Alkaptonuria is a rare inherited disorder for which there was no disease-modifying treatment. In order to develop a successful approved therapy of AKU multiple barriers had to be overcome. These included activities before the conduct of the study including deciding on the drug therapy, the dose of the drug to be used, clarify the nature of the disease, develop outcome measures likely to yield a positive outcome, have a strategy to ensure appropriate patient participation through identification, build a consortium of investigators, obtain regulatory approval for proposed investigation plan and secure funding. Significant barriers were overcome during the conduct of the multicentre study to ensure harmonisation. Mechanisms were put in place to recruit and retain patients in the study. Barriers to patient access following completion of the study and regulatory approval were resolved.

Management of osteoporosis in patients with alkaptonuria can be challenging. This is the first case report confirming the effectiveness of teriparatide following zoledronic acid therapy in treating osteoporosis and preventing fragility fractures in a patient with alkaptonuria.

Alkaptonuria (AKU) is an ultra-rare metabolic disease caused by the accumulation of homogentisic acid (HGA), an intermediate product of phenylalanine and tyrosine degradation. AKU patients carry variants within the gene coding for homogentisate-1,2-dioxygenase (HGD), which are responsible for reducing the enzyme catalytic activity and the consequent accumulation of HGA and formation of a dark pigment called the ochronotic pigment. In individuals with alkaptonuria, ochronotic pigmentation of connective tissues occurs, leading to inflammation, degeneration, and eventually osteoarthritis. The molecular mechanisms underlying the multisystemic development of the disease severity are still not fully understood and are mostly limited to the metabolic pathway segment involving HGA. In this view, untargeted metabolomics of biofluids in metabolic diseases allows the direct investigation of molecular species involved in pathways alterations and their interplay. Here, we present the untargeted metabolomics study of AKU through the nuclear magnetic resonance of urine from a cohort of Italian patients; the study aims to unravel molecular species and mechanisms underlying the AKU metabolic disorder. Dysregulation of metabolic pathways other than the HGD route and new potential biomarkers beyond homogentisate are suggested, contributing to a more comprehensive molecular signature definition for AKU and the development of future adjuvant treatment.

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.

Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis, which results on the long term in an early-onset and severe osteoarthropathy. HGD's quaternary structure is known to be easily disrupted by missense mutations, which makes them an interesting target for novel treatment strategies that aim to rescue enzyme activity. However, only prediction models are available providing information on a structural basis. Therefore, an E. coli based whole-cell screening was developed to evaluate HGD missense variants in 96-well microtiter plates. The screening principle is based on HGD's ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity could be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Optimized screening conditions comprised several E. coli expression strains, varied expression temperatures and varied substrate concentrations. In addition, plate uniformity, signal variability and spatial uniformity were investigated and optimized. Finally, eight HGD missense variants were generated via site-directed mutagenesis and evaluated with the developed high-throughput screening (HTS) assay. For the HTS assay, quality parameters passed the minimum acceptance criterion for Z' values > 0.4 and single window values > 2. We found that activity percentages versus wildtype HGD were 70.37 ± 3.08% (for M368V), 68.78 ± 6.40% (for E42A), 58.15 ± 1.16% (for A122V), 69.07 ± 2.26% (for Y62C), 35.26 ± 1.90% (for G161R), 35.86 ± 1.14% (for P230S), 23.43 ± 4.63% (for G115R) and 19.57 ± 11.00% (for G361R). To conclude, a robust, simple, and cost-effective HTS system was developed to reliably evaluate and distinguish human HGD missense variants by their HGA consumption ability. This HGA quantification assay may lay the foundation for the development of novel treatment strategies for missense variants in AKU.

Alkaptonuria is a rare autosomal recessive disorder that arises from a mutation of the homogentisate 1,2-dioxygenase (HGD) gene, resulting in a deficiency of the enzyme HGD.[1] This enzyme plays a role in the phenylalanine and tyrosine degradation pathways by catabolizing the tyrosine intermediate metabolite homogentisic acid (HGA) into maleylacetoacetic acid (Figure 1).[2] Deficiency of the HGD enzyme results in the abnormal accumulation of upstream precursors HGA, tyrosine, and phenylalanine. In particular, the buildup and deposition of HGA is the major contributor to the classic manifestations and consequences of the disease, including homogentisic aciduria, ochronosis, and ochronotic osteoarthropathy.[2] Alkaptonuria leads to dysfunctions in various tissues throughout the body; this article will broadly cover the prominent presentations and focus on ocular manifestations of the disorder in particular. Early identification and diagnosis of the disorder are imperative for management and treatment as complications can be sight-threatening, including astigmatism, central vein occlusion, glaucoma, and blindness.[3] The first case of alkaptonuria is believed to have presented in 1500 BC as found in the Egyptian mummy Harwa. Biopsy of the body’s hip cartilage and intervertebral discs in 1979 demonstrated extensive calcification and HGA pigmentation consistent with the process of ochronosis.[4]  Cases of the disease reported in the 16 century centered around the observation of dark urine, which continued to be the identifying feature and namesake of the “black urine disease” through the 19 century. In 1859, Dr. Carl Boedeker found that adding alkali to these patients’ urine produced the appearance of dark discoloration (later discovered to be due to HGA’s reducing power). He subsequently named the disease “alkaptonuria” in reference to “alkali” in Arabic, meaning “alkali,” “alkali” in Greek, meaning “to suck up oxygen greedily in alkali,” and “alkapton” in German, meaning “a reducing compound.”[1] Alkaptonuria is a disease that occupies a noteworthy place in the histories of biochemistry, genetics, and medicine. In 1902, it was the first medical disorder reported to adhere to the Mendelian principles of autosomal recessive inheritance by Sir Archibald Garrod. Garrod deduced that all cases of alkaptonuria reported at the time required certain genetic contributions from both parents. In other words, the spectrum of outward manifestations could all be traced back to a specific underlying genetic trait, emphasizing the impact of science and genetics on the human constitution. At the time, many debilitating diseases were already noted to be more prevalent in consanguineous marriages, but few reports addressed consanguinity from a strictly scientific perspective. By connecting alkaptonuria to a genetic basis and referring to the similar gametes passed on by consanguineous parents, Garrod provided scientific evidence for some deleterious effects of consanguineous unions.[2]  In his 1908 Croonian Lecture to the Royal College of Physicians, Garrod also later used alkaptonuria as an example to demonstrate his concept of “the inborn error of metabolism,” now a widely used term applied to genetic disorders of innate metabolic pathways such as alkaptonuria.[5]

Alkaptonuria (AKU) is a rare autosomal recessive disorder caused by mutations within a gene coding for homogentisate 1,2-dioxygenase (HGD). To date, 251 different variants of this gene have been reported. The metabolic disorder in AKU leads to the accumulation of homogentisic acid (HGA), resulting in ochronosis (pigmentation of the connective tissues) and severe ochronotic spondylo-arthropathy, which usually manifests in the mid-thirties. An earlier genotype-phenotype correlation study showed no differences in serum HGA levels, absolute urinary excretion of HGA, or in the clinical symptoms between patients carrying HGD variants leading to 1% or >30% residual HGD activity. Still, as reported previously, the variance of the excretion of the HGA was smaller within affected siblings that share a common genotype. The present study is the first ever to systematically analyze the baseline clinical data of 24 AKU sibling pairs/groups collected in the SONIA 2 (Suitability Of Nitisinone In Alkaptonuria 2) study to evaluate phenotypical differences between patients carrying the same HGD genetic variants. We show that even between siblings there was considerable variability in the disease severity. This indicates that some other yet unidentified genetic, biomechanical, or environmental modifying factors may contribute to accelerated pigmentation and connective tissue damage observed in some patients.

Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homogentisic acid (HGA), the active molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was performed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. In total, we observed 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2-16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2-0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia.

Changes in the phenylalanine (PHE)/tyrosine (TYR) pathway metabolites before and during homogentisic acid (HGA)-lowering by nitisinone in the Suitability of Nitisinone in Alkaptonuria (AKU) 2 (SONIA 2) study enabled the magnitude of the flux in the pathway to be examined. SONIA 2 was a 48-month randomised, open-label, evaluator-blinded, parallel-group study performed in the UK, France and Slovakia recruiting patients with confirmed AKU to receive either 10 mg nitisinone or no treatment. Site visits were performed at 3 months and yearly thereafter. Results from history, photographs of eyes/ears, whole body scintigraphy, echocardiography and abdomen/pelvis ultrasonography were combined to produce the Alkaptonuria Severity Score Index (cAKUSSI). PHE, TYR, hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and HGA metabolites were analysed by liquid chromatography/tandem mass spectrometry in 24 h urine and serum samples collected before and during nitisinone. Serum metabolites were corrected for total body water (TBW), and the sum of 24 h urine plus total body water metabolites of PHE, TYR, HPPA, HPLA and HGA were determined. The sum of urine metabolites (PHE, TYR, HPPA, HPLA and HGA) were similar pre- and peri-nitisinone. The sum of TBW metabolites and sum TBW + URINE metabolites were significantly higher peri-nitisinone (p < 0.001 for both) compared with pre-nitisinone baseline. Significantly higher concentrations of metabolites from the tyrosine metabolic pathway were observed during treatment with nitisinone. Arguments for unmasking of the ochronotic pathway and biliary elimination of HGA are put forward.

This review briefly discusses the discovery of the mode of action of the triketone herbicide, 2-(2-nitro-4-trifluormethylbenzoyl)-1,3-cyclohexanedione and its use as a drug Nitisinone for the treatment of inborn errors of tyrosine metabolism. Nitisinone is a potent reversible tight-binding inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase, involved in the catabolism of the amino acid tyrosine. Nitisinone is used to treat the rare disease hereditary tyrosinaemia type 1 where the last enzyme in the breakdown of tyrosine, fumarylacetoacetase is deficient. Nitisinone is also used to treat patients with alkaptonuria where the enzyme homogentisic acid oxidase is deficient. Articles in this issue discuss metabolites of tyrosine catabolism in healthy patients and those with alkaptonuria.

Nitisinone (NIT) produces inevitable but varying degree of tyrosinaemia. However, the understanding of the dynamic adaptive relationships within the tyrosine catabolic pathway has not been investigated fully. The objective of the study was to assess the contribution of protein intake, serum NIT (sNIT) and tyrosine pathway metabolites to nitisinone-induced tyrosinaemia in alkaptonuria (AKU). Samples of serum and 24-h urine collected during SONIA 2 (Suitability Of Nitisinone In Alkaptonuria 2) at months 3 (V2), 12 (V3), 24 (V4), 36 (V5) and 48 (V6) were included in these analyses. Homogentisic acid (HGA), tyrosine (TYR), phenylalanine (PHE), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and sNIT were analysed at all time-points in serum and urine. Total body water (TBW) metabolites were derived using 60% body weight. 24-h urine and TBW metabolites were summed to obtain combined values. All statistical analyses were post-hoc. 307 serum and 24-h urine sampling points were analysed. Serum TYR from V2 to V6, ranging from 478 to 1983 µmol/L were stratified (number of sampling points in brackets) into groups < 701 (47), 701-900 (105), 901-1100 (96) and > 1100 (59) µmol/L. The majority of sampling points had values greater than 900 µmol/L. sPHE increased with increasing sTYR (p < 0.001). Tyrosine, HPPA and HPLA in serum and TBW all increased with rising sTYR (p < 0.001), while HPLA/TYR ratio decreased (p < 0.0001). During NIT therapy, adaptive response to minimise TYR formation was demonstrated. Decreased conversion of HPPA to HPLA, relative to TYR, seems to be most influential in determining the degree of tyrosinaemia.

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in the body. Affected individuals lack functional levels of an enzyme required to breakdown HGA. Mutations in the homogentisate 1,2-dioxygenase (HGD) gene cause AKU and they are responsible for deficient levels of functional HGD, which, in turn, leads to excess levels of HGA. Although HGA is rapidly cleared from the body by the kidneys, in the long term it starts accumulating in various tissues, especially cartilage. Over time (rarely before adulthood), it eventually changes the color of affected tissue to slate blue or black. Here we report a comprehensive mutation analysis of 111 pathogenic and 190 non-pathogenic HGD missense mutations using protein structural information. Using our comprehensive suite of graph-based signature methods, mCSM complemented with sequence-based tools, we studied the functional and molecular consequences of each mutation on protein stability, interaction and evolutionary conservation. The scores generated from the structure and sequence-based tools were used to train a supervised machine learning algorithm with 89% accuracy. The empirical classifier was used to generate the variant phenotype for novel HGD missense mutations. All this information is deployed as a user friendly freely available web server called HGDiscovery (https://biosig.lab.uq.edu.au/hgdiscovery/).

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.

Alkaptonuria is a rare hereditary disease with a defective enzyme that results in increased homogentisic acid levels in the body. Homogentisic acid accumulates in multiple body parts and initializes tissue damage. Clinical manifestations such as pigmentation of the skin areas and joint destruction result in ochronosis. Nitisinone decreases serum and urinary homogentisic acid levels, improving morbidity by preventing and slowing the progression of alkaptonuria. Nitisinone-induced hypertyrosinemia causes keratopathy and mental ill effects, which can be managed by diet restriction and regular check-ups. A personalized approach is required for treatment by nitisinone. Low-dose oral nitisinone is associated with overall good results and a better safety profile.

Nitisinone (NIT) causes tyrosinaemia and corneal keratopathy (KP), especially in men. However, the adaptation within the phenylalanine (PHE)/tyrosine (TYR) catabolic pathway during KP is not understood. The objective of this study is to assess potential differences in the PHE/TYR pathway during KP and the influence of gender in NIT-induced tyrosinaemia in alkaptonuria (AKU). Samples of serum and 24 h urine collected from patients treated with NIT during a 4-year randomized study in NIT vs. no-treatment controls (SONIA 2; Suitability Of Nitisinone In Alkaptonuria 2; EudraCT no. 2013-001633-41) at months 3 (V2), 12 (V3), 24 (V4), 36 (V5) and 48 (V6) were included in these analyses. Homogentisic acid (HGA), TYR, PHE, hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and sNIT were analysed at all time-points in serum and urine in the NIT-group. All statistical analyses were post hoc. Keratopathy occurred in 10 out of 69 AKU patients, eight of them male. Thirty-five sampling points (serum and 24 h urine) were analysed in patients experiencing KP and 272 in those with no-KP (NKP) during NIT therapy. The KP group had a lower HPLA/TYR ratio and a higher TYR/PHE ratio compared with the NKP group (p < 0.05 for both). There were 24, 45, 100 and 207 sampling points (serum and 24 h urine) in the NIT group which were pre-NIT female, pre-NIT male, NIT female and NIT male, respectively. The PHE/TYR ratio and the HPLA/TYR ratio were lower in males (p < 0.001 and p < 0.01, respectively). In the KP group and in the male group during NIT therapy, adaptive responses to minimise TYR formation were impaired compared to NKP group and females, respectively.

Alkaptonuria is one of a rare autosomal recessive genetic disorder, which results from the deficiency of homogentisate 1,2 dioxygenase (HGD). HGD gene is expressed in the liver, kidney, prostate, small intestine, and colon.[1] This enzyme plays a role in the metabolism of tyrosine that converts homogentisic acid (HGA) into malate and acetoacetate. In the absence of HGD, homogentisic acid produced in excess by the liver oxidizes into ochronotic pigment polymer. Accumulation of this pigment in various tissues leads to systemic disease. This process is called ochronosis. Alkaptonuria was amongst the first genetic disorders in humans that found to follow the principles of Mendelian recessive inheritance.[2] Historically, in 1908 it was used by Archibald Garrod in his Croonian lectures to illustrate the principles behind "inborn errors of metabolism." However, the Egyptian mummy Harwa believed to be the first clinical case of Alkaptonuria dating back as far as 1500 BC.[2] The term alkaptonuria originated from the Arabic word "alkali." Also, Boedeker created the name in 1859 after he noticed unusual decreasing properties in patient urine.[2] In 1866 ochronosis was discovered by Virchow, who noticed under microscopy when HGA pigment appeared to be a pale brownish yellow color (ochre-like).[2] 

Ochronosis is named for the reddish-brown hue of tissue termed "ochre-like" that was first described by a young physician named Archibald Garrod in the early-19th century. In what he called an "inborn error of metabolism," Archibald Garrod changed history when he coined that term for the obscure "black urine" disease, alkaptonuria. He proposed that ochronosis was due to deposition of a phenolic compound – "alkaptons" polymerizing into connective tissue - because the enzyme that could cleave it was absent and was inherited as a "chemical individuality." This was insightful work given that this preceded the conceptualization of genes. The work of Dr. Garrod would build core foundations on how we understand biochemistry, genetics, and medicine today. Alkaptonuria is iconic for being the initial description of the prototypical autosomal recessive disease. Today we know that expression of the homogentisate 1,2 dioxygenase becomes stunted because of mutations arising in the HGD gene, described as Dr. Garrod's "chemical individuality." Serum levels of homogentisic acid (Alkaptons) concomitantly rise and polymerize into tissue over time. The sequestration effect is also termed as endogenous ochronosis. This article reviews this and the exogenous form of ochronosis, commonly seen as a side-effect of medications that competitively inhibit the same enzyme mentioned above. In contrast to endogenous ochronosis, the deposition is often limited to the skin.

Alkaptonuria (AKU) is a rare debilitating autosomal recessive disorder of tyrosine (TYR) metabolism which results in a deficiency of the enzyme homogentisate 1,2-dioxygenase activity. Several studies have reported the metabolic changes in homogentisic acid (HGA) concentrations and subsequent deposition of an ochronotic pigment in connective tissues, especially cartilage. Treatment with nitisinone (NTBC) reduces urinary and circulating HGA, but its mode of action results in hypertyrosinaemia. The effect of NTBC on other metabolites in the TYR pathway has not been reported. Modification of the current reverse phase liquid chromatography tandem mass spectrometry methods for serum and urine to include phenylalanine (PHE), hydroxyphenyllactate (HPLA) and hydroxyphenylpyruvate (HPPA) has been validated. HPPA and HPLA (negative ionisation) eluted at 2.8 and 2.9 min respectively on an Atlantis C18 column with PHE (positive ionisation) eluting earlier at 2.4 min. Intra- and inter-assay accuracy was between 96.3% and 100.3% for PHE; 96.6% and 110.5% for HPLA and 95.0% and 107.8% for HPPA in both urine and serum. Precision, both inter- and intra-assay, was <10% for all analytes in both serum and urine. No significant issues with carry-over, stability or matrix interferences were seen in either the urine or serum assays. Measurement of serum and urine from AKU patients has demonstrated a robust, fully validated assay, appropriate for monitoring of patients with AKU and for demonstrating metabolite changes, following NTBC therapy.