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


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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  1. 1.

    Löffler M, Fairbanks LD, Zameitat E, Marinaki AM, Simmonds HA (2005) Pyrimidine pathways in health and disease. Trends Mol Med 11:430–437

    PubMed  Article  Google Scholar 

  2. 2.

    Harden KK, Robinson JL (1987) Deficiency of UMP synthase in dairy cattle: a model for hereditary orotic aciduria. J Inherit Metab Dis 10:201–209

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Traut TW, Jones ME (1996) Uracil metabolism-UMP synthesis from orotic acid or uridine and conversion of uracil to β-alanine: enzymes and cDNAs. Prog Nucleic Acid Res Mol Biol 53:1–78

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Simmonds HA, Webster DR, Becroft DM (1980) Potter CF Purine and pyrimidine metabolism in hereditary orotic aciduria: some unexpected effects of allopurinol. Eur J Clin Investig 10:333–339

    CAS  Article  Google Scholar 

  5. 5.

    Bailey CJ (2009) Orotic aciduria and uridine monophosphate synthase A reappraisal. J Inherit Metab Dis 32:227–233

    Article  Google Scholar 

  6. 6.

    Mian A, Lee B (2002) Urea-cycle disorders as a paradigm for inborn errors of hepatocyte metabolism. Trends Mol Med 12:583–589

    Article  Google Scholar 

  7. 7.

    Gennip AHV, Bree-Blom EJV, Grift J, DeBree PK, Wadman SK (1980) Urinary purines and pyrimidines in patients with hyperammonemia of various origins. Clin Chim Acta 104:227–239

    PubMed  Article  Google Scholar 

  8. 8.

    Bachmann C, Colombo JP (1980) Diagnostic value of orotic acid excretion in heritable disorders of the urea cycle and in hyperammonemia due to organic acidurias. Eur J Pediatr 134:109–113

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Asai M, Sumi S, Kidouchi K, Imaeda H, Togari H, Wada Y (2000) Urinary pyrimidine analysis in healthy newborns, infants, children, adults and patients with congenital metabolic diseases. Pediatr Int 42:499–503

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Ferrari V, Giordano G, Cracco AT, Dussini N, Chiandetti L, Zacchello F (1989) Determination of urinary orotate excretion by high-performance liquid chromatography. J Chromatogr 497:101–107

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Ohba S, Kidouchi K, Katoh T, Kibe T, Kobayashi M, Wada Y (1991) Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching. J Chromatogr 568:325–332

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Salerno C, Crifò C (2002) Diagnostic value of urinary orotic acid levels: applicable separation methods. J Chromatogr B Analyt Technol Biomed Life Sci 781:57–71

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Rashed MS, Jacob M, Al-Amoudi M, Rahbeeni Z, Al-Sayed MA, Al-Ahaidib L, Saadallah AA, Legaspi S (2003) Rapid determination of orotic acid in urine by liquid chromatography-electrospray tandem mass spectrometry. Clin Chem 49:499–501

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    McCann MT, Thompson MM, Gueron IC, Tuchman M (1995) Quantification of orotic acid in dried filter paper urine samples by stable isotope dilution. Clin Chem 41:739–743

    CAS  PubMed  Google Scholar 

  15. 15.

    Shibata T, Kawasaki S, Fujita J, Kabashima T, Kai M (2010) A novel and specific fluorescence reaction for uracil. Anal Chim Acta 674:234–238

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Dragusha S, Shibata T, Yin S, Fujita J, Kabashima T, Kai M (2014) Selective, sensitive and fluorometric determination of urinary cytosine with 4-trifluoromethylbenzamidoxime and N, N-dimethylformamide. Clin Chim Acta 429:123–128

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Yavolovskii AA, Ivanov EI (2004) Conversion of amino group in position 6 of uracil. Chem Heterocycl Compd 40:361–363

    CAS  Article  Google Scholar 

  18. 18.

    Yavolovskii AA, Ivanov EI, Mazepa AV, Ivanov YE (2003) Synthesis of 1,2,3-triazolo [4,5-d] pyrimidine-3-oxides. Russ J Gen Chem 73:486–1488

    Google Scholar 

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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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Correspondence to Masaaki Kai.

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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).

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  • Orotic acid
  • Fluorogenic reaction
  • Quantitative determination
  • Human urine
  • HeLa cells