title: Quick Diagnosis of Alkaptonuria by Homogentisic Acid Determination in Urine Paper Spots.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100, Siena, Italy. gabriella.iacomelli@unisi.it.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Jacomelli
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100, Siena, Italy.
authors.forename: Vanna
authors.initials: V
authors.lastname: Micheli
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100, Siena, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Bernardini
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100, Siena, Italy.
authors.forename: Lia
authors.initials: L
authors.lastname: Millucci
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100, Siena, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Santucci