Alkaptonuria Research Articles

Nitisinone causes acquired tyrosinosis in alkaptonuria.

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 C9 ]tyrosine and l-[d8 ]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.

Homogentisic acid is not only eliminated by glomerular filtration and tubular secretion but also produced in the kidney in alkaptonuria.

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 (CLHGA ) (P < 0.001) and FEHGA (P < 0.001). CLHGA and FEHGA 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.

Alkaptonuric Ochronosis Superimposed With Septic Arthritis in a Middle-aged Man

Alkaptonuria is a rare inborn metabolic disease, in which an enzymatic deficiency accumulates alkapton in different tissues, causing darkness and injury, especially in spine and large cartilages, called ochronosis. The urine darkness can be a key to early diagnosis in childhood, but some cases are missed until adulthood and gradual damage to cartilages causes disability and impairs the patients’ quality of life. Here, a 49-year old male patient is presented with a 2 week history of left knee pain and swelling, who underwent arthrotomy, and the macro- and microscopic evaluation revealed ochronosis, superimposed by septic arthritis. Diagnosis of this rare disease should be considered in differential diagnoses of common joint disorders, like septic arthritis and osteoarthritis, so that appropriate management of the disease can prevent further damages.

Homogentisic acid affects human osteoblastic functionality by oxidative stress and alteration of the Wnt/β-catenin signaling pathway.

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.

Intradural Disc Extrusion in a Dog

Background: Intervertebral disc extrusion is an important cause of spinal cord dysfunction in dogs. Intradural localization of the extruded disc material is rare, and is generally associated with a traumatic event or with recurrence of disc extrusion at a previously affected site. We report the clinical presentation, diagnostic workup, and treatment of a dog with intradural intervertebral disc extrusion not preceded by a traumatic event.Case: A 6-year-old male Dachshund was referred for neurological evaluation due to acute onset of hind-end paralysis preceded by claudication of the left hindlimb. The patient had been receiving conservative treatment to no effect. Neurological examination revealed asymmetric non-ambulatory paraparesis, absence of postural reactions and decreased muscle tone in both hindlimbs, a bilaterally diminished patellar reflex, and a hindlimb withdrawal reflex which was normal on the right and greatly diminished to absent on the left. The lower back was tender to epaxial palpation. Plain radiographs of the lumbar spine in the lateral projection showed calcified material within the spinal canal between the third and fourth lumbar vertebrae. Myelography was suggestively abnormal at the same level, with epidural leakage of contrast at L3-L4. Considering the clinical history, breed, age, neurological signs, and radiographic findings, intervertebral disc disease was suspected despite the inconclusive myelography findings. A dorsolateral lumbar hemilaminectomy was performed. Intraoperatively, the diagnosis was confirmed by visualization of a discolored spinal cord and absence of extradural material. The intradural space was accessed via durotomy. A firm, straw-yellow material was seen compressing the spinal cord and removed. Subsequent histopathological examination confirmed that this material consisted of extruded intervertebral disc contents. Postoperatively, the patient underwent physiotherapy and achieved a satisfactory recovery.Discussion: The most common cause of paraparesis in chondrodystrophic dog breeds is intervertebral disc extrusion. Intradural extrusion of the intervertebral disc is a rare phenomenon, often associated with vigorous exercise that causes laceration of the dura mater, allowing penetration of disc material into the intradural space. Although there were no classic signs of intervertebral disc disease on plain radiography, radiopaque material was visible within the spinal canal, which can occur in cases of calcified intervertebral disc extrusion. Myelography was inconclusive, but the decision was made to operate nevertheless, considering that the patient had not responded to conservative treatment and that surgicaltreatment is the most suitable approach for dogs with non-ambulatory paraparesis or paraplegia secondary to intervertebral disc extrusion. The surgical technique consisted of a hemilaminectomy and durotomy. Our diagnostic suspicion was confirmed intraoperatively, as in most cases of intradural disc extrusion in humans. Intradural disc extrusion is anuncommon phenomenon in dogs, and the diagnosis is usually only established intraoperatively. This unusual variant of intravertebral disc disease should be included in the differential diagnosis of spinal cord dysfunction in chondrodystrophic breeds, even in the absence of a history of trauma or preexisting intervertebral disc disease. Clinical treatment appears ineffective in these cases. Conversely, surgical treatment can yield good outcomes, and even functional recovery.

Dietary restriction of tyrosine and phenylalanine lowers tyrosinemia associated with nitisinone therapy of alkaptonuria.

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.

Alkaptonuria: Current Perspectives.

The last 15 years have been the most fruitful in the history of research on the metabolic disorder alkaptonuria (AKU). AKU is caused by a deficiency of homogentisate dioxygenase (HGD), the enzyme involved in metabolism of tyrosine, and is characterized by the presence of dark ochronotic pigment in the connective tissue that is formed, due to high levels of circulating homogentisic acid. Almost 120 years ago, Sir Archibald Garrod used AKU to illustrate the concept of Mendelian inheritance in man. In January 2019, the phase III clinical study SONIA 2 was completed, which tested the effectiveness and safety of nitisinone in the treatment of AKU. Results were positive, and they will serve as the basis for the application for registration of nitisinone for treatment of AKU at the European Medicines Agency. Therefore, AKU might become a rare disease for which a cure will be found by 2020. We understand the natural history of the disease and the process of ochronosis much more, but at the same time there are still unanswered questions. One of them is the issue of the factors influencing the varying severity of the disease, since our recent genotype–phenotype study did not show that differences in residual homogentisic acid activity caused by the different mutations was responsible. Although nitisinone has proved to arrest the process of ochronosis, it has some unwanted effects and does not cure the disease completely. As such, enzyme replacement or gene therapy might become a new focus of AKU research, for which a novel suitable mouse model of AKU is available already. We believe that the story of AKU is also a story of effective collaboration between scientists and patients that might serve as an example for other rare diseases.

Lumbar intervertebral disc calcification in adult

Intervertebral disc calcification (IVDC) is a rare cause of spinal pain. Both children and adults can be involved, however this affection is more common in cervical and thoracic spine than lumbar column.

Cell and tissue models of alkaptonuria

Alkaptonuria (AKU) is a rare metabolic disease of historical and medical interest. Despite the identification of gene and protein defects leading to the accumulation of homogentisic acid (HGA), little is known on how HGA is transformed into an ochronotic pigment (the hallmark of the disease) leading to a range of clinical manifestations. Major obstacles in tackling the pathological features of AKU are the rarity of biological samples, the invasiveness of sampling techniques and the intrinsic difficulties of studying the pigmented tissues. This review provides an overview of the in vitro and ex vivo cell and tissue models that were recently developed and characterized to fill the above-mentioned gaps in the knowledge of AKU.

The Spectrum of Underlying Diseases in Children with Torticollis.

To investigate the underlying conditions in children with torticollis. Between May 2016 and December 2019, 24 patients (10 girls and 14 boys; mean age, 8 years) presenting with twisted neck, neck pain, weakness of extremities, imbalance, and gait disorder were evaluated retrospectively. Five of the patients had cranial pathologies (cerebellar anaplastic ependymoma and medulloblastoma, brain stem glioma, atypical teratoid rhabdoid tumor, and acute disseminated encephalomyelitis), and five of the patients had spinal pathologies (idiopathic intervertebral disc calcification, vertebral hemangiomatosis, compression fracture, multiple hereditary exostoses, and Langerhans cell histiocytosis at C4). Six of the patients had ocular pathologies (strabismus, Duane syndrome, and Brown syndrome each in two patients). Four patients had otorhinolaryngological infections (Sandifer syndrome, esophageal atresia, reflux, and spasmus nutans, with one patient each). Detailed clinical physical examination and necessary laboratory investigation were performed for all patients. Torticollis is a sign that is not always innocent and may herald an underlying severe disease. Misdiagnosis can lead to wrong and unnecessary surgical procedures and treatments, and sometimes, the results can be damaging due to underlying severe conditions if diagnosed late. In addition, we first report a case of vertebral hemangiomatosis and temporomandibular joint ankylosis that presented with torticollis in the English medical literature.

Passing Strategies and Performative Identities: Coping with (In)Visible Chronic Diseases

In this article I consider the role of passing and performance in the everyday lives of alkaptonuria (AKU) and vitiligo patients. Race, LGBTQ, gender and disability scholars have long used the term passing to describe sub-groups of people within marginal populations who intentionally manipulate their bodies or alter their behaviour in order to claim identities that are not socially assigned to them at birth. In this paper I demonstrate the effectiveness of the passing strategies that patients use in order to mitigate their disease symptoms and render them invisible, thus enabling them to pass as “healthy” or unaffected by their condition. I further consider how patients who choose not to pass utilise resistance strategies in order to generate awareness of their disease and encourage funding for it. I conclude by assessing the effectiveness of these strategies in determining whether or not patients can pass, and the ways in which this is aided or hindered by their social and economic status.

Hedgehog proteins and parathyroid hormone-related protein are involved in intervertebral disc maturation, degeneration, and calcification.

Parathyroid hormone‐related protein (PTHrP) and hedgehog signaling play an important role in chondrocyte development, (hypertrophic) differentiation, and/or calcification, but their role in intervertebral disc (IVD) degeneration is unknown. Better understanding their involvement may provide therapeutic clues for low back pain due to IVD degeneration. Therefore, this study aimed to explore the role of PTHrP and hedgehog proteins in postnatal canine and human IVDs during the aging/degenerative process. The expression of PTHrP, hedgehog proteins and related receptors was studied during the natural loss of the notochordal cell (NC) phenotype during IVD maturation using tissue samples and de‐differentiation in vitro and degeneration by real‐time quantitative polymerase chain reaction (RT‐qPCR) and immunohistochemistry. Correlations between their expression and calcification levels (Alizarin Red S staining) were determined. In addition, the effect of PTHrP and hedgehog proteins on canine and human chondrocyte‐like cells (CLCs) was determined in vitro focusing on the propensity to induce calcification. The expression of PTHrP, its receptor (PTHR1) and hedgehog receptors decreased during loss of the NC phenotype. N‐terminal (active) hedgehog (Indian hedgehog/Sonic hedgehog) protein expression did not change during maturation or degeneration, whereas expression of PTHrP, PTHR1 and hedgehog receptors increased during IVD degeneration. Hedgehog and PTHR1 immunopositivity were increased in nucleus pulposus tissue with abundant vs no/low calcification. In vitro, hedgehog proteins facilitated calcification in CLCs, whereas PTHrP did not affect calcification levels. In conclusion, hedgehog and PTHrP expression is present in healthy and degenerated IVDs. Hedgehog proteins had the propensity to induce calcification in CLCs from degenerated IVDs, indicating that in the future, inhibiting hedgehog signaling could be an approach to inhibit calcification during IVD degeneration.

A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors

Hereditary tyrosinemia type 1 (HT1) and alkaptonuria (AKU) are inherited metabolic disorders caused by defective enzymes involved in tyrosine catabolism. Nitisinone, an ex-herbicide and member of the β-triketone family, is therapeutically applied to prevent accumulation of toxic metabolites in patients by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD). Here, we developed a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of human HPD inhibitors in a high-throughput and a robust fashion. The principle of our screening system is based on the degradation of tyrosine through 4-hydroxyphenylpyruvate into homogentisate by human HPD expressed in E. coli and subsequent production of a soluble melanin-like pigment. With the aim to optimise the assay, we tested different E. coli strains, expression and reaction temperatures, and time-points for supplementing the substrate. We found that in our assay the addition of prototypical β-triketone HPD inhibitors decreases pigment production in a dose-dependent manner with increasing inhibitor concentrations. In addition, plate uniformity, signal variability and spatial uniformity assessment showed that we have developed a robust high-throughput screening assay that is simple to use, cost-effective and enables identification and evaluation of novel therapeutic human HPD inhibitors for the treatment of tyrosine-related metabolic disorders.

Black bone disease of the foot: a case study of minocycline-induced bone pigmentation

Black bone disease of the foot: a case study of minocycline-induced bone pigmentation

Interactive alkaptonuria database: investigating clinical data to improve patient care in a rare disease.

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.

Ochronotic pigmentation is caused by homogentisic acid and is the key event in alkaptonuria leading to the destructive consequences of the disease-A review.

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.

Ochronotic arthritis and ochronotic Achilles tendon rupture in alkaptonuria

Introduction:Alkaptonuria (AKU) is a rare disease caused by deficiency of homogentisate 1,2-dioxygenase which results in deposition of homogentisic acid (HGA). Ochronotic arthritis, the deposition of excess oxidized HGA in the connective tissues, causes pigmentation and degeneration of the joint tissues ultimately resulting in chronic inflammation and osteoarthritis. The ochronotic arthritis has similar clinical features with osteoarthritis. There is currently no specific treatment for AKU and management is usually symptomatic. In severe cases, total joint arthroplasty is the major treatment approaches. It is rarely reported in China.Patient concerns:Here we reported a case of a patient with bilateral knee pain for more than 1 year. He complained of a 20-year history of chronic, nonspecific low back pain and stiffness. His urine was black since he was a child. Six years after the knee surgery, his Achilles tendon ruptured.Diagnosis:Specific radiographic and magnetic resonance imaging manifestations were observed. Darkly pigmented full-thickness cartilage and subchondral bone were found during the operation. Histological investigation also manifested dark stains in meniscus and synovial tissues. Black-denatured tendon tissue was also found during the operation. The patient was diagnosed as AKU.Interventions:Total knee arthroplasty and Achilles tendon repair were operated separately after the disease was diagnosed.Outcomes:The patient recovered very well after the second surgery. He returned to full activities, described no knee pain, and presented to the clinic walking without any aid. Physical examination revealed 0 to 20 of plantar flexion and 0 to 15 of dorsiflexion of the ankle.Conclusions:Ochronosis is a very rare disease in Asia. This paper supplies new information for study of this disease. The mechanism is still unknown right now. Further studies will be necessary.

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

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

Interference of hydroxyphenylpyruvic acid, hydroxyphenyllactic acid and tyrosine on routine serum and urine clinical chemistry assays; implications for biochemical monitoring of patients with alkaptonuria treated with nitisinone

ObjectivesWe 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 methodsHPPA, 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. ResultsNo 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. ConclusionsIn 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.

Evaluation of the serum metabolome of patients with alkaptonuria before and after two years of treatment with nitisinone using LC-QTOF-MS.

BackgroundThe homogentisic acid‐lowering therapy nitisinone is being evaluated for the treatment of alkaptonuria (AKU) at the National Centre for AKU. Beyond hypertyrosinemia, the wider metabolic consequences of its use are largely unknown. The aim of this work was to evaluate the impact of nitisinone on the serum metabolome of patients with AKU after 12 and 24 months of treatment.MethodsDeproteinized serum from 25 patients with AKU (mean age[±SD] 51.1 ± 14.9 years, 12 male) was analyzed using the 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time‐of‐flight mass spectrometry (Agilent, UK). Raw data were processed using a batch targeted feature extraction algorithm and an accurate mass retention time database containing 469 intermediary metabolites (MW 72‐785). Matched entities (±10 ppm theoretical accurate mass and ±0.3 minutes retention time window) were filtered based on their frequency and variability (<25% CV) in group quality control samples, and repeated measures statistical significance analysis with Benjamini‐Hochberg false discovery rate adjustment was used to assess changes in metabolite abundance.ResultsEight metabolites increased in abundance (log2 fold change [FC] 2.1‐15.2, P < .05); 7 of 8 entities were related to tyrosine metabolism, and 13 decreased in abundance (log2 FC 1.5‐15.5, P < .05); including entities related to tyrosine (n = 2), tryptophan (n = 3), xanthine (n = 2), and citric acid cycle metabolism (n = 2).ConclusionsEvaluation of the serum metabolome of patients with AKU showed a significant difference in the abundance of several metabolites following treatment with nitisinone, including a number that have not been previously reported; several of these were not related to the tyrosine metabolic pathway.SynopsisNitisinone therapy has a significant impact on several metabolites beyond the tyrosine metabolic pathway, several of which appear to be related to the redox state of the cell.