title: Sensitive and Selective Determination of Orotic Acid in Biological Specimens Using a Novel Fluorogenic Reaction.
abstract: Orotic acid is an intermediate in the synthesis pathway of uridine-5'-monophosphate, and increases in body fluids of patients suffering from hereditary disorders such as orotic aciduria and hyperammonemia. In this study, we developed a spectrofluorometric method with or without high-performance liquid chromatography for the selective and sensitive quantification of orotic acid in human biological specimens, using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. This reagent provided intensive fluorescence for only orotic acid amongst 62 compounds including structurally related bio-substances such as nucleic acid bases, nucleosides, nucleotides, amino acids, vitamins, bilirubin, uric acid, urea, creatine, creatinine and sugars. Under optimized reaction conditions, orotic acid was reacted with 4-TFMBAO, K3[Fe(CN)6] and K2CO3 in an aqueous solution. The fluorescence produced from the orotic acid derivative was measured at an excitation of 340 nm and an emission of 460 nm. A concentration of 1.2 μM orotic acid per 1.0 mM creatinine in normal urine and 0.64 nmol orotic acid per 5.0 × 10(5) HeLa cells were determined by this method. The present method permitted the facile quantification of orotic acid in healthy human urine and cultured HeLa cells by spectrofluorometry and/or high-performance liquid chromatography.
authors.affiliation: Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki, 852-8521, Japan.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yin
authors.forename: Shpend
authors.initials: S
authors.lastname: Dragusha
authors.forename: Valon
authors.initials: V
authors.lastname: Ejupi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shibata
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kabashima
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kai
mesh_terms: D000328:Adult; D001550:Benzamidines / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000737:chemistry*; D007202:Indicators and Reagents; D008297:Male; D009963:Orotic Acid / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D016482:Urinalysis / Q000379:methods*
chemical_list: C000591998:4-trifluoromethylbenzamidoxime; D001550:Benzamidines; D006845:Hydrocarbons, Fluorinated; D007202:Indicators and Reagents; D009963:Orotic Acid