Abstract
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 S-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.
Highlights
COMT (EC 2.1.1.6) is an enzyme that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to one of the hydroxyls of a catechol in the presence of Mg2+
COMT assays are performed under physiological conditions
To simplify the assay we have focused on the detection of membrane-bound COMT (MB-COMT) activity and the optimal conditions established for MB-COMT were used
Summary
COMT (EC 2.1.1.6) is an enzyme that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to one of the hydroxyls of a catechol in the presence of Mg2+ (for review see Männistö and Kaakkola, 1999; Pihlavisto and Reenilä, 2002). COMT can be found in most mammalian tissues (e.g., in brain; regulating the amounts of norepinephrine, dopamine, and epinephrine), with highest activities in liver, kidney, and intestinal tract, where COMT may modulate the dopaminergic tone. S-COMT is located in the cytosol and MB-COMT is anchored to the rough endoplasmic reticulum (Ulmanen et al, 1997; Myöhänen et al, 2010). MB-COMT has higher affinity for catechol substrates and for S-adenosylL-methionine coenzyme (Lotta et al, 1995). This enzyme has been detected in extracellular vesicles (EVs) secreted by hepatocytes (Conde-Vancells et al, 2008) and in serum circulating vesicles (Rodríguez-Suárez et al, 2014) suggesting a possible role of these vesicles in the metabolism of catecholamine neurotransmitters and catecholestrogens metabolisms
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