Measurements of Homogentisic Acid Levels in Alkaptonuria Patients Using an Optimized and Validated Gas Chromatography Method / Mass Spectrometry

Abstract

Alkaptonuria is a very rare genetic disorder, characterized by a lack of homogentisate dioxygenase and causes accumulation of homogentisic acid. Clinical manifestations include dark urine, dark-black pigmentation of connective tissues (ochronosis), and arthritis of large joints and spine. The disease is usually diagnosed in adulthood by assessment of signs and symptoms of ochronosis. Confirmation of suspected diagnosis can be achieved by quantitative measurements of homogentisic acid levels. In this study, homogentisic acid was analyzed in 17 alkaptonuria patients. After liquid-liquid extraction, the analyte was determined by Gas Chromatography/ Mass Spectrometry (GC-MS) method. For quantitation purposes, external calibration was applied first, regression coefficient of ≥ 0.995 indicated the linearity in the concentration range of 1-100 ng/µl. The instrumental detection limit (IDL) and lower limit of quantitation were 3.82 and 12.7 µg/L, respectively. Recovery rate was ≥ 89%. Precision given as relative standard deviation (RSD) ranged from 3 -10 %. The results showed that the concentration of homogentisic acid ranged from 0.46 to 1.5 g/24 hours.

Alkaptonuria in an adolescent boy

“…Ferric chloride and benedict’s test are few biochemical tests to screen the presence of HGA in urine. The basis of confirmational test for diagnosis of alkaptonuria depends on the amount of HGA in urine by gas chromatography–mass spectrometry analysis and high-performance liquid chromatography 22. Identification of pathogenic variants in HGD gene on molecular genetic analysis confirms the diagnosis.…”

Melanins as Sustainable Resources for Advanced Biotechnological Applications

“…In humans, urine of patients with alkaptonuria becomes dark due to the oxidation of HGA to benzoquinone acetic acid (BQA), which is a common means of diagnosis and the reason it is often known as black urine disease. A variety of different mass spectrometry techniques have been applied for the analysis of HGA and oxidation products thereof in samples of patient's bodily fluids including EI‐MS, [ 147 ] gas chromatography coupled to MS (GC‐MS), [ 148,149 ] liquid chromatography coupled to MS (LC/TOF‐MS in ESI mode), [ 150 ] which also enables studies of the binding of HGA and BQA to amyloids, [ 151 ] which can potentially offer insight into the natural melanin formation process, [ 152,153 ] and give insight into potential therapeutic opportunities for removing the damaging pigment in this condition. More advanced MS setups have facilitated various studies, including LC–tandem mass spectrometry (LC–MS/MS in ESI mode) to quantify tyrosine and HGA in clinical trial samples to determine the efficacy and response to nitisinone in the treatment of AKU, [ 154 ] mixtures of homovanillic acid, vanillylmandelic acid, orotic acid, and HGA, [ 155 ] LC‐QTOF‐MS was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with AKU, [ 156 ] and the products of polymerization of tyrosine and HGA have been studied by MALDI‐TOF; [ 17,157 ] all of which serve to highlight the importance of MS techniques to study melanins for red biotechnology applications.…”

Urological Aspects (Dynamic Bladder and Urethral Function, Urinary Tract Stones and Calcifications) in Patients with Alkaptonuria: A Case Control Study

“…Diagnosis of alkaptonuria is con rmed by gas chromatography-mass spectrometry analysis after identifying a signi cant amount of HGA in the urine (7). The amount of HGA excreted per day in individuals with alkaptonuria is usually 1-8 grams.…”