Simultaneous analysis of catechol‐O‐methyl transferase activity, S‐adenosylhomocysteine and adenosine

Reenilä, Ilkka; Rauhala, Pekka

doi:10.1002/bmc.1288

Cited by 5 publications

(3 citation statements)

References 21 publications

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“…To date, a variety of COMT enzyme assays have been developed (Pihlavisto and Reenilä, 2002; Aoyama et al, 2005; Masuda et al, 2006; Hirano et al, 2007; Reenilä and Rauhala, 2010; Kimos et al, 2016). However, to our knowledge, none of them uses ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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“…Activity assay of recombinant Fc-COMT using HPLC-MS: Using DHBAc as substrate, the enzymatic reaction products of Vanillic Acid (VA) and Isovanillic Acid (IVA) were detected by HPLC-MS according to methods previously described (Li et al, 2004;Reenila and Rauhala, 2009;Reenila et al, 1995).…”

Section: Expression and Purification Of Recombinant Fc-comtmentioning

confidence: 99%

Expression, Purification and Activity Assay of the Recombinant Protein of Catechol-O-Methyltransferase from Chinese White Shrimp (Fenneropenaeus chinensis)

Li¹,

Du²,

Zhao³

et al. 2010

American Journal of Biochemistry and Biotechnology

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Problem statement: We have previously cloned a gene of Chinese white shrimp Catechol O-Methyltransferase (designated Fc-COMT) and characterized the gene expression pattern. In this study, expression and purification as well as activity assay of the recombinant Fc-COMT was further conducted. Approach: Using pET-30a (+) as a prokaryotic expression vector, the recombinant Fc- COMT was expressed in the supernatant of Escherichia coli lysate and easily purified by His-Bind resin chromatography. SDS-PAGE analysis showed that the molecular mass of recombinant Fc-COMT was approximately 30,000 Da, in good agreement with the software-predicted molecular weight. The enzymatic activity of recombinant Fc-COMT was tested using Dihydroxybenzoic Acid (DHBAc) as a substrate. Results: The methyl products of DHBAc, Vanillic Acid (VA) and Isovanillic Acid (IVA), were detected in the enzymatic reaction mixture with recombinant Fc-COMT by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS). Conclusion: The recombinant Fc-COMT has catalytic activity of transferring methyl group from S-Adenosyl-L-Methionine (SAM) to the 3 hydroxyl or 4 hydroxyl group of benzyl ring of DHBAc

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“…Many of crucial psychiatric disorders such as schizophrenia, alcoholism, bipolar and and obsessive‐compulsive disorder are caused by SNP in a single nucleotide polymorphism (Val108/158 Met) in the catechol‐ O ‐methyl transferase (COMT) gene 20, 21. COMT enzyme catalyzes the O ‐methylation of substrates which has a catechol moiety 22. COMT has a basic role for the catabolism of endogenous catecholamine neurotransmitters such as epinephrine, dopamine 23.…”

Section: Introductionmentioning

confidence: 99%

Gold(I)‐Catalyzed Hydration of Alkynylphosphonates: Efficient Access to β‐Ketophosphonates

Xie¹,

Yuan²,

Wang³

et al. 2014

Eur J Org Chem

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Simultaneous analysis of catechol‐O‐methyl transferase activity, S‐adenosylhomocysteine and adenosine

Cited by 5 publications

References 21 publications

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

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

Expression, Purification and Activity Assay of the Recombinant Protein of Catechol-O-Methyltransferase from Chinese White Shrimp (<i>Fenneropenaeus chinensis</i>)

Gold(I)‐Catalyzed Hydration of Alkynylphosphonates: Efficient Access to β‐Ketophosphonates

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