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 × 105 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.
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This work was supported by Grants-in-Aid for scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and also supported partially by the Global Center of Excellence Program at Nagasaki University.
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Yin, S., Dragusha, S., Ejupi, V. et al. Sensitive and Selective Determination of Orotic Acid in Biological Specimens Using a Novel Fluorogenic Reaction. J Fluoresc 25, 1005–1011 (2015). https://doi.org/10.1007/s10895-015-1584-3
- Orotic acid
- Fluorogenic reaction
- Quantitative determination
- Human urine
- HeLa cells