Search

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by mutations in the homogentisate 1,2-dioxygenase (HGD) gene, leading to the accumulation of homogentisic acid (HGA), causing severe inflammatory conditions. Recently, the presence of serum amyloid A (SAA) has been reported in AKU tissues, classifying AKU as novel secondary amyloidosis; AA amyloidosis is characterized by the extracellular tissue deposition of fibrils composed of fragments of SAA. AA amyloidosis may complicate several chronic inflammatory conditions, like rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, chronic infections, neoplasms, etc. Treatments of AA amyloidosis relieve inflammatory disorders by reducing SAA concentrations; however, no definitive therapy is currently available. SAA regulation is a crucial step to improve AA secondary amyloidosis treatments. Here, applying a comprehensive in vitro and in silico approach, we provided evidence that HGA is a disruptor modulator of SAA, able to enhance its polymerization, fibril formation, and aggregation upon SAA/SAP colocalization. In silico studies deeply dissected the SAA misfolding molecular pathway and SAA/HGA binding, suggesting novel molecular insights about it. Our results could represent an important starting point for identifying novel therapeutic strategies in AKU and AA secondary amyloidosis-related diseases.

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., (© 2024 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Alkaptonuria (AKU) is a genetic disorder that affects connective tissues of several body compartments causing cartilage degeneration, tendon calcification, heart problems, and an invalidating, early-onset form of osteoarthritis. The molecular mechanisms underlying AKU involve homogentisic acid (HGA) accumulation in cells and tissues. HGA is highly reactive, able to modify several macromolecules, and activates different pathways, mostly involved in the onset and propagation of oxidative stress and inflammation, with consequences spreading from the microscopic to the macroscopic level leading to irreversible damage. Gaining a deeper understanding of AKU molecular mechanisms may provide novel possible therapeutical approaches to counteract disease progression. In this review, we first describe inflammation and oxidative stress in AKU and discuss similarities with other more common disorders. Then, we focus on HGA reactivity and AKU molecular mechanisms. We finally describe a multi-purpose digital platform, named ApreciseKUre, created to facilitate data collection, integration, and analysis of AKU-related data., Competing Interests: A.S. was employed by Competence Center ARTES 4.0 and SienabioACTIVE—SbA. M.G. was employed by SienabioACTIVE—SbA. The other authors declare no conflicts of interest.

Despite urgent warnings about the spread of multidrug-resistant bacteria, the antibiotic development pipeline has remained sparsely populated. Naturally occurring antibacterial compounds may provide novel chemical starting points for antibiotic development programs and should be actively sought out. Evaluation of homogentisic acid (HGA), an intermediate in the tyrosine degradation pathway, showed that the compound had innate activity against Gram-positive and Gram-negative bacteria, which was lost following conversion into the degradation product benzoquinone acetic acid (BQA). Anti-staphylococcal activity of HGA can be attributed to effects on bacterial membranes. Despite an absence of haemolytic activity, the compound was cytotoxic to human HepG2 cells. We conclude that the antibacterial activity and in vitro safety profile of HGA render it more suitable for use as a topical agent or for inclusion in a small-molecule medicinal chemistry program.

Alkaptonuria (AKU, OMIM, No. 203500) is a rare, slow-progressing, irreversible, multisystemic disease resulting from a deficiency of the homogentisate 1,2-dioxygenase enzyme, which leads to the accumulation of homogentisic acid (HGA) and subsequent deposition as pigment in connective tissues called ochronosis. As a result, severe arthropathy of large joints and spondyloarthropathy with frequent fractures, ligament ruptures, and osteoporosis develops in AKU patients. Since 2020, the first-time treatment with nitisinone has become available in the European Union. Nitisinone significantly reduces HGA production and arrests ochronosis in AKU patients. However, blocking of the tyrosine metabolic pathway by the drug leads to tyrosine plasma and tissue concentrations increase. The nitisinone-induced hypertyrosinemia can lead to the development of corneal keratopathy, and once it develops, the treatment needs to be interrupted. A decrease in overall protein intake reduces the risk of the keratopathy during nitisinone-induced hypertyrosinemia in AKU patients. The low-protein diet is not only poorly tolerated by patients, but over longer periods, leads to a severe muscle loss and weight gain due to increased energy intake from carbohydrates and fats. Therefore, the development of novel nutritional approaches is required to prevent the adverse events due to nitisinone-induced hypertyrosinemia and the negative impact on skeletal muscle metabolism in AKU patients., (© 2023 Richard Imrich et al., published by Sciendo.)

Alkaptonuria (AKU) is a rare genetic autosomal recessive disorder characterized by elevated serum levels of homogentisic acid (HGA). In this disease, tyrosine metabolism is interrupted because of the alterations in homogentisate dioxygenase (HGD) gene. The patient suffers from ochronosis, fractures, and tendon ruptures. To date, no medicine has been approved for the treatment of AKU. However, physiotherapy and strong painkillers are administered to help mitigate the condition. Recently, nitisinone, an FDA-approved drug for type 1 tyrosinemia, has been given to AKU patients in some countries and has shown encouraging results in reducing the disease progression. However, this drug is not the targeted treatment for AKU, and causes keratopathy. Therefore, the foremost aim of this study is the identification of potent and druggable inhibitors of AKU with no or minimal side effects by targeting 4-hydroxyphenylpyruvate dioxygenase. To achieve our goal, we have performed computational modelling using BioSolveIT suit. The library of ligands for molecular docking was acquired by fragment replacement of reference molecules by ReCore. Subsequently, the hits were screened on the basis of estimated affinities, and their pharmacokinetic properties were evaluated using SwissADME. Afterward, the interactions between target and ligands were investigated using Discovery Studio. Ultimately, compounds c and f were identified as potent inhibitors of 4-hydroxyphenylpyruvate dioxygenase.

Alkaptonuria (AKU) is an ultra-rare inherited inborn error of metabolism that afflicts the tyrosine metabolic pathway, resulting in the accumulation of homogentisic acid (HGA) in the circulation, and significant excretion in urine. Clinical manifestations, typically observed from the third decade of life, are lifelong and significantly affect the quality of life. This review provides a comprehensive overview of the natural history of AKU, including clinical, biochemical and genetic perspectives. An update on the major advances on studies in murine models and human subjects, providing mechanistic insight into the molecular and biochemical processes that underlie pathophysiology and its response to treatment are presented. The impact of treatment with nitisinone is also presented with a specific emphasis on hypertyrosinemia, as uncertainty on this topic remains. Future perspectives are explored, such as novel approaches to treat hypertyrosinemia including the use of binding agents and amino acid transporter inhibitors, as well as advanced potentially curative gene and cell therapy initiatives., (Copyright © 2023. Published by Elsevier Inc.)

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs, which occurs because the homogentisate 1,2-dioxygenase (HGD) enzyme is not functional due to gene variants. Over time, HGA oxidation and accumulation cause the formation of the ochronotic pigment, a deposit that provokes tissue degeneration and organ malfunction. Here, we report a comprehensive review of the variants so far reported, the structural studies on the molecular consequences of protein stability and interaction, and molecular simulations for pharmacological chaperones as protein rescuers. Moreover, evidence accumulated so far in alkaptonuria research will be re-proposed as the bases for a precision medicine approach in a rare disease., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Alkaptonuria is a disease often forgotten because of its rarity. Its pathogenic mechanism is the deficiency of one of the enzymes of the tyrosine degradation pathway-homogentisate-1, 2-dioxygenase, which sequelae is accumulation and deposition of its metabolite homogentisic acid in connective tissues and urine. Alkaptonuria presents as a clinical triad-darkening urine upon prolonged exposure to air, pigmentation of connective tissues and debilitating arthropathy. We present a case report of a 67-year old patient with alkaptonuria who presented with the clinical triad, but was mistakenly diagnosed as having ankylosing spondylitis in the past. Currently there is no treatment for the disease hence the management strategy was focused on symptoms control with analgesics, physical therapy, dietary modification, vitamin C supplementation, and joint arthroplasty. Alkaptonuria's clinical features are extensively described in the literature and despite the fact that it is a rare disease, due to the similar radiographic changes with spondyloarthropathies, it should be included in the differential diagnosis in young patients presenting with severe joint involvement. Early recognition of the disease is necessary since its natural evolution is joint destruction leading to significant reduction in the quality of life. Alkaptonuria's articular features in the spine and peripheral tissues are well described using the classical imaging techniques. Musculoskeletal ultrasonography shows a characteristic set of findings in the soft tissues, including synovium, cartilage, tendons and entheses., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nitisinone (NTBC) was recently approved to treat alkaptonuria (AKU), but there is no information on its impact on oxidative stress and inflammation, which are observed in AKU. Therefore, serum samples collected during the clinical studies SONIA1 (40 AKU patients) and SONIA2 (138 AKU patients) were tested for Serum Amyloid A (SAA), CRP and IL-8 by ELISA; Advanced Oxidation Protein Products (AOPP) by spectrophotometry; and protein carbonyls by Western blot. Our results show that NTBC had no significant effects on the tested markers except for a slight but statistically significant effect for NTBC, but not for the combination of time and NTBC, on SAA levels in SONIA2 patients. Notably, the majority of SONIA2 patients presented with SAA > 10 mg/L, and 30 patients in the control group (43.5%) and 40 patients (58.0%) in the NTBC-treated group showed persistently elevated SAA > 10 mg/L at each visit during SONIA2. Higher serum SAA correlated with lower quality of life and higher morbidity. Despite no quantitative differences in AOPP, the preliminary analysis of protein carbonyls highlighted patterns that deserve further investigation. Overall, our results suggest that NTBC cannot control the sub-clinical inflammation due to increased SAA observed in AKU, which is also a risk factor for developing secondary amyloidosis., Competing Interests: The authors declare that they have no conflicts of interest. The sponsors had no role in the design, execution, interpretation, or writing of the study.

Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Introduction: Nitisinone used in alkaptonuria (AKU) can result in keratopathy due to strongly increased tyrosine levels., Methods: This study aimed to investigate nutritional status and changes in plasma tyrosine and phenylalanine and urinary homogentisic acid (u-HGA) levels in 8 adult AKU patients (mean age, 56.3 ± 4.7 years) who were on tyrosine/phenylalanine-restricted diet together with 2 mg/day nitisinone., Results: The treatment period was 23.4 ± 6.9 months. Daily dietary protein intake was restricted to 0.8-1.0 g/kg/day. Daily tyrosine intake was restricted to 260-450 mg/day for females and 330-550 mg/day for males. Tyrosine/phenylalanine-free amino acid supplements accounted for an average of 56.1% of daily protein intake. The following assessments were performed: anthropometric and plasma tyrosine level measurements every 2 months; ophthalmological examination every 6 months, and nutritional laboratory analyses and measurements of plasma amino acids and u-HGA once in a year. It was targeted to keep the plasma tyrosine level <500 μmol/L. The plasma tyrosine level was <100 μmol/L before the treatment in all patients and around a mean of 582.5 ± 194.8 μmol/L during the treatment. The diet was rearranged if a plasma tyrosine level of >700 μmol/L was detected. The u-HGA level before and after the 1st year of treatment was 1,429.3 ± 1,073.4 mmol/mol creatinine and 33.6 ± 9.5 mmol/mol creatinine, respectively. None of the patients developed keratopathy or experienced weight loss and protein or micronutrient deficiency., Conclusion: AKU patients should receive tyrosine/phenylalanine-restricted diet for reducing plasma tyrosine level to the safe range. Tyrosine/phenylalanine-free amino acid supplements can be safely used to enhance dietary compliance. Keratopathy and nutrient deficiency should be frequently monitored., (© 2021 S. Karger AG, Basel.)

From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation., (© 2021 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Alkaptonuria (AKU) is an ultra-rare disease caused by the deficient activity of homogentisate 1,2-dioxygenase enzyme, leading the accumulation of homogentisic acid (HGA) in connective tissues implicating the formation of a black pigmentation called "ochronosis." Although AKU is a multisystemic disease, the most affected tissue is the articular cartilage, which during the pathology appears to be highly damaged. In this study, a model of alkaptonuric chondrocytes and cartilage was realized to investigate the role of HGA in the alteration of the extracellular matrix (ECM). The AKU tissues lost its architecture composed of collagen, proteoglycans, and all the proteins that characterize the ECM. The cause of this alteration in AKU cartilage is attributed to a degeneration of the cytoskeletal network in chondrocytes caused by the accumulation of HGA. The three cytoskeletal proteins, actin, vimentin, and tubulin, were analyzed and a modification in their amount and disposition in AKU chondrocytes model was identified. Cytoskeleton is involved in many fundamental cellular processes; therefore, the aberration in this complex network is involved in the manifestation of AKU disease., (© 2021 Wiley Periodicals LLC.)

A large alkaptonuria (AKU) cohort was studied to better characterize the poorly understood spondyloarthropathy of rare disease AKU. Eighty-seven patients attended the National Alkaptonuria Centre (NAC) between 2007 and 2020. Seven only attended once. Fifty-seven attended more than once and received nitisinone 2 mg daily. Twenty-three attended at least twice without receiving nitisinone. Assessments included questionnaire analysis, 18F Positron emission tomography computerised tomography (PETCT), as well as photographs of ochronotic pigment in eyes and ears at baseline when 2 mg nitisinone was commenced and yearly thereafter. Blood and urine samples were collected for chemical measurement. The prevalence of ochronosis, as well as pain, PETCT and combined pain and PETCT scores, was greatly increased at 90.5%, 85.7%, 100%, and 100%, respectively. Joint pain scores were greatest in proximal joints in upper and lower limbs. PETCT joint scores were higher in proximal joints in upper limb but higher in distal joints in the lower limb. Spine pain scores were highest in lumbar, followed by cervical, thoracic, and cervical regions at 77.4%, 59.5%, 46.4%, and 25%, respectively. PETCT spine scores were highest in thoracic followed by lumbar, cervical, and sacroiliac regions at 74.4%, 70.7%, 64.6%, and 47.8% respectively; ochronosis associated closely with spondyloarthropathy scores (R = .65; P < .0001). Nitisinone reversed ochronosis significantly, with a similar pattern of decreased joint and spine disease. Spondyloarthropathy is a highly prevalent feature in this NAC cohort. Ochronosis appears to be associated with spondyloarthropathy. Nitisinone decreases ochronosis and had a similar nonsignificant effect pattern on spondyloarthropathy., (© 2021 SSIEM.)

A 52-year-old men suffered from osteoarthritis of the knee. During knee replacement surgery, the remaining cartilage appeared black. This discoloration and early degeneration of the cartilage is characteristic for the metabolic disorder alkaptonuria in which homogentisic acid accumulates in the body.

Alkaptonuria (AKU) is a rare disease correlated with deficiency of the enzyme homogentisate 1,2 dioxygenase, which causes homogentisic acid (HGA) accumulation. HGA is subjected to oxidation/polymerization reactions, leading to the production of a peculiar melanin-like pigmentation (ochronosis) after chronic inflammation, which is considered as a triggering event for the generation of oxidative stress. Clinical manifestations of AKU are urine darkening, sclera pigmentation, early severe osteoarthropathy, and cardiovascular and renal complication. Despite major clinical manifestations of AKU being observed in the bones and skeleton, the molecular and functional parameters are so far unknown in AKU. In the present study, we used human osteoblasts supplemented with HGA as a AKU cellular model. We observed marked oxidative stress, and for the first time, we were able to correlate HGA deposition with an impairment in the Wnt/β-catenin signaling pathway, opening a range of possible therapeutic strategies for a disease still lacking a known cure., (© 2020 Wiley Periodicals, Inc.)

Alkaptonuria (AKU) is a rare metabolic disease correlated with the deficiency of homogentisate 1,2-dioxygenase and leading to an accumulation of the metabolite homogentisic acid (HGA) which can be subjected to oxidation and polymerization reactions. These events are considered a trigger for the induction of oxidative stress in AKU but, despite the large description of an altered redox status, the underlying pathogenetic processes are still unstudied. In the present study, we investigated the molecular mechanisms responsible for the oxidative damage present in an osteoblast-based cellular model of AKU. Bone, in fact, is largely affected in AKU patients: severe osteoclastic resorption, osteoporosis, even for pediatric cases, and an altered rate of remodeling biomarkers have been reported. In our AKU osteoblast cell model, we found a clear altered redox homeostasis, determined by elevated hydrogen peroxide (H 2 O 2 ) levels and 4HNE protein adducts formation. These findings were correlated with increased NADPH oxidase (NOX) activity and altered mitochondrial respiration. In addition, we observed a decreased activity of superoxide dismutase (SOD) and reduced levels of thioredoxin (TRX) that parallel the decreased Nrf2-DNA binding. Overall, our results reveal that HGA is able to alter the cellular redox homeostasis by modulating the endogenous ROS production via NOX activation and mitochondrial dysfunctions and impair the cellular response mechanism. These findings can be useful for understanding the pathophysiology of AKU, not yet well studied in bones, but which is an important source of comorbidities that affect the life quality of the patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Alkaptonuria is a rare, genetic, multisystem disease characterised by the accumulation of homogentisic acid (HGA). No HGA-lowering therapy has been approved to date. The aim of SONIA 2 was to investigate the efficacy and safety of once-daily nitisinone for reducing HGA excretion in patients with alkaptonuria and to evaluate whether nitisinone has a clinical benefit., Methods: SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study done at three sites in the UK, France, and Slovakia. Patients aged 25 years or older with confirmed alkaptonuria and any clinical disease manifestations were randomly assigned (1:1) to receive either oral nitisinone 10 mg daily or no treatment. Patients could not be masked to treatment due to colour changes in the urine, but the study was evaluator-blinded as far as possible. The primary endpoint was daily urinary HGA excretion (u-HGA 24 ) after 12 months. Clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) score was assessed at 12, 24, 36, and 48 months. Efficacy variables were analysed in all randomly assigned patients with a valid u-HGA 24 measurement at baseline. Safety variables were analysed in all randomly assigned patients. The study was registered at ClinicalTrials.gov (NCT01916382)., Findings: Between May 7, 2014, and Feb 16, 2015, 139 patients were screened, of whom 138 were included in the study, with 69 patients randomly assigned to each group. 55 patients in the nitisinone group and 53 in the control group completed the study. u-HGA 24 at 12 months was significantly decreased by 99·7% in the nitisinone group compared with the control group (adjusted geometric mean ratio of nitisinone/control 0·003 [95% CI 0·003 to 0·004], p<0·0001). At 48 months, the increase in cAKUSSI score from baseline was significantly lower in the nitisinone group compared with the control group (adjusted mean difference -8·6 points [-16·0 to -1·2], p=0·023). 400 adverse events occurred in 59 (86%) patients in the nitisinone group and 284 events occurred in 57 (83%) patients in the control group. No treatment-related deaths occurred., Interpretation: Nitisinone 10 mg daily was well tolerated and effective in reducing urinary excretion of HGA. Nitisinone decreased ochronosis and improved clinical signs, indicating a slower disease progression., Funding: European Commission Seventh Framework Programme., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

For over two decades, nitisinone (NTBC) has been successfully used to manipulate the tyrosine degradation pathway and save the lives of many children with hereditary tyrosinaemia type 1. More recently, NTBC has been used to halt homogentisic acid accumulation in alkaptonuria (AKU) with evidence suggesting its efficacy as a disease modifying agent. NTBC-induced hypertyrosinaemia has been associated with cognitive impairment and potentially sight-threatening keratopathy. In the context of a non-lethal condition (ie, AKU), these serious risks call for an evaluation of the wider impact of NTBC on the tyrosine pathway. We hypothesised that NTBC increases the tyrosine pool size and concentrations in tissues. In AKU mice tyrosine concentrations of tissue homogenates were measured before and after treatment with NTBC. In humans, pulse injection with l-[ 13 C 9 ]tyrosine and l-[d 8 ]phenylalanine was used along with compartmental modelling to estimate the size of tyrosine pools before and after treatment with NTBC. We found that NTBC increased tyrosine concentrations in murine tissues by five to nine folds. It also significantly increased the tyrosine pool size in humans (P < .001), suggesting that NTBC increases tyrosine not just in serum but also in tissues (ie, acquired tyrosinosis). This study provides, for the first time, the experimental proof for the magnitude of NTBC-related acquired tyrosinosis which should be overcome to ensure the safe use of NTBC in AKU., (© 2020 SSIEM.)

Alkaptonuria (AKU) is a rare disease characterized by high levels of homogentisic acid (HGA); patients suffer from tissue ochronosis: dark brown pigmentation, especially of joint cartilage, leading to severe early osteoarthropathy. No molecular mechanism links elevated HGA to ochronosis; the pigment's chemical identity is still not known, nor how it induces joint cartilage degradation. Here we give key insight on HGA-derived pigment composition and collagen disruption in AKU cartilage. Synthetic pigment and pigmented human cartilage tissue both showed hydroquinone-resembling NMR signals. EPR spectroscopy showed that the synthetic pigment contains radicals. Moreover, we observed intrastrand disruption of collagen triple helix in pigmented AKU human cartilage, and in cartilage from patients with osteoarthritis. We propose that collagen degradation can occur via transient glycyl radicals, the formation of which is enhanced in AKU due to the redox environment generated by pigmentation., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P < 0.001), and was significantly associated with decreased HGA clearance (CL HGA ) (P < 0.001) and FE HGA (P < 0.001). CL HGA and FE HGA were increased beyond the theoretical maximum renal plasma flow, confirming renal production and emphasising the greater contribution of net tubular secretion than glomerular filtration to renal elimination of HGA. The kidneys are crucial to elimination of HGA. Elimination of HGA is impaired with age resulting in worsening disease over time. The kidney is an important site for production of HGA. Tubular secretion of HGA contributes more to elimination of HGA in AKU than glomerular filtration., (© 2019 SSIEM.)

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.

Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700 μmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. Elevated tyrosine (813 μmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3, 274.8, and 144.3 μmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3, 530.2, and 656.2 μmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (P = .002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine <700 μmol/L. Tyrosine/phenylalanine dietary restriction significantly reduced nitisinone-induced tyrosinemia in mice, with phenylalanine restriction alone proving ineffective. Similarly, protein restriction significantly reduced circulating tyrosine in AKU patients., (© 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a -/- mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Alkaptonuria (AKU) is an ultrarare autosomal recessive disorder (MIM 203500) that is caused byby a complex set of mutations in homogentisate 1,2-dioxygenasegene and consequent accumulation of homogentisic acid (HGA), causing a significant protein oxidation. A secondary form of amyloidosis was identified in AKU and related to high circulating serum amyloid A (SAA) levels, which are linked with inflammation and oxidative stress and might contribute to disease progression and patients' poor quality of life. Recently, we reported that inflammatory markers (SAA and chitotriosidase) and oxidative stress markers (protein thiolation index) might be disease activity markers in AKU. Thanks to an international network, we collected genotypic, phenotypic, and clinical data from more than 200 patients with AKU. These data are currently stored in our AKU database, named ApreciseKUre. In this work, we developed an algorithm able to make predictions about the oxidative status trend of each patient with AKU based on 55 predictors, namely circulating HGA, body mass index, total cholesterol, SAA, and chitotriosidase. Our general aim is to integrate the data of apparently heterogeneous patients with AKUAKU by using specific bioinformatics tools, in order to identify pivotal mechanisms involved in AKU for a preventive, predictive, and personalized medicine approach to AKU.-Cicaloni, V., Spiga, O., Dimitri, G. M., Maiocchi, R., Millucci, L., Giustarini, D., Bernardini, G., Bernini, A., Marzocchi, B., Braconi, D., Santucci, A. Interactive alkaptonuria database: investigating clinical data to improve patient care in a rare disease.

Ochronosis is the process in alkaptonuria (AKU) that causes all the debilitating morbidity. The process involves selective deposition of homogentisic acid (HGA)-derived pigment in tissues altering the properties of these tissues, leading to their failure. Some tissues like cartilage are more easily affected by ochronosis while others such as the liver and brain are unaffected for reasons that are still not understood. In vitro and mouse models of ochronosis have confirmed the dose relationships between HGA and ochronosis and also their modulation by p-hydroxyphenylpyruvate dioxygenase inhibition. Ochronosis cannot be fully reversed and is a key factor in influencing treatment decisions. Earlier detection of ochronosis preferably by noninvasive means is desirable. A cause-effect relationship between HGA and ochronosis is discussed. The similarity in AKU and familial hypercholesterolaemia is explored and lessons learnt. More research is needed to more fully understand the crucial nature of ochronosis., (© 2019 SSIEM.)

Objectives: We have assessed the effect of elevated concentrations of hydroxyphenylpyruvic acid (HPPA), hydroxyphenyllactic acid (HPLA) and tyrosine, on a range of chemistry tests in serum and urine to explore the potential for chemical interference on routine laboratory analyses in patients with alkaptonuria (AKU) treated with nitisinone and similarly implications for patients with hereditary tyrosinemia type 1 (HT-1)., Materials and Methods: HPPA, HPLA and tyrosine were added separately to pooled serum from subjects without AKU in a range of assays with Roche Modular chemistries. Effects on urine were assessed by changes in urine strip chemistries after mixing a positive control urine with various amounts of the test compounds and reading on a Siemens urine strip meter., Results: No significant effect (p > 0.1) was observed up to 225 μmol/L of HPPA and HPLA, and up to 5000 μmol/L tyrosine, on any of the serum-based assays including those with peroxidase-coupled reaction systems of enzymatic creatinine, urate, total cholesterol, HDL cholesterol and triglyceride. Both the monohydroxy HPPA, and the dihydroxy homogentisic acid (HGA), at increased urine concentrations typical of nitisinone-treated AKU and non-treated AKU respectively, did however show marked negative interference in strip assays for glucose and leucocytes; i.e. those with peroxide-linked endpoints. The effect of increased HPLA was less marked., Conclusions: In patients with AKU or on nitisinone treatment and HT-1 patients on nitisinone, urine strip chemistry testing should be used sparingly, if at all, to avoid false negative reporting. It is recommended that urine assays should be organised with a suitable specialist laboratory., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Alkaptonuria (AKU) is a disease caused by a deficient homogentisate 1,2-dioxygenase activity leading to systemic accumulation of homogentisic acid (HGA), that forms a melanin-like polymer that progressively deposits onto connective tissues causing a pigmentation called "ochronosis" and tissue degeneration. The effects of AKU and ochronotic pigment on the biomechanical properties of articular cartilage need further investigation. To this aim, AKU cartilage was studied using thermal (thermogravimetry and differential scanning calorimetry) and rheological analysis. We found that AKU cartilage had a doubled mesopore radius compared to healthy cartilage. Since the mesoporous structure is the main responsible for maintaining a correct hydrostatic pressure and tissue homoeostasis, drastic changes of thermal and rheological parameters were found in AKU. In particular, AKU tissue lost its capability to enhance chondrocytes metabolism (decreased heat capacity) and hence the production of proteoglycans. A drastic increase in stiffness and decrease in dissipative and lubricant role ensued in AKU cartilage. Multiphoton and scanning electron microscopies revealed destruction of cell-matrix microstructure and disruption of the superficial layer. Such observations on AKU specimens were confirmed in HGA-treated healthy cartilage, indicating that HGA is the toxic responsible of morphological and mechanical alterations of cartilage in AKU., (© 2018 Wiley Periodicals, Inc.)

Background: Identification of unknown chemical entities is a major challenge in metabolomics. To address this challenge, we developed a comprehensive targeted profiling strategy, combining 3 complementary liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) techniques and in-house accurate mass retention time (AMRT) databases established from commercial standards. This strategy was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with alkaptonuria (AKU). Because hypertyrosinemia is a known consequence of nitisinone therapy, we investigated the wider metabolic consequences beyond hypertyrosinemia., Methods: A total of 619 standards (molecular weight, 45-1354 Da) covering a range of primary metabolic pathways were analyzed using 3 liquid chromatography methods-2 reversed phase and 1 normal phase-coupled to QTOF-MS. Separate AMRT databases were generated for the 3 methods, comprising chemical name, formula, theoretical accurate mass, and measured retention time. Databases were used to identify chemical entities acquired from nontargeted analysis of AKU urine: match window theoretical accurate mass ±10 ppm and retention time ±0.3 min., Results: Application of the AMRT databases to data acquired from analysis of urine from 25 patients with AKU (pretreatment and after 3, 12, and 24 months on nitisinone) and 18 HGD -/- mice (pretreatment and after 1 week on nitisinone) revealed 31 previously unreported statistically significant changes in metabolite patterns and abundance, indicating alterations to tyrosine, tryptophan, and purine metabolism after nitisinone administration., Conclusions: The comprehensive targeted profiling strategy described here has the potential of enabling discovery of novel pathways associated with pathogenesis and management of AKU., (© 2019 American Association for Clinical Chemistry.)

Aim: Alkaptonuria is a disorder of tyrosine metabolism where elevated circulating homogentisic acid causes progressive dysfunction of collagenous tissues. Logistical, financial and patient experience concerns with collection and transport of specimens to central laboratories makes evaluation of microsampling attractive., Methodology: Volumetric absorptive microsampling devices were evaluated for accuracy, precision and drying time. Elution methods were evaluated for several urinary metabolites of interest and stability assessed under multiple conditions. Comparison was performed between dried and liquid specimens via LC-MS/MS., Conclusion: Volumetric absorptive microsampling was found to be highly accurate and precise. Elution methods showed good recovery and reproducibility. Dried and liquid samples compared favorably. Analyte stability was variable, presenting barriers to implementation into routine use in this context.

Question: Does Nitisinone prevent the clinical progression of the Alkaptonuria?, Findings: In this observational study on 39 patients, 2 mg of daily nitisinone inhibited ochronosis and significantly slowed the progression of AKU over a three-year period., Meaning: Nitisinone is a beneficial therapy in Alkaptonuria., Background: Nitisinone decreases homogentisic acid (HGA), but has not been shown to modify progression of Alkaptonuria (AKU)., Methods: Thirty-nine AKU patients attended the National AKU Centre (NAC) in Liverpool for assessments and treatment. Nitisinone was commenced at V1 or baseline. Thirty nine, 34 and 22 AKU patients completed 1, 2 and 3 years of monitoring respectively (V2, V3 and V4) in the VAR group. Seventeen patients also attended a pre-baseline visit (V0) in the VAR group. Within the 39 patients, a subgroup of the same ten patients attended V0, V1, V2, V3 and V4 visits constituting the SAME Group. Severity of AKU was assessed by calculation of the AKU Severity Score Index (AKUSSI) allowing comparison between the pre-nitisinone and the nitisinone treatment phases., Results: The ALL (sum of clinical, joint and spine AKUSSI features) AKUSSI rate of change of scores/patient/month, in the SAME group, was significantly lower at two (0.32 ± 0.19) and three (0.15 ± 0.13) years post-nitisinone when compared to pre-nitisinone (0.65 ± 0.15) (p < .01 for both comparisons). Similarly, the ALL AKUSSI rate of change of scores/patient/month, in the VAR group, was significantly lower at one (0.16 ± 0.08) and three (0.19 ± 0.06) years post-nitisinone when compared to pre-nitisinone (0.59 ± 0.13) (p < .01 for both comparisons). Combined ear and ocular ochronosis rate of change of scores/patient/month was significantly lower at one, two and three year's post-nitisinone in both VAR and SAME groups compared with pre-nitisinone (p < .05)., Conclusion: This is the first indication that a 2 mg dose of nitisinone slows down the clinical progression of AKU. Combined ocular and ear ochronosis progression was arrested by nitisinone., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: The aim of this work was to assess baseline serum levels of established biomarkers related to inflammation and oxidative stress in samples from alkaptonuric subjects enrolled in SONIA1 (n = 40) and SONIA2 (n = 138) clinical trials (DevelopAKUre project)., Methods: Baseline serum levels of Serum Amyloid A (SAA), IL-6, IL-1β, TNFα, CRP, cathepsin D (CATD), IL-1ra, and MMP-3 were determined through commercial ELISA assays. Chitotriosidase activity was assessed through a fluorimetric method. Advanced Oxidation Protein Products (AOPP) were determined by spectrophotometry. Thiols, S-thiolated proteins and Protein Thiolation Index (PTI) were determined by spectrophotometry and HPLC. Patients' quality of life was assessed through validated questionnaires., Results: We found that SAA serum levels were significantly increased compared to reference threshold in 57.5% and 86% of SONIA1 and SONIA2 samples, respectively. Similarly, chitotriosidase activity was above the reference threshold in half of SONIA2 samples, whereas CRP levels were increased only in a minority of samples. CATD, IL-1β, IL-6, TNFα, MMP-3, AOPP, thiols, S-thiolated protein and PTI showed no statistically significant differences from control population. We provided evidence that alkaptonuric patients presenting with significantly higher SAA, chitotriosidase activity and PTI reported more often a decreased quality of life. This suggests that worsening of symptoms in alkaptonuria (AKU) is paralleled by increased inflammation and oxidative stress, which might play a role in disease progression., Conclusions: Monitoring of SAA may be suggested in AKU to evaluate inflammation. Though further evidence is needed, SAA, chitotriosidase activity and PTI might be proposed as disease activity markers in AKU., (Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Alkaptonuria (AKU) is an ultra-rare genetic disease, in which the accumulation of a toxic metabolite, homogentisic acid (HGA) leads to the systemic development of ochronotic aggregates. These aggregates cause severe complications mainly at the level of joints with extensive degradation of the articular cartilage. Primary cilia have been demonstrated to play an essential role in development and the maintenance of articular cartilage homeostasis, through their involvement in mechanosignaling and Hedgehog signaling pathways. Hedgehog signaling has been demonstrated to be activated in osteoarthritis (OA) and to drive cartilage degeneration in vivo. The numerous similarities between OA and AKU suggest that primary cilia Hedgehog signaling may also be altered in AKU. Thus, we characterized an AKU cellular model in which healthy chondrocytes were treated with HGA (66 µM) to replicate AKU cartilage pathology. We investigated the degree of activation of the Hedgehog signaling pathway and how treatment with inhibitors of the receptor Smoothened (Smo) influenced Hedgehog activation and primary cilia structure. The results obtained in this work provide a further step in the comprehension of the pathophysiological features of AKU, suggesting a potential therapeutic approach to modulate AKU cartilage degradation processes through manipulation of the Hedgehog pathway., (© 2016 Wiley Periodicals, Inc.)

Alkaptonuria (AKU) is an inborn error of metabolism where mutation of homogentisate 1,2-dioxygenase (HGD) gene leads to a deleterious or misfolded product with subsequent loss of enzymatic degradation of homogentisic acid (HGA) whose accumulation in tissues causes ochronosis and degeneration. There is no licensed therapy for AKU. Many missense mutations have been individuated as responsible for quaternary structure disruption of the native hexameric HGD. A new approach to the treatment of AKU is here proposed aiming to totally or partially rescue enzyme activity by targeting of HGD with pharmacological chaperones, i.e. small molecules helping structural stability. Co-factor pockets from oligomeric proteins have already been successfully exploited as targets for such a strategy, but no similar sites are present at HGD surface; hence, transient pockets are here proposed as a target for pharmacological chaperones. Transient pockets are detected along the molecular dynamics trajectory of the protein and filtered down to a set of suitable sites for structural stabilization by mean of biochemical and pharmacological criteria. The result is a computational workflow relevant to other inborn errors of metabolism requiring rescue of oligomeric, misfolded enzymes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog-related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU., (© 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.)

Background: Alkaptonuria (AKU) is an ultra-rare disease associated to the lack of an enzyme involved in tyrosine catabolism. This deficiency results in the accumulation of homogentisic acid (HGA) in the form of ochronotic pigment in joint cartilage, leading to a severe arthropathy. Secondary amyloidosis has been also unequivocally assessed as a comorbidity of AKU arthropathy. Composition of ochronotic pigment and how it is structurally related to amyloid is still unknown., Methods: We exploited Synchrotron Radiation Infrared and X-Ray Fluorescence microscopies in combination with conventional bio-assays and analytical tools to characterize chemical composition and morphology of AKU cartilage., Results: We evinced that AKU cartilage is characterized by proteoglycans depletion, increased Sodium levels, accumulation of lipids in the peri-lacunar regions and amyloid formation. We also highlighted an increase of aromatic compounds and oxygen-containing species, depletion in overall Magnesium content (although localized in the peri-lacunar region) and the presence of calcium carbonate fragments in proximity of cartilage lacunae., Conclusions: We highlighted common features between AKU and arthropathy, but also specific signatures of the disease, like presence of amyloids and peculiar calcifications. Our analyses provide a unified picture of AKU cartilage, shedding a new light on the disease and opening new perspectives., General Significance: Ochronotic pigment is a hallmark of AKU and responsible of tissue degeneration. Conventional bio-assays have not yet clarified its composition and its structural relationship with amyloids. The present work proposes new strategies for filling the aforementioned gap that encompass the integration of new analytical approaches with standardized analyses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism characterized by homogentisic acid (HGA) accumulation due to a deficient activity of the homogentisate 1.2-dioxygenase (HGD) enzyme. This leads to the production of dark pigments that are deposited onto connective tissues, a condition named 'ochronosis' and whose mechanisms are not completely clear. Recently, the potential role of hitherto unidentified proteins in the ochronotic process was hypothesized, and the presence of Serum Amyloid A (SAA) in alkaptonuric tissues was reported, allowing the classification of AKU as a novel secondary amyloidosis., Methods: Gel electrophoresis, Western Blot, Congo Red-based assays and electron microscopy were used to investigate the effects of HGA on the aggregation and fibrillation propensity of amyloidogenic proteins and peptides [Aβ(1-42), transthyretin, atrial natriuretic peptide, α-synuclein and SAA]. LC/MS and in silico analyses were undertaken to identify possible binding sites for HGA (or its oxidative metabolite, a benzoquinone acetate or BQA) in SAA., Results: We found that HGA might act as an amyloid aggregation enhancer in vitro for all the tested proteins and peptides in a time- and dose- dependent fashion, and identified a small crevice at the interface between two HGD subunits as a candidate binding site for HGA/BQA., Conclusions: HGA might be an important amyloid co- component playing significant roles in AKU amyloidosis., General Significance: Our results provide a possible explanation for the clinically verified onset of amyloidotic processes in AKU and might lay the basis to setup proper pharmacological approaches to alkaptonuric ochronosis, which are still lacking., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: Alkaptonuria (AKU) is a rare autosomal recessive disease resulting from a single enzyme deficiency in tyrosine metabolism. As a result, homogentisic acid cannot be metabolized, causing systemic increases. Over time, homogentisic acid polymerizes and deposits in collagenous tissues, leading to ochronosis. Typically, this occurs in joint cartilages, leading to an early onset, rapidly progressing osteoarthropathy. The aim of this study was to examine tissue turnover in cartilage affected by ochronosis and its role in disease initiation and progression., Methods: With informed patient consent, hip and knee cartilages were obtained at surgery for arthropathy due to AKU (n = 6; 2 knees/4 hips) and OA (n = 12; 5 knees/7 hips); healthy non-arthritic (non-OA n = 6; 1 knee/5 hips) cartilages were obtained as waste from trauma surgery. We measured cartilage concentrations (normalized to dry weight) of racemized aspartate, GAG, COMP and deamidated COMP (D-COMP). Unpaired AKU, OA and non-OA samples were compared by non-parametric Mann-Whitney U test., Results: Despite more extractable total protein being obtained from AKU cartilage than from OA or non-OA cartilage, there was significantly less extractable GAG, COMP and D-COMP in AKU samples compared with OA and non-OA comparators. Racemized Asx (aspartate and asparagine) was significantly enriched in AKU cartilage compared with in OA cartilage., Conclusions: These novel data represent the first examination of cartilage matrix components in a sample of patients with AKU, representing almost 10% of the known UK alkaptonuric population. Compared with OA and non-OA, AKU cartilage demonstrates a very low turnover state and has low levels of extractable matrix proteins., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of AKU treatment is palliative and little is known about its physiopathology. Neovascularization is involved in the pathogenesis of systemic inflammatory rheumatic diseases, a family of related disorders that includes AKU. Here, we investigated the presence of neoangiogenesis in AKU synovium and healthy controls. Synovium from AKU patients, who had undergone total joint replacement or arthroscopy, or from healthy patients without any history of rheumatic diseases, who underwent surgical operation following sport trauma was subjected to hematoxylin and eosin staining. Histologic grades were assigned for clinical disease activity and synovitis based on cellular content of the synovium. By immunofluorescence microscopy, using different endothelial cell markers, we observed large vascularization in AKU but not in healthy synovium. Moreover, Western blotting and quantification analyses confirmed strong expression of endothelial cell markers in AKU synovial tissues. Importantly, AKU synovium vascular endothelium expressed high levels of β-dystroglycan, a protein previously involved in the regulation of angiogenesis in osteoarthritic synovium. This is the first report providing experimental evidences that new blood vessels are formed in AKU synovial tissues, opening new perspectives for AKU therapy.

Alkaptonuria is a rare autosomal recessive condition resulting from inability to breakdown homogentisic acid (HGA), an intermediate in tyrosine degradation. The condition has a triad of clinical features, the most damaging of which is ochronotic osteoarthropathy. HGA is elevated from birth, but pigmentation takes many years. We hypothesise that interleukins play a role in initiation and progression of ochronotic osteoarthropathy. C20/A4 cells were cultured and maintained in 9-cm petri dishes containing either HGA at 0.33 mM, a single interleukin (IL-1β, IL-6 or IL-10) at 1 ng/ml or a combination of HGA and a single interleukin. Statistical analysis of pigment deposits and cell viability was performed using analysis of variance with Newman-Keuls post-test. All cultures containing HGA showed a significant increase in pigment deposition compared to control and IL cultures alone. The cultures containing HGA and IL-6 showed a significant increase in pigment deposits compared to HGA alone. The cell viability counts across all cultures on day 10 demonstrated a significant decrease in cultures containing HGA compared to those which did not. There was no significant difference between cultures containing just HGA or those combined with an interleukin. This work demonstrates a role for cytokines present in the joint(s) in the pigmentation process, particularly IL-6, and that the presence of HGA in joint tissues appears more detrimental to chondrocytes than the presence of any of the interleukins found in response to joint injury, trauma and osteoarthritis (OA). This further supports the evidence that the arthropathy in alkaptonuria is much more severe and rapidly progressing.

Alkaptonuria (AKU) is a rare multisystem metabolic disease caused by deficient activity of homogentisate 1,2-dioxygenase (HGD), which leads to the accumulation of homogentisic acid (HGA). Currently, there is no treatment for AKU. The sole drug with some beneficial effects is the herbicide nitisinone (1), an inhibitor of p-hydroxyphenylpyruvate dioxygenase (4-HPPD). 1 has been used as a life-saving drug in infants with type I tyrosinemia despite severe side effects due to the buildup of tyrosine. Four clinical trials of nitisinone to treat AKU have shown that 1 consistently decreases HGA levels, but also caused the accumulation of tyrosine in blood serum. Moreover, the human preclinical toxicological data for 1 are incomplete. In this work, we performed pharmacodynamics and toxicological evaluations of 1, providing the first report of LD50 values in human cells. Intracellular tyrosinemia was also evaluated. Three additional 4-HPPD inhibitors with a more favorable profile than that of 1 in terms of IC50, LD50, and tyrosine accumulation were also identified among commercially available compounds. These may be promising starting points for the development of new therapeutic strategies for the treatment of AKU., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Alkaptonuria (AKU) is a rare inherited metabolic disorder of tyrosine metabolism that results from a defect in an enzyme called homogentisate 1,2-dioxygenase. The result of this is that homogentisic acid (HGA) accumulates in the body. HGA is central to the pathophysiology of this disease and the consequences observed; these include spondyloarthropathy, rupture of ligaments/muscle/tendons, valvular heart disease including aortic stenosis and renal stones. While AKU is considered to be a chronic progressive disorder, it is clear from published case reports that fatal acute metabolic complications can also occur. These include oxidative haemolysis and methaemoglobinaemia. The exact mechanisms underlying the latter are not clear, but it is proposed that disordered metabolism within the red blood cell is responsible for favouring a pro-oxidant environment that leads to the life threatening complications observed. Herein the role of red blood cell in maintaining the redox state of the body is reviewed in the context of AKU. In addition previously reported therapeutic strategies are discussed, specifically with respect to why reported treatments had little therapeutic effect. The potential use of nitisinone for the management of patients suffering from the acute metabolic decompensation in AKU is proposed as an alternative strategy.

Alkaptonuria is a rare inborn error of metabolism, identified over a century ago. But its basic pathomechanism (i.e. ochronosis) is still not completely explained. Though clinical onset of osteoarthropathy and complications from other organs (including: heart and blood vessels, skin, eyes, kidneys) occurs at adult age, the symptoms are progressive, cause severe pains and significantly limit everyday life of the patients. Until now no effective therapeutic methods have been known in alkaptonuria. Recently, thanks to an initiative of the international patient organization for alkaptonuria, a hope for a potential treatment availability, appears. So, alkaptonuria is an example of a role of multidysciplinary care, cooperation and ongoing progress in the area of rare diseases.