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Determination of Dopamine-β-hydroxylase Activity in Human Serum Using UHPLC-PDA Detection

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Abstract

Dopamine-β-hydroxylase (DBH, EC 1.14.17.1) is an enzyme with implications in various neuropsychiatric and cardiovascular diseases and is a known drug target. There is a dearth of cost effective and fast method for estimation of activity of this enzyme. A sensitive UHPLC based method for the estimation of DBH activity in human sera samples based on separation of substrate tyramine from the product octopamine in 3 min is described here. In this newly developed protocol, a Solid Phase Extraction (SPE) sample purification step prior to LC separation, selectively removes interferences from the reaction cocktail with almost no additional burden on analyte recovery. The response was found to be linear with an r2 = 0.999. The coefficient of variation for assay precision was < 10% and recovery > 90%. As a proof of concept, DBH activity in sera from healthy human volunteers (n = 60) and schizophrenia subjects (n = 60) were successfully determined using this method. There was a significant decrease in sera DBH activity in subjects affected by schizophrenia (p < 0.05) as compared to healthy volunteers. This novel assay employing SPE to separate octopamine and tyramine from the cocktail matrix may have implications for categorising subjects into various risk groups for Schizophrenia, Parkinson’s disease as well as in high throughput screening of inhibitors.

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Acknowledgements

We gratefully acknowledge M/s Waters (India) Private limited for technical assistance; Dr. Nipender Singh & Mr. Rajeev Kumar of Central Instrumentation Facility (CIF), University of Delhi South Campus for assistance throughout this work. This work was supported by Department of Biotechnology, Government of India, New Delhi Grant# BT/01/COE/07UDSC and National Institute of Health Grant# D43TW06167; junior and senior research fellowship Grant # DBT-JRF/07–08/264 from Dept. of Biotechnology, Govt. of India, New Delhi and UGC research fellowship in sciences for meritorious students, Dept. of Genetics, University of Delhi South campus, New Delhi to TJP.

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Authors and Affiliations

  1. Department of Genetics, University of Delhi, South Campus, Benito Juarez Road, New Delhi, 110021, India

    Toyanji Joseph Punchaichira & B. K. Thelma

  2. Department of Psychiatry, Postgraduate Institute of Medical Education and Research-Dr. Ram Manohar Lohia Hospital, Baba Kharak Singh Marg, Connaught Place, New Delhi, 110001, India

    Smita Neelkanth Deshpande

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  1. Toyanji Joseph Punchaichira
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Correspondence to B. K. Thelma.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical clearance for this work was obtained (Letter No.18–62/06-RMLH(HA-I)/VOL II/63, dated 30.11.2008) from the Institutional ethics committee RMLH, located at Dr.R.M.L. Hospital, Baba Kharak Singh Marg, New Delhi. This article does not contain any studies with animals performed by any of the authors.

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Punchaichira, T.J., Deshpande, S.N. & Thelma, B.K. Determination of Dopamine-β-hydroxylase Activity in Human Serum Using UHPLC-PDA Detection. Neurochem Res 43, 2324–2332 (2018). https://doi.org/10.1007/s11064-018-2653-1

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  • DOI: https://doi.org/10.1007/s11064-018-2653-1

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