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Journal of Fluorescence

, Volume 25, Issue 4, pp 1005–1011 | Cite as

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

  • Sheng Yin
  • Shpend Dragusha
  • Valon Ejupi
  • Takayuki Shibata
  • Tsutomu Kabashima
  • Masaaki Kai
ORIGINAL PAPER

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 × 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.

Keywords

Orotic acid Fluorogenic reaction Quantitative determination Human urine HeLa cells 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sheng Yin
    • 1
  • Shpend Dragusha
    • 1
  • Valon Ejupi
    • 1
  • Takayuki Shibata
    • 1
  • Tsutomu Kabashima
    • 1
  • Masaaki Kai
    • 1
  1. 1.Faculty of Pharmaceutical Sciences, Graduate School of Biomedical SciencesNagasaki UniversityNagasakiJapan

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