Quantitative radioisotopic determination of histidine decarboxylase using high-performance liquid chromatography.
We have developed a procedure for the accurate measurement of histidine decarboxylase in tissues expressing low levels of enzymatic activity. Briefly, histamine is enzymatically synthesized from [3H]-labeled histidine, followed by purification using high-performance liquid chromatography (HPLC) and quantitation by liquid scintillation counting. This method presents three advantages over previous techniques. First, prior to HPLC purification, excess precursor [3H]histidine is removed on an anion-exchange resin. Second, purification by HPLC is considerably more selective than that of classical cation-exchange gravity columns or organic solvent extractions. Finally, the accuracy of this method is improved by including non-radiolabeled histamine as internal standard, which is quantified by ultraviolet detection. This simple procedure allows highly sensitive and accurate determinations of histamine synthesis.
Ortiz
J
J
Department of Biochemistry and Molecular Biology, School of Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain. jortiz@servet.uab.es
Gómez
J
J
Torrent
A
A
Aldavert
M
M
Blanco
I
I
D000818:Animals; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D006640:Histidine Decarboxylase / Q000378:metabolism*; D011874:Radiometry / Q000379:methods*; D051381:Rats; D012015:Reference Standards; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D013347:Subcellular Fractions / Q000201:enzymology; D014316:Tritium
D014316:Tritium; D006640:Histidine Decarboxylase