Quick Diagnosis of Alkaptonuria by Homogentisic Acid Determination in Urine Paper Spots

  • Gabriella JacomelliEmail author
  • Vanna Micheli
  • Giulia Bernardini
  • Lia Millucci
  • Annalisa Santucci
Research Report
Part of the JIMD Reports book series (JIMD, volume 31)


Objectives: Two methods are described for homogentisic acid (HGA) determination in dried urine spots (DUS) on paper from Alkaptonuria (AKU) patients, devised for quick early diagnosis. AKU is a rare autosomal recessive disorder caused by deficiency of homogentisate 1,2-dioxygenase, yielding in accumulation of HGA. Its massive excretion causes urine darkening by exposure to air or alkalinization, and is a diagnostic marker. The deposition of polymers produced after HGA oxidation within the connective tissues causes ochronotic arthritis, a degenerative joint disease manifesting in adulthood and only rarely in childhood. No early diagnosis is usually accomplished, awareness following symptom development.

Design and methods: Two methods were designed for HGA determination in DUS: (1) a rapid semi-quantitative reliable method based on colour development in alkali and quantification by comparison with dried paper spots from HGA solutions of known concentration and (2) a quantitative and sensitive HPLC-linked method, previously devised for purine and pyrimidine analysis in urine and plasma.

Results: Colour intensity developed by DUS after alkali addition was proportional to HGA concentration, and calculated amounts were in good agreement with quantitative analysis performed by RP-HPLC on DUS and on urines as such.

Conclusions: DUS, often used for different diagnostic purpose, are easily prepared and safely delivered. The simple and quick colour method proposed provides reliable HGA assessment and is fit for large screening. HGA concentration determined in 10 AKU patient DUS by both methods 1 and 2 was in agreement with direct urine assay and in the range reported by literature.

A reliable HGA quantification based on colour development in paper urine spots is validated by HPLC-linked HGA quantification, and proposed as a quick diagnostic tool for AKU patients.


Colour Development Homogentisic Acid Alkali Addition Ochronotic Arthropathy Reliable Analytical Tool 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work has been supported by Telethon Italy grant GGP10058. The authors also thank Toscana Life Sciences Orphan_1 project, Fondazione Monte dei Paschi di Siena and aimAKU – Associazione Italiana Malati di Alcaptonuria (ORPHA263402).


  1. Barbas C, Garcìa A, de Miguel L, Simo C (2002) Evaluation of filter paper collection of urine samples for detection and measurement of organic acidurias by capillary electrophoresis. J Chromatogr B 780:73–82CrossRefGoogle Scholar
  2. Bory C, Boulieu R, Chantin C, Mathieu M (1989) Homogentisic acid in biological fluids by HPLC. Clin Chem 35(2):321–322PubMedGoogle Scholar
  3. Bradley DM (1975) Screening for inherited metabolic disease in Wales using urine-impregnated filter paper. Arch Dis Child 50:264–268CrossRefPubMedPubMedCentralGoogle Scholar
  4. Hughes AT, Milan AM, Christensen P et al (2014) Urine homogentisic acid and tyrosine: simultaneous analysis by liquid chromatography tandem mass spectrometry. J Chromatogr B 963:106–112CrossRefGoogle Scholar
  5. Hughes AT, Milan AM, Davison AS et al (2015) Serum markers in alkaptonuria: simultaneous analysis of homogentisic acid, tyrosine and nitisinone by liquid chromatography tandem mass spectrometry. Ann Clin Biochem 52(Pt 5):597–605CrossRefPubMedGoogle Scholar
  6. Koska L, Srsen S (1977) A simple urine screening test for alkaptonuria. N Engl J Med 297:1471–1472PubMedGoogle Scholar
  7. Mannoni A, Selvi E, Lorenzini S et al (2004) Alkaptonuria, ochronosis, and ochronotic arthropathy. Semin Arthritis Rheum 39(4):239–248CrossRefGoogle Scholar
  8. Mathieu P, Prevel A, David L et al (1997) Screening infants for neuroblastoma: discovery of alkaptonuria in one case. Clin Chim Acta 264:255–259CrossRefPubMedGoogle Scholar
  9. Micheli V, Sestini S, Rocchigiani M et al (1999) Hypoxanthine-guanine phosphoribosyl transferase deficiency and erythrocyte synthesis of pyridine coenzymes. Life Sci 64:2479–2487CrossRefPubMedGoogle Scholar
  10. Millucci L, Braconi D, Bernardini G et al (2015) Amyloidosis in alkaptonuria. J Inherit Metab Dis 38(5):797–805CrossRefPubMedGoogle Scholar
  11. Pecker E, Yonden Z, Sogut S (2008) From darkening urine to early diagnosis of alkaptonuria. Indian J Dermatol Venereol Leprol 74:700–704CrossRefGoogle Scholar
  12. Ranganath LR, Jarvis JC, Gallagher JA (2013) Recent advances in management of alkaptonuria. J Clin Pathol 66:367–373CrossRefPubMedGoogle Scholar
  13. Ranganath LR, Milan AM, Hughes AT et al (2016) Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment. Ann Rheum Dis 75(2):362–367CrossRefPubMedGoogle Scholar
  14. Tokuhara Y, Shukuya K, Tanaka M et al (2014) Detection of novel visible-light region absorbance peaks in the urine after alkalization in patients with alkaptonuria. PLoS One 9(1):e86606CrossRefPubMedPubMedCentralGoogle Scholar
  15. Zhao BH, Chen BC, Shao DC, Zhang Q (2009) Osteoarthritis? Ochronotic arthritis! Knee Surg Sports Traumatol Arthrosc 17:778–781CrossRefPubMedGoogle Scholar
  16. Zibolen M, Srsnova K, Srsen S (2000) Increased urolithiasis in patients with alkaptonuria in childhood “Letter to the Editor”. Clin Genet 58:79–80CrossRefPubMedGoogle Scholar

Copyright information

© SSIEM and Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Gabriella Jacomelli
    • 1
    Email author
  • Vanna Micheli
    • 1
  • Giulia Bernardini
    • 1
  • Lia Millucci
    • 1
  • Annalisa Santucci
    • 1
  1. 1.Department of Biotechnology, Chemistry and PharmacyUniversity of SienaSienaItaly

Personalised recommendations