catecholestrogens metabolisms. In addition, the presence of COMT activity in extracellular vesicles opens new possibilities to develop tools to evaluate personalized drug response in a low invasive manner.
显然需要根据个体患者的特征选择药物治疗方法，以提高疗效并减少药物不良反应的数量和严重程度。药物基因组学中需要考虑的主要酶之一是儿茶酚-O-甲基转移酶（COMT），它催化甲基从S-腺苷甲硫氨酸转移到儿茶酚和儿茶酚胺，如神经递质多巴胺、肾上腺素和去甲肾上腺素。虽然这种酶的大部分与细胞内囊泡相关，但最近也在肝细胞分泌的细胞外囊泡和血清循环囊泡中检测到。COMT与许多神经和精神疾病有关，如帕金森病、慢性疲劳、疼痛反应、精神分裂症和双相情感障碍。值得注意的是，COMT的基因变异会影响其活性，并与从精神疾病到雌激素诱导的癌症等各种人类疾病相关。因此，建立评估COMT活性的新方法是研究这种药物代谢酶生物学的一个重要方面。在此，我们开发了一种灵敏且选择性的方法来测定COMT活性。我们首先优化了用两种不同底物（去甲肾上腺素和多巴胺）孵育大鼠肝脏中的活性。酶促形成的产物（分别为去甲变肾上腺素和3-甲氧基酪胺）通过使用弱阳离子交换柱的固相萃取进行提取，进行色谱分离，并使用质谱仪进行检测和定量。两种产物的定量范围为0.005至25μg/mL。该方法对两种酶促产物均具有可接受的回收率（≥75%）以及良好的准确度和精密度（≤15%）。3-甲氧基酪胺和去甲变肾上腺素的定量下限分别为0.01和0.005μM。重要的是，这种灵敏的检测方法能够检测到肝细胞分泌的细胞外囊泡中COMT活性的存在，支持了这些囊泡在儿茶酚胺和儿茶酚雌激素代谢中的潜在作用。此外，细胞外囊泡中COMT活性的存在为开发以低侵入性方式评估个性化药物反应的工具开辟了新的可能性。
