Hepatobiliary circulation and dominant urinary excretion of homogentisic acid in a mouse model of alkaptonuria.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean [±SD] = 1003[±410] µmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] µmol/L). Biliary tyrosine, 3(4-hydroxyphenyl)pyruvic acid (HPPA) and 3(4-hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model.

Detection of homogentisic acid by electrospray ionization mass spectrometry.

OBJECTIVE: Homogentisic acid (HGA) is excreted in excessive amounts in the urine of patients with alkaptonuria, which is a hereditary metabolic disorder of phenylalanine and tyrosine. Therefore, the detection of HGA in urine is useful for the diagnosis of alkaptonuria. To evaluate the detection of HGA, we confirmed the color shift of HGA solutions and analyzed them by electrospray ionization mass spectrometry (ESI-MS). METHODS: We observed the color change of the HGA solutions under different pH conditions (pH 6.0, 7.0, and 8.0) and examined the influences of adding potassium hydroxide (KOH) and ascorbic acid (AA) to the HGA solutions. Then, we analyzed the chemical reaction in HGA solutions using ESI-MS. RESULTS: The HGA solution at pH 8.0 became brown after incubation at room temperature for 24 h and became darker brown with the addition of KOH; however, HGA solutions at pH 6.0 and 7.0 showed no color changes. The brown color change of the HGA solution at pH 8.0 was also inhibited by AA. Moreover, all HGA sample solutions showed the deprotonated molecular ion peak at m/z 167.035 in the negative ion mode after incubation at room temperature for 24 h and with the addition of KOH and AA. CONCLUSION: We identified the molecular ion of HGA in all sample solutions by ESI-MS, regardless of different pH conditions, color changes, or the presence of AA. These results suggest that spectral analysis by ESI-MS is suitable for the detection of HGA and the diagnosis of alkaptonuria.

Bacterial quality of urinary tract in patients with alkaptonuria.

BACKGROUND: The aim of the current study was to determine whether there is an association between alkaptonuria (AKU) and urinary tract infection (UTI) by exploring the bacterial quality of the urinary tract, as most of the patients with AKU present with frequent occurrence of urinary tract symptoms such as incomplete emptying of urinary bladder, dysuria and nocturia. METHODS: Study samples were collected from 22 participants; 9 from patients with AKU, 9 from individuals who were AKU carriers, and 4 people served as control. Confirmation of AKU diagnosis was established by the ferric chloride test and quantitative determination of urinary homogentisic acid (HGA) levels. RESULTS: In the ferric chloride test, the urine samples of AKU patients showed a characteristic black ring upon addition of few drops of ferric chloride solution. During urinary HGA determination, patients with AKU had increased levels of urinary HGA as compared to carriers and controls. The following 10 bacterial species were isolated from the urinary tract of AKU patients, carriers and controls: Sphingomonas paucimobilis, Escherichia coli, Francisella tularensis, Staphylococcus hominis, Staphylococcus haemolyticus, Leuconostoc mesenteroides, Dermacoccus nishinomiyaensis, Kytococcus sedentarius, Serratia fonticola and Granulicatella adiacens. The presence of S. paucimobilis was found in three male patients, and one female each from the carrier and control groups. Almost all study samples were positive for D. nishinomiyaensis and K. sedentarius. S. fonticola and G. adiacens were found only in AKU carrier females. CONCLUSIONS: The results deduced that males show symptoms of arthritis early and more severely than females and by this it appears that there is an association between these symptoms and the percentage of bacterial infection in males that requires more accurate diagnosis and treatment to clarify such relationship. In the current study, males (patients, carriers, and controls) were more likely to have bacterial infections than females (64% vs. 36%). The 16 and 2 bacterial isolates, detected in 7 males and 2 females AKU patients, respectively, revealed that male AKU patients had a 2.3-fold greater rate of bacterial infection than female AKU patients. Therefore, further studies are warranted to investigate if there's any relationship between higher incidence of bacterial infections and development of AKU-related clinical symptoms in the male population.

Alkaptonuria in Russia.

Alkaptonuria is characterized by the accumulation of homogentisic acid (HGA), part of which is excreted in the urine but the excess HGA forms a dark brown ochronotic pigment that deposits in the connective tissue (ochronosis), eventually leading to early-onset severe arthropathy. We analyzed a cohort of 48 Russian AKU families by sequencing all 14 exons (including flanking intronic sequences) of the homogentisate 1,2-dioxygenase gene (HGD) and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. Nine novel likely pathogenic HGD variants were identified, which have not been reported previously in any other country. Recently, Bychkov et al. [1] reported on the variant spectrum in another cohort of 49 Russian AKU patients. Here we summarize complete data from both cohorts that include 82 Russian AKU families. Taken together, 31 different HGD variants were found in these patients, of which 14 are novel and found only in Russia. The most common variant was c.481G>A (p.(Gly161Arg)), present in almost 54% of all AKU alleles.

A molecular spectroscopy approach for the investigation of early phase ochronotic pigment development in Alkaptonuria.

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene. Over time, HGA accumulation causes the formation of the ochronotic pigment, a dark deposit that leads to tissue degeneration and organ malfunction. Such behaviour can be observed also in vitro for HGA solutions or HGA-containing biofluids (e.g. urine from AKU patients) upon alkalinisation, although a comparison at the molecular level between the laboratory and the physiological conditions is lacking. Indeed, independently from the conditions, such process is usually explained with the formation of 1,4-benzoquinone acetic acid (BQA) as the product of HGA chemical oxidation, mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the corresponding para-benzoquinone. To test such correlation, a comprehensive, comparative investigation on HGA and BQA chemical behaviours was carried out by a combined approach of spectroscopic techniques (UV spectrometry, Nuclear Magnetic Resonance, Electron Paramagnetic Resonance, Dynamic Light Scattering) under acid/base titration both in solution and in biofluids. New insights on the process leading from HGA to ochronotic pigment have been obtained, spotting out the central role of radical species as intermediates not reported so far. Such evidence opens the way for molecular investigation of HGA fate in cells and tissue aiming to find new targets for Alkaptonuria therapy.

HPLC with electrochemical detection for determining homogentisic acid and its application to urine from rats fed tyrosine-enriched food.

A highly sensitive method for determining urine homogentisic acid (HGA) is required to provide adequate diagnosis and therapy for alkaptonuria in early stages. In this study, we developed a highly sensitive high-performance liquid chromatography with electrochemical detection (HPLC-ECD) for determining HGA in urine. In order to obtain a chromatogram of HGA by HPLC-ECD, an oxidation current was monitored at +0.5 V vs. Ag/AgCl. The peak heights of HGA showed linearity (r = 0.999) ranging from 4.2 ng/mL to 168 ng/mL, and the detection limit was 1.2 ng/mL (signal-to-noise ratio, S/N = 3). In recovery tests using human control urine spiked with an HGA standard, the recoveries of HGA were more than 93.2 %, and the relative standard deviations (n = 6) were less than 1.9 %. As an in vivo application using male Wistar rats, the level of urine HGA, which was metabolized from tyrosine in tyrosine-enriched food, was determined by this HPLC-ECD method. The determination of HGA in urine by this HPLC-ECD method requires only 0.1 mL of a rat urine specimen and simple sample preparation consisting of dilution and filtration.

Assessment of Thyroid Function in Patients With Alkaptonuria.

Importance: Alkaptonuria is an autosomal recessive disorder caused by pathogenic variants in the HGD gene. Deficiency of the HGD enzyme leads to tissue deposition of homogentisic acid (HGA), causing severe osteoarthropathies and cardiac valve degeneration. Although HGD is vital for the catabolism of tyrosine, which provides the basis for thyroid hormone synthesis, the prevalence of thyroid dysfunction in alkaptonuria is unknown. Objective: To assess thyroid structure and function in patients with alkaptonuria. Design, Setting, and Participants: A single-center cohort study was conducted in a tertiary referral center including patients with alkaptonuria followed up for a median of 93 (interquartile range, 48-150) months between February 1, 2000, and December 31, 2018. The alkaptonuria diagnosis was based on clinical presentation and elevated urine HGA levels. A total of 130 patients were considered for participation. Main Outcomes and Measures: Prevalence of thyroid dysfunction in adults with alkaptonuria compared with the general population. Thyrotropin and free thyroxine levels were measured by immunoassay and repeated in each patient a median of 3 (interquartile range, 2-22) times. Neck ultrasonographic scans were analyzed in a subset of participants. Logistic regression was used to test the association of thyroid dysfunction with age, sex, thyroid peroxidase (TPO) antibodies, serum tyrosine levels, and urine HGA levels. Results: Of the 130 patients, 5 were excluded owing to thyroidectomy as the cause of hypothyroidism. The study cohort consisted of 125 patients; the median age was 45 (interquartile range, 35-51) years. Most of the patients were men (72 [57.6%]). The prevalence of primary hyperthyroidism was 0.8% (1 of 125 patients), similar to 0.5% observed in the general population (difference, 0.003; 95% CI, -0.001 to 0.04; P = .88). The prevalence of primary hypothyroidism was 16.0% (20 of 125 patients), which is significantly higher than 3.7% reported in the general population (difference, 0.12; 95% CI, 0.10-0.24; P < .001). Women were more likely to have primary hypothyroidism than men (odds ratio, 10.99; 95% CI, 3.13-38.66; P < .001). Patients with TPO antibodies had a higher likelihood of primary hypothyroidism than those without TPO antibodies (odds ratio, 7.36; 95% CI, 1.89-28.62; P = .004). There was no significant difference in the prevalence of thyroid nodules between patients in this study (29 of 49 [59.2%]) vs the general population (68%) (difference, 0.088; 95% CI, -0.44 to 0.73; P = .20) or of cancer (7% vs 5%; difference, 0.01; 95% CI, -0.01 to 0.17; P = .86). Conclusions and Relevance: The high prevalence of primary hypothyroidism noted in patients with alkaptonuria in this study suggests that serial screening in this population should be considered and prioritized.

Quantification of the flux of tyrosine pathway metabolites during nitisinone treatment of Alkaptonuria.

Nitisinone decreases homogentisic acid (HGA) in Alkaptonuria (AKU) by inhibiting the tyrosine metabolic pathway in humans. The effect of different daily doses of nitisinone on circulating and 24 h urinary excretion of phenylalanine (PA), tyrosine (TYR), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and HGA in patients with AKU was studied over a four week period. Forty AKU patients, randomised into five groups of eight patients, received doses of 1, 2, 4 or 8 mg of nitisinone daily, or no drug (control). Metabolites were analysed by tandem mass spectrometry in 24 h urine and serum samples collected before and after nitisinone. Serum metabolites were corrected for total body water and the sum of 24 hr urine plus total body water metabolites of PA, TYR, HPPA, HPLA and HGA were determined. Body weight and urine urea were used to check on stability of diet and metabolism over the 4 weeks of study. The sum of quantities of urine metabolites (PA, TYR, HPPA, HPLA and HGA) were similar pre- and post-nitisinone. The sum of total body water metabolites were significantly higher post-nitisinone (p < 0.0001) at all doses. Similarly, combined 24 hr urine:total body water ratios for all analytes were significantly higher post-nitisinone, compared with pre-nitisinone baseline for all doses (p = 0.0002 - p < 0.0001). Significantly higher concentrations of metabolites from the tyrosine metabolic pathway were observed in a dose dependant manner following treatment with nitisinone and we speculate that, for the first time, experimental evidence of the metabolite pool that would otherwise be directed towards pigment formation, has been unmasked.

Glaucoma With Alkaptonuria as a Result of Pigment Accumulation.

PURPOSE: To report a case of alkaptonuria (AKU) in a patient with bilateral conjunctival and scleral black colorization who was diagnosed with glaucoma thereafter. METHODS: This is a single case report. RESULTS: A 67-year-old male patient with bilateral black colorization of conjunctiva and sclera was referred to our hospital. In the biomicroscopic examination, globular dark pigmentation was observed in the conjunctiva, sclera, and limbal cornea. The patient was diagnosed with a nuclear cataract in both eyes. He also had gray skin pigmentation at his nose and paranasal area. Corneal topography examination revealed irregular astigmatism. Intraocular pressure values were 29 and 31 mm Hg, in the right and left eye, respectively, with Goldmann applanation tonometry. The diagnosis of AKU was made after pathologic assessment of conjunctival biopsy by the internal medicine department. CONCLUSIONS: AKU is characterized by the accumulation of homogentisic acid in the connective tissues of many organs including the eye. Patients should be carefully examined in ophthalmology clinics in order to not miss systemic diagnoses. It should be kept in mind that AKU may cause iridocorneal angle pigmentation, which leads to glaucoma, and patients should be treated with proper medication when presenting with elevated intraocular pressure values.

Endogenous ochronosis: when clinical suspicion prevails over histopathology.

Endogenous ochronosis (EO) or alkaptonuria is an inherited autosomal recessive disease caused by the insufficiency of the enzyme homogentisic acid dioxygenase. This disturbance causes an accumulation and increased renal excretion of homogentisic acid (AHG), which manifests as dark urine when it oxidizes on contact with air. Other clinical manifestations of OE are the result of the deposit of AHG in the form of ochronotic pigment at the level of collagen in the skin and cartilage, where it causes blue-gray cutaneous hyperpigmentation, degenerative arthropathy, valvular disease, and other multisystem effects. Despite the progressive and irreversible nature of OE and the lack of a curative treatment, the life expectancy is preserved. We report a new case of EO with cutaneous and joint involvement, in which a high clinical suspicion, confirmed by elevated AHG in urine was the key in the diagnosis.

Determination of homogentisic acid in urine for diagnosis of alcaptonuria: Capillary electrophoretic method optimization using experimental design.

Homogentisic acid (HGA) is a diagnostic metabolite that accumulates in the urine and tissues of patients with alkaptonuria which is a rare autosomal recessive disease. HGA is a specific metabolite in urine and serum, which is used for diagnosis of alkaptonuria. This study presents an inexpensive and easy capillary electrophoretic method for the quantitative determination of HGA in urine samples. The method was optimized using full factorial experimental design. The optimal separation electrolyte and separation voltage were revealed as 45 mmol/L phosphate buffer at pH 7.0 and 22 kV, respectively. Under these conditions the presence of HGA was detected in 6 min. Repeatability of migration times and corrected peak areas of HGA (as RSD) were 0.37 and 1.99, respectively. The detection limit was 0.56 µg/mL, 3 times of the average noise, and the quantification limit was 1.85 µg/mL, 10 times the average noise for HGA. Urine samples were directly injected to the capillary without any pretreatment step.

The effect of nitisinone on homogentisic acid and tyrosine: a two-year survey of patients attending the National Alkaptonuria Centre, Liverpool.

Background Alkaptonuria is a rare, debilitating autosomal recessive disorder affecting tyrosine metabolism. Deficiency of homogentisate 1,2-dioxygenase leads to increased homogentisic acid which is deposited as ochronotic pigment. Clinical sequelae include severe early onset osteoarthritis, increased renal and prostate stone formation and cardiac complications. Treatment has been largely based on analgaesia and arthroplasty. The National Alkaptonuria Centre in Liverpool has been using 2 mg nitisinone (NTBC) off-license for all patients in the United Kingdom with alkaptonuria and monitoring the tyrosine metabolite profiles. Methods Patients with confirmed alkaptonuria are commenced on 2 mg dose (alternative days) of NTBC for three months with daily dose thereafter. Metabolite measurement by LC-MS/MS is performed at baseline, day 4, three-months, six-months and one-year post-commencing NTBC. Thereafter, monitoring and clinical assessments are performed annually. Results Urine homogentisic acid concentration decreased from a mean baseline 20,557 µmol/24 h (95th percentile confidence interval 18,446-22,669 µmol/24 h) by on average 95.4% by six months, 94.8% at one year and 94.1% at two year monitoring. A concurrent reduction in serum homogentisic acid concentration of 83.2% compared to baseline was also measured. Serum tyrosine increased from normal adult reference interval to a mean ± SD of 594 ± 184 µmol /L at year-two monitoring with an increased urinary excretion from 103 ± 81 µmol /24 h at baseline to 1071 ± 726 µmol /24 h two years from therapy. Conclusions The data presented represent the first longitudinal survey of NTBC use in an NHS service setting and demonstrate the sustained effect of NTBC on the tyrosine metabolite profile.

Long-term result of arthroplasty in the treatment of a case of ochronotic arthropathy.

Alkaptonuria is a rare metabolic disease caused by a partial or total deficiency of homogentisic acid oxidase, which results in excess homogentisic acid (HGA) levels. Homogentisic acid and its oxidation products can accumulate in hyaline cartilage, tendons, and ligaments. A 55-year-old male was admitted complaining of worsening chronic pain in his left knee. A radiographic evaluation showed tricompartmental end- stage osteoarthritis. A cemented total knee replacement was performed. At the 10-year follow-up, he had returned to full activity, had no knee pain, and was very satisfied with the outcome. No abnormality was observed in the femoral, tibial, or patellar components on radiography. We believe that total knee replacement is a good option in a patient with marked degenerative arthritis secondary to ochronotic arthritis.

Alkaptonuria Presenting with Impressive Osteoarticular Changes and Severe Aortic Stenosis.

Alkaptonuria, or ochronosis, a rare autosomal recessive metabolic disorder, causes an excess of homogentisic acid that results in dark pigmentation, calcification, and inflammation of cartilaginous and other tissues. Cardiovascular complications are also typical of the disease. We report the case of a 78-year-old male who presented with impressive osteoarticular changes and aortic stenosis associated with alkaptonuria.

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.

BACKGROUND: Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. METHODS: Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA24) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. FINDINGS: A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA24 was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA24 of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. CONCLUSIONS: In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. TRIAL REGISTRATION NUMBER: EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463.

Renal and prostate stones composition in alkaptonuria: a case report.

Alkaptonuria is a genetic disorder characterized by an accumulation of homogentisic acid due to an enzymatic defect of homogentisate 1,2 dioxygenase. The homogentisic acid is excreted exclusively by both glomerular filtration and tubular secretion leading to the renal parenchyma being exposed to high concentrations of homogentisic acid. The alkaptonuric patients are at higher risk of renal stones (and of prostate stones for males), usually in the later stages of the disease. We describe the case of a 51-year-old man whose renal and prostate stones were analyzed by X-ray diffraction and infrared spectroscopy, respectively. We review the cases of alkaptonuria (AKU) patients reported in the literature for whom the composition of kidney or prostate stones was assessed with physical or chemical techniques. In this paper, we also discuss the advantages and drawbacks of the different methodologies.