Determination of tetrahydrobiopterin in murine locus coeruleus by HPLC with fluorescence detection

• Published in: Brain research. Brain research protocols Vol. 8; no. 1; pp. 25 - 31
• Main Authors: Kaneko, Yoko S, Mori, Keiji, Nakashima, Akira, Nagatsu, Ikuko, Nagatsu, Toshiharu, Ota, Akira
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2001
• Subjects: Animals; Biopterins - analogs & derivatives; Biopterins - metabolism; Chromatography, High Pressure Liquid - methods; Differential oxidation; Fluorescence; Fluorescence detection; GTP cyclohydrolase I; High-performance liquid chromatography (HPLC); Locus coeruleus; Locus Coeruleus - metabolism; Mice; Mice, Inbred C3H; Oxidation-Reduction; Sensitivity and Specificity; Tetrahydrobiopterin; Time Factors; guanosine 5′-triphosphate; GTP; EDTA, ethylenediaminetetraacetic acid; High-performance liquid chromatography (HPLC); LC, locus coeruleus; Neurotransmitters, modulators, transporters, and receptors; Fluorescence detection; CNS, central nervous system; GTP cyclohydrolase I; Tetrahydrobiopterin; i.p., intraperitoneal; TH, tyrosine hydroxylase; Tris, 2-amino-2-hydroxymethyl-1,3-propanediol; BH4, (6 R)- l- erythro-dihydroxypropyl-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridin; Differential oxidation; Locus coeruleus
• ISSN: 1385-299X
• DOI: 10.1016/S1385-299X(01)00081-2

Tetrahydrobiopterin in the murine locus coeruleus was measured as its fully oxidized form, biopterin, using a HPLC coupled to a fluorescence detector, because tetrahydrobiopterin itself cannot be detected by such means. The differential oxidization method distinguished tetrahydrobiopterin-derived biopterin and dihydrobiopterin-derived biopterin. The protocol reported here is a rapid and sensitive method that facilitates the measurement of tissue and/or cellular tetrahydrobiopterin. Using this assay protocol, we were able to detect and quantify variations in the tetrahydrobiopterin content in the murine locus coeruleus.