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

Abstract

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.

Primary hip arthroplasty for the treatment of alkaptonuric hip arthritis: 3- to 24-year follow-ups

“…The synovium was thickened, inflamed, and pigmented. HGA was detected using dried urine spots [ 9 ] in all patients. In all cases, there was a macroscopic layer of black pigmented articular cartilage, which was more severe in elderly patients.…”

“…All patients reported gross limitation of daily routine activities and needed assisted ambulation. A dried urine spot (DUS) [ 9 ] detects HGA in all patients. Other laboratory parameters were within the normal ranges.…”

The dried blood spot sampling method in the laboratory medicine

“…This type of sample collection is suitable for all analytes excreted in urine. It can be used for example in the diagnosis of different diseases or for drug screening (100)(101)(102)(103)(104)(105).…”

Serendipity in inborn errors of metabolism: Combining two genetic mutations in a single patient

“…The brown color intensity obtained on the paper is proportional to HGA concentration from 1.4 mmol/L to 46 mmol/L. Such method fits very well for Alkaptonuria screening even before the detection of dark urine because of its safety, reliability, and ease of use 10 . After screening, additional confirmatory testing is necessary that include measuring enzymes activities and/or molecular analysis for gene mutations.…”