Sensitive and Selective Determination of Orotic Acid in Biological Specimens Using a Novel Fluorogenic Reaction

• Published in: Journal of fluorescence Vol. 25; no. 4; pp. 1005 - 1011
• Main Authors: Yin, Sheng, Dragusha, Shpend, Ejupi, Valon, Shibata, Takayuki, Kabashima, Tsutomu, Kai, Masaaki
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.07.2015
• Subjects: Adult; Analytical Chemistry; Benzamidines - chemistry; Biochemistry; Biological; Biological and Medical Physics; Biomedical and Life Sciences; Biomedicine; Biophysics; Biotechnology; Chromatography, High Pressure Liquid - methods; Creatinine; Fluorescence; HeLa Cells; Human; Humans; Hydrocarbons, Fluorinated - chemistry; Indicators and Reagents; Liquid chromatography; Male; Nucleosides; Original Paper; Orotic Acid - analysis; Spectrometry, Fluorescence - methods; Urinalysis - methods; Urine; Vitamins; Fluorogenic reaction; HeLa cells; Orotic acid; Human urine; Quantitative determination
• ISSN: 1053-0509; 1573-4994
• DOI: 10.1007/s10895-015-1584-3

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, K 3 [Fe(CN) 6 ] and K 2 CO 3 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.