A Novel Sensitive Method to Measure Catechol-O-Methyltransferase Activity Unravels the Presence of This Activity in Extracellular Vesicles Released by Rat Hepatocytes

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.

Abundance of Cytochromes in Hepatic Extracellular Vesicles Is Altered by Drugs Related With Drug‐Induced Liver Injury

“…Remarkably, Cyp2d1 was highly enriched in the same fraction, strongly suggesting that this enzyme was associated with extracellular vesicles. We have demonstrated in hepatocyte‐secreted EVs the presence of not only the protein but also the activity for the Arg1, Ces1d, and Comt enzymes . Here, we addressed the question of whether the Cyp2d1 enzyme, detected in EVs, is active.…”

“…In addition, a common feature to all treatments related to amino acid metabolism was the increased level of the enzyme catecholamine methyl transferase (Comt) (Fig. and Supporting Table ), which is involved in the metabolization of tyrosine‐derived stress hormones (e.g., epinephrine) and shown to be active in EVs …”

“…5 and Supporting Table S1), which is involved in the metabolization of tyrosine-derived stress hormones (e.g., epinephrine) and shown to be active in EVs. (25) Another common feature detected by our proteomics analysis was a decrease in the EV levels of some transporters (i.e., Atp1a, Slc2a2, Slc22a7, and Slco1a2) and their intracellular regulators (basigin and beta-subunit of coatomer) ( Fig. 5 and Supporting Table S1) involved in the regulation of glucose, prostaglandin, and bile acid metabolisms.…”

“…Importantly, the different compounds acted differentially to the abundance of EVs of different xenobiotic‐related enzymes, including Ces1d and Comt. We have shown that these enzymes are active in EVs . Here, we demonstrated that another xenobiotic‐related activity, Cyp2d1, is also active in hepatocyte‐secreted EVs and, as occurs with the previous enzymes, pharmaceutical compounds altered its abundance in EVs.…”

“…We have demonstrated in hepatocyte-secreted EVs the presence of not only the protein but also the activity for the Arg1, Ces1d, and Comt enzymes. (11,23,25) Here, we addressed the question of whether the Cyp2d1 enzyme, detected in EVs, is active. For this purpose, we used a commercial enzymatic assay that is available for CYP2D6, the human orthologous of the rat Cyp2d1.…”