Nitisinone Treatment Affects Biomarkers of Bone and Cartilage Remodelling in Alkaptonuria Patients.

Nitisinone has been approved for treatment of alkaptonuria (AKU). Non-invasive biomarkers of joint tissue remodelling could aid in understanding the molecular changes in AKU pathogenesis and how these can be affected by treatment. Serological and urinary biomarkers of type I collagen and II collagen in AKU were investigated in patients enrolled in the randomized SONIA 2 (NCT01916382) clinical study at baseline and yearly until the end of the study (Year 4). The trajectories of the biomarkers over time were observed. After treatment with nitisinone, the biomarkers of type I collagen remodelling increased at Year 1 (19% and 40% increase in CTX-I and PRO-C1, respectively), which was potentially reflected in the higher degree of mobility seen following treatment. The biomarkers of type II collagen remodelling decreased over time in the nitisinone group: C2M showed a 9.7% decline at Year 1, and levels then remained stable over the following visits; CTX-II showed a 26% decline at Year 3 and 4 in the nitisinone-treated patients. Nitisinone treatment induced changes in biomarkers of bone and cartilage remodelling. These biomarkers can aid patient management and deepen our knowledge of the molecular mechanisms of this rare disease.

Conditional targeting in mice reveals that hepatic homogentisate 1,2-dioxygenase activity is essential in reducing circulating homogentisic acid and for effective therapy in the genetic disease alkaptonuria.

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.

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.

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.

Evaluation of the 25-hydroxy vitamin D assay on a fully automated liquid chromatography mass spectrometry system, the Thermo Scientific Cascadion SM Clinical Analyzer with the Cascadion 25-hydroxy vitamin D assay in a routine clinical laboratory.

Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers advantages over immunoassay due to its increased specificity and ability to multiplex metabolites within a single run. Wide scale adoption of LC-MS/MS in routine clinical laboratories is restricted in part due to the high level of technical expertise required. The Thermo Scientific™ Cascadion™ SM Clinical Analyzer is the first fully automated, random access clinical analyser that utilises LC-MS/MS technology. We report an analytical validation of the 25-hydroxy vitamin D2 and D3 assays on the Cascadion Analyzer and an assessment of its performance within a routine clinical laboratory. Methods Analyser usability was assessed by staff with no previous experience of LC-MS/MS. An analytical validation included analysis of 154 patient samples on two different Cascadion Analyzers and a four-way method comparison of 146 patient samples on Roche and Siemens immunoassays and an in-house LC-MS/MS method. Accuracy was assessed using external quality assurance and reference materials. Seven third party IQC materials were tested on Cascadion. Results Cascadion proved easy to use by scientific and non-scientific staff. The assay passed all validation criteria. Excellent agreement was demonstrated between two different Cascadions (y = 0.97x + 3.9 nmol/L, r2 > 0.99). A method comparison demonstrated no significant difference (p > 0.05) between the Cascadion and the Roche immunoassay. A significant difference (p < 0.0001) was observed between the Cascadion and an LC-MS/MS and Siemens methods. Results obtained from EQA and reference material showed a mean bias of +3.09% and all samples were within ±10% of assigned concentrations. All third party IQC samples tested were compatible for use with Cascadion. Conclusions The Cascadion Analyzer is a fully automated LC-MS/MS system that requires no prior LC-MS/MS expertise. The vitamin D assays demonstrated excellent performance with high levels of accuracy.

A Comprehensive LC-QTOF-MS Metabolic Phenotyping Strategy: Application to Alkaptonuria.

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.

Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.

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

Vitamin D assays: past and present debates, difficulties, and developments.

Clinical interest in Vitamin D and its purported roles not only in calcium and bone metabolism but in several other medical conditions (diabetes, cardiovascular disease, multiple sclerosis, cancer, psychiatric disorders, neuro-muscular disease) has led to a surge in laboratory requests for 25 hydroxy vitamin D and 1,25 dihydroxy vitamin D measurement. Circulating 25 hydroxy vitamin D concentration is routinely used as the best indicator of vitamin D status, but measurement of other metabolites, especially the physiologically active 1,25 dihyroxy vitamin D, are of clinical value. Over the last 40 years the development of assays for vitamin D and its metabolites from early competitive binding assays through to immunoassay and liquid chromatography aligned to mass spectrometry have demonstrated various analytical challenges, the advantages and disadvantages of each method are constantly changing with new technological developments. Immunoassay remains the predominant mode of measurement for 25-hydroxy vitamin D although problems with equimolar recovery of the D2 and D3 metabolites remain an issue. Standardisation of all assays has been improved but not resolved with the currently available reference materials as evidenced by the international vitamin D external quality assurance scheme, DEQAS. The choice of method for each laboratory remains a balance mainly between turn around time, convenience, cost and the specificity and accuracy of the information obtained. With increasing discussion and clinical interest surrounding other vitamin D metabolites the vitamin D assay debate is set to continue.

Increased prevalence of Parkinson's disease in alkaptonuria.

Amongst a cohort of 88 alkaptonuria (AKU) patients attending the United Kingdom National Alkaptonuria Centre (NAC), four unrelated patients had co-existing Parkinson's disease (PD). Two of the NAC patients developed PD before receiving nitisinone (NIT) while the other two developed overt PD during NIT therapy. NIT lowers redox-active homogentisic acid (HGA) and profoundly increases tyrosine (TYR). A further unpublished case of a Dutch patient with AKU and PD on deep brain stimulation is included in this report. A Pubmed search revealed a further five AKU patients with PD, all without NIT usage. The prevalence of PD in AKU in the NAC appears to be nearly 20-times higher than in the non-AKU population (p < 0.001) even when adjusted for age. We propose that life-long exposure to redox-active HGA may account for the higher prevalence of PD in AKU. Furthermore, the appearance of PD in AKU patients during NIT therapy may be due to unmasking dopamine deficiency in susceptible individuals, as a result of the tyrosinaemia during NIT therapy inhibiting the rate-limiting brain tyrosine hydroxylase.

Method development and validation for analysis of phenylalanine, 4-hydroxyphenyllactic acid and 4-hydroxyphenylpyruvic acid in serum and urine.

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.

Impact of Nitisinone on the Cerebrospinal Fluid Metabolome of a Murine Model of Alkaptonuria.

Background: Nitisinone-induced hypertyrosinaemia is well documented in Alkaptonuria (AKU), and there is uncertainty over whether it may contribute to a decline in cognitive function and/or mood by altering neurotransmitter metabolism. The aim of this work was to evaluate the impact of nitisinone on the cerebrospinal fluid (CSF) metabolome in a murine model of AKU, with a view to providing additional insight into metabolic changes that occur following treatment with nitisinone. Methods: 17 CSF samples were collected from BALB/c Hgd−/− mice (n = 8, treated with nitisinone­4 mg/L and n = 9, no treatment). Samples were diluted 1:1 with deionised water and analysed using a 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass spectrometry (Agilent, Cheadle, UK). Raw data were processed using a targeted feature extraction algorithm and an established in-house accurate mass retention time database. Matched entities (±10 ppm theoretical accurate mass and ±0.3 min retention time window) were filtered based on their frequency and variability. Experimental groups were compared using a moderated t-test with Benjamini−Hochberg false-discovery rate adjustment. Results: L-Tyrosine, N-acetyl-L-tyrosine, γ-glutamyl-L-tyrosine, p-hydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)lactic acid were shown to increase in abundance (log2 fold change 2.6−6.9, 3/5 were significant p < 0.05) in the mice that received nitisinone. Several other metabolites of interest were matched, but no significant differences were observed, including the aromatic amino acids phenylalanine and tryptophan, and monoamine metabolites adrenaline, 3-methoxy-4-hydroxyphenylglycol, and octopamine. Conclusions: Evaluation of the CSF metabolome of a murine model of AKU revealed a significant increase in the abundance of a limited number of metabolites following treatment with nitisinone. Further work is required to understand the significance of these findings and the mechanisms by which the altered metabolite abundances occur.

Comparing nitisinone 2 mg and 10 mg in the treatment of alkaptonuria-An approach using statistical modelling.

BACKGROUND: Outcomes from studies employing nitisinone 10 mg and 2 mg in alkaptonuria were compared. PATIENTS AND METHODS: Sixty-nine patients in each of the nitisinone (10 mg daily) and controls of suitability of nitisinone in alkaptonuria 2 (SONIA 2), as well as 37 and 23 in nitisinone (2 mg daily) and control cohorts at the National Alkaptonuria Centre (NAC), respectively, were followed up for 4 years. Severity of alkaptonuria (AKU) was assessed by the AKU Severity Score Index (AKUSSI). 24-h urine homogentisic acid (uHGA24), serum HGA (sHGA), serum tyrosine (sTYR) and serum nitisinone (sNIT) were also analysed at each time point. Dietetic support was used in the NAC, but not in SONIA 2. Safety outcomes were also compared. All statistical analyses were post hoc. RESULTS: The slope of the AKUSSI was 0.55, 0.19, 0.30, and 0.06 per month in the control NAC, nitisinone NAC, control SONIA 2, and nitisinone SONIA 2 cohorts, respectively. The intersection of the slopes on the x-axis was -132, -411, -295, and - 1460 months, respectively. The control and nitisinone slope comparisons were statistically significant both in the NAC (p < 0.001) and the SONIA 2 (p < 0.001). Corneal keratopathy occurred in 3 and 10 patients in the NAC and SONIA 2, respectively. DISCUSSION: The nitisinone 10 mg dose decreased disease progression more than the 2 mg dose although the incidence of corneal keratopathy was 14.5% and 4.9%, respectively. CONCLUSION: Nitisinone 10 mg decreased urine and serum HGA, increased serum tyrosine, and decreased disease progression more than 2 mg. Low-protein dietetic support may be needed to mitigate tyrosinaemia following nitisinone. HIGHLIGHTS: Nitisinone 10 mg apparently slows alkaptonuria disease progression more than 2 mg in adults.Corneal keratopathy during nitisinone therapy was more common in men.Serum nitisinone concentrations increased significantly over time.Nitisinone may inhibit cytochrome P450 self catabolism.

Comparing the Phenylalanine/Tyrosine Pathway and Related Factors between Keratopathy and No-Keratopathy Groups as Well as between Genders in Alkaptonuria during Nitisinone Treatment.

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.

Revisiting Quantification of Phenylalanine/Tyrosine Flux in the Ochronotic Pathway during Long-Term Nitisinone Treatment of Alkaptonuria.

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.

Metabolomic studies in the inborn error of metabolism alkaptonuria reveal new biotransformations in tyrosine metabolism.

Alkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of active enzyme homogentisate 1,2-dioxygenase (HGD). The primary consequence of HGD deficiency is increased circulating homogentisic acid (HGA), the main agent in the pathology of AKU disease. Here we report the first metabolomic analysis of AKU homozygous Hgd knockout (Hgd -/-) mice to model the wider metabolic effects of Hgd deletion and the implication for AKU in humans. Untargeted metabolic profiling was performed on urine from Hgd -/- AKU (n = 15) and Hgd +/- non-AKU control (n = 14) mice by liquid chromatography high-resolution time-of-flight mass spectrometry (Experiment 1). The metabolites showing alteration in Hgd -/- were further investigated in AKU mice (n = 18) and patients from the UK National AKU Centre (n = 25) at baseline and after treatment with the HGA-lowering agent nitisinone (Experiment 2). A metabolic flux experiment was carried out after administration of 13C-labelled HGA to Hgd -/-(n = 4) and Hgd +/-(n = 4) mice (Experiment 3) to confirm direct association with HGA. Hgd -/- mice showed the expected increase in HGA, together with unexpected alterations in tyrosine, purine and TCA-cycle pathways. Metabolites with the greatest abundance increases in Hgd -/- were HGA and previously unreported sulfate and glucuronide HGA conjugates, these were decreased in mice and patients on nitisinone and shown to be products from HGA by the 13C-labelled HGA tracer. Our findings reveal that increased HGA in AKU undergoes further metabolism by mainly phase II biotransformations. The data advance our understanding of overall tyrosine metabolism, demonstrating how specific metabolic conditions can elucidate hitherto undiscovered pathways in biochemistry and metabolism.

Vitamin D, vitamin D-binding protein, free vitamin D and COVID-19 mortality in hospitalized patients.

BACKGROUND: Vitamin D deficiency has been associated with worse coronavirus disease 2019 (COVID-19) outcomes, but circulating 25-hydroxyvitamin D [25(OH)D] is largely bound to vitamin D-binding protein (DBP) or albumin, both of which tend to fall in illness, making the 25(OH)D status hard to interpret. Because of this, measurements of unbound ("free") and albumin-bound ("bioavailable") 25(OH)D have been proposed. OBJECTIVES: We aimed to examine the relationship between vitamin D status and mortality from COVID-19. METHODS: In this observational study conducted in Liverpool, UK, hospitalized COVID-19 patients with surplus sera available for 25(OH)D analysis were studied. Clinical data, including age, ethnicity, and comorbidities, were extracted from case notes. Serum 25(OH)D, DBP, and albumin concentrations were measured. Free and bioavailable 25(OH)D were calculated. Relationships between total, free, and bioavailable 25(OH)D and 28-day mortality were analyzed by logistic regression. RESULTS: There were 472 patients with COVID-19 included, of whom 112 (23.7%) died within 28 days. Nonsurvivors were older (mean age, 73 years; range, 34-98 years) than survivors (mean age, 65 years; range, 19-95 years; P = 0.003) and were more likely to be male (67%; P = 0.02). The frequency of vitamin D deficiency [25(OH)D < 50 nmol/L] was similar between nonsurvivors (71/112; 63.4%) and survivors (204/360; 56.7%; P = 0.15) but, after adjustments for age, sex, and comorbidities, increased odds for mortality were present in those with severe deficiency [25(OH)D < 25 nmol/L: OR, 2.37; 95% CI, 1.17-4.78] or a high 25(OH)D (≥100 nmol/L; OR, 4.65; 95% CI, 1.51-14.34) compared with a 25(OH)D value of 50-74 nmol/L (reference). Serum DBP levels were not associated with mortality after adjustments for 25(OH)D, age, sex, and comorbidities. Neither free nor bioavailable 25(OH)D values were associated with mortality. CONCLUSIONS: Vitamin D deficiency, as commonly defined by serum 25(OH)D levels (<50 nmol/L), is not associated with increased mortality from COVID-19, but extremely low (<25 nmol/L) and high (>100 nmol/L) levels may be associated with mortality risks. Neither free nor bioavailable 25(OH)D values are associated with mortality risk. The study protocol was approved by the London-Surrey Research Ethics Committee (20/HRA/2282).

Comprehensive Biotransformation Analysis of Phenylalanine-Tyrosine Metabolism Reveals Alternative Routes of Metabolite Clearance in Nitisinone-Treated Alkaptonuria.

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.

Reversal of ochronotic pigmentation in alkaptonuria following nitisinone therapy: Analysis of data from the United Kingdom National Alkaptonuria Centre.

BACKGROUND: Increased homogentisic acid (HGA) causes ochronosis. Nitisinone decreases HGA. The aim was to study the effect of nitisinone on the ochronosis progression. METHODS: Photographs of the eyes and ears were acquired from patients attending the National Alkaptonuria Centre (NAC) at V-1 (pre-baseline visit), V0 (baseline visit when 2 mg nitisinone was commenced), and yearly at V1, V2, and V3 visits. Photographs were inspected for evolution of ochronotic pigment and also scored categorically to derive eye, ear, and combined ochronosis scores. An ear cartilage biopsy was also carried out at V0 and one year after V3 (V4) and ochronotic pigment was assessed and quantitated. Visits were compared for changes in pigment. Fasting blood and 24-hour urine samples were collected for measurement of HGA. RESULTS: There were 80 AKU patients at V0, and 52, 47, and 40 at V1, V2, and V3 in the group with variable numbers (VAR Group) respectively; 23 patients attended once before V0, in the V-1 visit. Photographs of patients show increase in eye pigment between V-1 and V0, followed by decrease post-nitisinone at V1, V2, and V3. Ear and combined ochronosis semiquantitative scoring showed an increase between V-1 and V0 (P < .01), followed by a decrease at V1, V2, and V3, in the VAR group (P < .01). Ochronotic pigment in ear biopsy between V0 and V4 showed a 19.1% decrease (P < .05). CONCLUSIONS: Nitisinone decreases HGA and partially reverses ochronosis.