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

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          Abstract

          Background

          The 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.

          Methods

          Deproteinized 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.

          Results

          Eight metabolites increased in abundance (log 2 fold change [FC] 2.1‐15.2, P < .05); 7 of 8 entities were related to tyrosine metabolism, and 13 decreased in abundance (log 2 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).

          Conclusions

          Evaluation 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.

          Synopsis

          Nitisinone 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.

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          Most cited references38

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          Natural history of alkaptonuria.

          Alkaptonuria, caused by mutations in the HGO gene and a deficiency of homogentisate 1,2-dioxygenase, results in an accumulation of homogentisic acid (HGA), ochronosis, and destruction of connective tissue. There is no effective therapy for this disorder, although nitisinone inhibits the enzyme that produces HGA. We performed a study to delineate the natural history of alkaptonuria. We evaluated 58 patients with alkaptonuria (age range, 4 to 80 years), using clinical, radiographic, biochemical, and molecular methods. A radiographic scoring system was devised to assess the severity of spinal and joint damage. Two patients were treated with nitisinone for 10 and 9 days, respectively. Life-table analyses showed that joint replacement was performed at a mean age of 55 years and that renal stones developed at 64 years, cardiac-valve involvement at 54 years, and coronary-artery calcification at 59 years. Linear regression analysis indicated that the radiographic score for the severity of disease began increasing after the age of 30 years, with a more rapid increase in men than in women. Twenty-three new HGO mutations were identified. In a 51-year-old woman, urinary HGA excretion fell from 2.9 to 0.13 g per day after a 10-day course of nitisinone (7 days at a dose of 0.7 mg per day and 3 days at 2.8 mg per day). In a 59-year-old woman, urinary HGA fell from 6.4 g to 1.7 g per day after nine days of treatment with nitisinone (0.7 mg per day). Plasma tyrosine levels in these patients rose from approximately 1.1 mg per deciliter (60 micromol per liter) in both to approximately 12.8 mg per deciliter (700 micromol per liter) and 23.6 mg per deciliter (1300 micromol per liter), respectively, with no clinical signs or symptoms. The reported data on the natural history of alkaptonuria provide a basis for the evaluation of long-term therapies. Although nitisinone can reduce HGA production in humans with homogentisate 1,2-dioxygenase deficiency, the long-term safety and efficacy of this treatment require further evaluation. Copyright 2002 Massachusetts Medical Society
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            Untargeted UPLC-MS Profiling Pipeline to Expand Tissue Metabolome Coverage: Application to Cardiovascular Disease

            Metabolic profiling studies aim to achieve broad metabolome coverage in specific biological samples. However, wide metabolome coverage has proven difficult to achieve, mostly because of the diverse physicochemical properties of small molecules, obligating analysts to seek multiplatform and multimethod approaches. Challenges are even greater when it comes to applications to tissue samples, where tissue lysis and metabolite extraction can induce significant systematic variation in composition. We have developed a pipeline for obtaining the aqueous and organic compounds from diseased arterial tissue using two consecutive extractions, followed by a different untargeted UPLC-MS analysis method for each extract. Methods were rationally chosen and optimized to address the different physicochemical properties of each extract: hydrophilic interaction liquid chromatography (HILIC) for the aqueous extract and reversed-phase chromatography for the organic. This pipeline can be generic for tissue analysis as demonstrated by applications to different tissue types. The experimental setup and fast turnaround time of the two methods contributed toward obtaining highly reproducible features with exceptional chromatographic performance (CV % < 0.5%), making this pipeline suitable for metabolic profiling applications. We structurally assigned 226 metabolites from a range of chemical classes (e.g., carnitines, α-amino acids, purines, pyrimidines, phospholipids, sphingolipids, free fatty acids, and glycerolipids) which were mapped to their corresponding pathways, biological functions and known disease mechanisms. The combination of the two untargeted UPLC-MS methods showed high metabolite complementarity. We demonstrate the application of this pipeline to cardiovascular disease, where we show that the analyzed diseased groups (n = 120) of arterial tissue could be distinguished based on their metabolic profiles.
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              A 3-year randomized therapeutic trial of nitisinone in alkaptonuria.

              Alkaptonuria is a rare, autosomal recessive disorder of tyrosine degradation due to deficiency of the third enzyme in the catabolic pathway. As a result, homogentisic acid (HGA) accumulates and is excreted in gram quantities in the urine, which turns dark upon alkalization. The first symptoms, occurring in early adulthood, involve a painful, progressively debilitating arthritis of the spine and large joints. Cardiac valvular disease and renal and prostate stones occur later. Previously suggested therapies have failed to show benefit, and management remains symptomatic. Nitisinone, a potent inhibitor of the second enzyme in the tyrosine catabolic pathway, is considered a potential therapy; proof-of-principle studies showed 95% reduction in urinary HGA. Based on those findings, a prospective, randomized clinical trial was initiated in 2005 to evaluate 40 patients over a 36-month period. The primary outcome parameter was hip total range of motion with measures of musculoskeletal function serving as secondary parameters. Biochemically, this study consistently demonstrated 95% reduction of HGA in urine and plasma over the course of 3 years. Clinically, primary and secondary parameters did not prove benefit from the medication. Side effects were infrequent. This trial illustrates the remarkable tolerability of nitisinone, its biochemical efficacy, and the need to investigate its use in younger individuals prior to development of debilitating arthritis. Published by Elsevier Inc.
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                Author and article information

                Contributors
                andrew.davison@rlbuht.nhs.uk
                Journal
                JIMD Rep
                JIMD Rep
                10.1002/(ISSN)2192-8312
                JMD2
                JIMD Reports
                John Wiley & Sons, Inc. (Hoboken, USA )
                2192-8304
                2192-8312
                31 May 2019
                July 2019
                : 48
                : 1 ( doiID: 10.1002/jmd2.v48.1 )
                : 67-74
                Affiliations
                [ 1 ] Department of Clinical Biochemistry and Metabolic Medicine, Liverpool Clinical Laboratories Royal Liverpool University Hospitals Trust Liverpool UK
                [ 2 ] Musculoskeletal Biology I, Institute of Ageing and Chronic Disease University of Liverpool, Liverpool Health Partners Liverpool UK
                [ 3 ] Agilent Technologies Cheadle UK
                Author notes
                [*] [* ] Correspondence

                Andrew S. Davison, Department of Clinical Biochemistry and Metabolic Medicine, Liverpool Clinical Laboratories, Royal Liverpool University Hospitals Trust, Liverpool, L7 8XP, UK.

                Email: andrew.davison@ 123456rlbuht.nhs.uk

                Author information
                https://orcid.org/0000-0001-5501-4475
                Article
                JMD212042
                10.1002/jmd2.12042
                6606987
                31392115
                6f1a13b5-5a13-4b5f-93d9-16b2a6a80c1d
                © 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 18 March 2019
                : 26 April 2019
                : 02 May 2019
                Page count
                Figures: 2, Tables: 1, Pages: 8, Words: 5937
                Funding
                Funded by: Research Trainees Coordinating Centre
                Award ID: DRF‐2014‐05‐009
                Funded by: National Institute for Health Research
                Award ID: DRF‐2014‐05‐009
                Categories
                Research Report
                Research Reports
                Custom metadata
                2.0
                jmd212042
                July 2019
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.6.5 mode:remove_FC converted:03.07.2019

                alkaptonuria,metabolomics,nitisinone,tyrosine
                alkaptonuria, metabolomics, nitisinone, tyrosine

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