An investigation of dopaminergic metabolites in the striatum and in the substantia nigra in vivo utilising radiolabelled L-DOPA and high performance liquid chromatography: a new approach in the search for transmitter metabolites.
Although the major routes of dopamine metabolism seem to be established, at least in terminal regions such as the striatum, it is important to search for previously unknown metabolites and to investigate the relevance of previously suggested minor alternative pathways. An urgent issue is to verify and quantify the transformation of dopamine to putative toxic species, another is to further explore metabolism of dopamine located in cell bodies/dendrites, e.g. in the substantia nigra. We have developed a new method in order to widen the search for alternative metabolites of dopamine. The method is based on systemic injection of tritiated L-DOPA to rats in vivo. Brain tissue was homogenised and centrifuged and the resulting supernatant fractioned following passage through a liquid chromatography system. The radioactivity of each fraction was measured using a scintillation system. By identifying fractions containing major catecholamines and metabolites, according to a standard solution, novel metabolites can be searched for in the remaining fractions. It was possible to obtain sufficient radioactivity in separate fractions of supernatant of homogenised tissue, even from such a small brain nucleus as substantia nigra. Radioactivity was obtained in those fractions that contained the major catecholamines and their metabolites, as well as in other fractions where it may represent previously unknown metabolites of L-DOPA/dopamine. The method was used to evaluate the possibility that cytochrome P450 2E1 is involved in the metabolism of dopamine in the substantia nigra. Significant changes in the radioactivity pattern were induced by inhibition of the enzyme but conclusions about whether cytochrome P450 2E1 is involved in the metabolism of dopamine or not requires further studies. The method can be used to study the metabolism of dopamine and can be extended, by using other radiolabelled precursors, also to evaluate metabolism of other transmitters, e.g. serotonin.
Niazi Shahabi
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Department of Pharmacology, Institute of Physiology and Pharmacology, Göteborg University, Box 431, 405 30, Göteborg, Sweden.
Bergquist
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Nissbrandt
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D015102:3,4-Dihydroxyphenylacetic Acid / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism*; D004298:Dopamine / Q000031:analogs & derivatives* / Q000378:metabolism*; D015259:Dopamine Agents / Q000493:pharmacokinetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006719:Homovanillic Acid / Q000378:metabolism; D017879:Isothiocyanates / Q000494:pharmacology; D007980:Levodopa / Q000493:pharmacokinetics*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013378:Substantia Nigra / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D014316:Tritium / Q000493:pharmacokinetics
D015259:Dopamine Agents; D004791:Enzyme Inhibitors; D017879:Isothiocyanates; D014316:Tritium; D015102:3,4-Dihydroxyphenylacetic Acid; D007980:Levodopa; C058305:phenethyl isothiocyanate; C001746:3-methoxytyramine; D004298:Dopamine; D006719:Homovanillic Acid