Summary of Study ST002297
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001471. The data can be accessed directly via it's Project DOI: 10.21228/M8KH70 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST002297 |
| Study Title | Comprehensive biotransformation analysis of phenylalanine-tyrosine metabolism reveals alternative routes of metabolite clearance in nitisinone-treated alkaptonuria (Urine metabolomic analysis) |
| Study Type | Urine metabolomic analysis (study 2 of 2) |
| Study Summary | Background: Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homo-gentisic 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. Methods: 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 per-formed 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. Results: In total, we ob-served 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. Conclusions: 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. |
| Institute | University of Liverpool Institute of Life Course & Medical Sciences |
| Department | Department of Musculoskeletal & Ageing Science |
| Last Name | Brendan |
| First Name | Norman |
| Address | William Henry Duncan Building, 6 West Derby Street, Liverpool, UK. L7 8TX |
| bnorman@liverpool.ac.uk | |
| Phone | +447809606497 |
| Submit Date | 2022-09-28 |
| Num Groups | 2 |
| Total Subjects | 103 |
| Num Males | 65 |
| Num Females | 38 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | d |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-10-14 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001471 |
| Project DOI: | doi: 10.21228/M8KH70 |
| Project Title: | Metabolomic analysis in SONIA 2, a phase 3 international randomised-controlled trial of nitisinone in alkaptonuria (AKU) |
| Project Summary: | Abstract from main SONIA 2 publication: Background Alkaptonuria is a rare, genetic, multisystem disease characterised by the accumulation of homogentisic acid (HGA). No HGA-lowering therapy has been approved to date. The aim of SONIA 2 was to investigate the efficacy and safety of once-daily nitisinone for reducing HGA excretion in patients with alkaptonuria and to evaluate whether nitisinone has a clinical benefit. Methods SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study done at three sites in the UK, France, and Slovakia. Patients aged 25 years or older with confirmed alkaptonuria and any clinical disease manifestations were randomly assigned (1:1) to receive either oral nitisinone 10 mg daily or no treatment. Patients could not be masked to treatment due to colour changes in the urine, but the study was evaluator-blinded as far as possible. The primary endpoint was daily urinary HGA excretion (u-HGA24) after 12 months. Clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) score was assessed at 12, 24, 36, and 48 months. Efficacy variables were analysed in all randomly assigned patients with a valid u-HGA24 measurement at baseline. Safety variables were analysed in all randomly assigned patients. The study was registered at ClinicalTrials.gov (NCT01916382). Findings Between May 7, 2014, and Feb 16, 2015, 139 patients were screened, of whom 138 were included in the study, with 69 patients randomly assigned to each group. 55 patients in the nitisinone group and 53 in the control group completed the study. u-HGA24 at 12 months was significantly decreased by 99·7% in the nitisinone group compared with the control group (adjusted geometric mean ratio of nitisinone/control 0·003 [95% CI 0·003 to 0·004], p<0·0001). At 48 months, the increase in cAKUSSI score from baseline was significantly lower in the nitisinone group compared with the control group (adjusted mean difference –8·6 points [–16·0 to –1·2], p=0·023). 400 adverse events occurred in 59 (86%) patients in the nitisinone group and 284 events occurred in 57 (83%) patients in the control group. No treatment-related deaths occurred. Interpretation Nitisinone 10 mg daily was well tolerated and effective in reducing urinary excretion of HGA. Nitisinone decreased ochronosis and improved clinical signs, indicating a slower disease progression. |
| Institute: | University of Liverpool Institute of Life Course & Medical Sciences |
| Department: | Department of Musculoskeletal & Ageing Science |
| Last Name: | Brendan |
| First Name: | Norman |
| Address: | William Henry Duncan Building, 6 West Derby Street, Liverpool, UK. L7 8TX |
| Email: | bnorman@liverpool.ac.uk |
| Phone: | +447809606497 |
| Funding Source: | European Commission for the Framework 7 grant award (DevelopAKUre, project number: 304985), Alkaptonuria Society (BPN is funded by the Alkaptonuria Society through a Sireau Fellowship Award). |
Subject:
| Subject ID: | SU002383 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatedrial arm |
|---|---|---|
| SA220649 | P03_V4 | Treated |
| SA220650 | P03_V1 | Treated |
| SA220651 | P03_V6 | Treated |
| SA220652 | P07_V1 | Treated |
| SA220653 | P07_V4 | Treated |
| SA220654 | P02_V6 | Treated |
| SA220655 | P02_V4 | Treated |
| SA220656 | L40_V1 | Treated |
| SA220657 | L40_V4 | Treated |
| SA220658 | L40_V6 | Treated |
| SA220659 | P02_V1 | Treated |
| SA220660 | P07_V6 | Treated |
| SA220661 | P11_V4 | Treated |
| SA220662 | P18_V6 | Treated |
| SA220663 | P18_V4 | Treated |
| SA220664 | P22_V1 | Treated |
| SA220665 | P22_V4 | Treated |
| SA220666 | P22_V6 | Treated |
| SA220667 | P18_V1 | Treated |
| SA220668 | P15_V6 | Treated |
| SA220669 | L35_V6 | Treated |
| SA220670 | P11_V6 | Treated |
| SA220671 | P15_V1 | Treated |
| SA220672 | P15_V4 | Treated |
| SA220673 | P11_V1 | Treated |
| SA220674 | L35_V1 | Treated |
| SA220675 | L16_V6 | Treated |
| SA220676 | L16_V4 | Treated |
| SA220677 | L18_V1 | Treated |
| SA220678 | L18_V4 | Treated |
| SA220679 | L18_V6 | Treated |
| SA220680 | L16_V1 | Treated |
| SA220681 | L14_V6 | Treated |
| SA220682 | L12_V4 | Treated |
| SA220683 | L12_V6 | Treated |
| SA220684 | L14_V1 | Treated |
| SA220685 | L14_V4 | Treated |
| SA220686 | L22_V1 | Treated |
| SA220687 | L22_V4 | Treated |
| SA220688 | L33_V1 | Treated |
| SA220689 | L30_V6 | Treated |
| SA220690 | L33_V4 | Treated |
| SA220691 | L33_V6 | Treated |
| SA220692 | P23_V1 | Treated |
| SA220693 | L30_V4 | Treated |
| SA220694 | L30_V1 | Treated |
| SA220695 | L22_V6 | Treated |
| SA220696 | L28_V1 | Treated |
| SA220697 | L28_V4 | Treated |
| SA220698 | L28_V6 | Treated |
| SA220699 | F01_V1 | Treated |
| SA220700 | P23_V4 | Treated |
| SA220701 | P48_V4 | Treated |
| SA220702 | P48_V1 | Treated |
| SA220703 | P48_V6 | Treated |
| SA220704 | P52_V1 | Treated |
| SA220705 | P52_V4 | Treated |
| SA220706 | P47_V6 | Treated |
| SA220707 | P47_V4 | Treated |
| SA220708 | P46_V1 | Treated |
| SA220709 | P46_V4 | Treated |
| SA220710 | P46_V6 | Treated |
| SA220711 | P47_V1 | Treated |
| SA220712 | P52_V6 | Treated |
| SA220713 | P55_V1 | Treated |
| SA220714 | P62_V6 | Treated |
| SA220715 | P62_V4 | Treated |
| SA220716 | P64_V1 | Treated |
| SA220717 | P64_V4 | Treated |
| SA220718 | P64_V6 | Treated |
| SA220719 | P62_V1 | Treated |
| SA220720 | P60_V6 | Treated |
| SA220721 | P55_V4 | Treated |
| SA220722 | P55_V6 | Treated |
| SA220723 | P60_V1 | Treated |
| SA220724 | P60_V4 | Treated |
| SA220725 | P44_V6 | Treated |
| SA220726 | P44_V4 | Treated |
| SA220727 | P31_V1 | Treated |
| SA220728 | P30_V6 | Treated |
| SA220729 | P31_V4 | Treated |
| SA220730 | P31_V6 | Treated |
| SA220731 | P34_V1 | Treated |
| SA220732 | P30_V4 | Treated |
| SA220733 | P30_V1 | Treated |
| SA220734 | P23_V6 | Treated |
| SA220735 | P26_V1 | Treated |
| SA220736 | P26_V4 | Treated |
| SA220737 | P26_V6 | Treated |
| SA220738 | P34_V4 | Treated |
| SA220739 | P34_V6 | Treated |
| SA220740 | P43_V1 | Treated |
| SA220741 | P43_V4 | Treated |
| SA220742 | P43_V6 | Treated |
| SA220743 | P44_V1 | Treated |
| SA220744 | P40_V6 | Treated |
| SA220745 | P40_V4 | Treated |
| SA220746 | P39_V1 | Treated |
| SA220747 | P39_V4 | Treated |
| SA220748 | P39_V6 | Treated |
Collection:
| Collection ID: | CO002376 |
| Collection Summary: | Serum and urine samples were collected at the three study sites; Liverpool, Paris and Piešťany. Samples collected at Paris and Piešťany were transported frozen to the Royal Liverpool University Hospital for metabolite analysis and storage at -80 °C. Urine was collected over 24-h into 2.5 L bottles containing 30 mL of 5 mol/L sulphuric acid which was subsequently aliquoted and stored at -80 °C. |
| Sample Type: | Urine |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002395 |
| Treatment Summary: | Samples studied were from patients partaking in the SONIA 2 clinical trial of nitisinone in AKU. SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study undertaken at three study sites; Liverpool (UK), Paris (France) and Piešťany (Slovakia). The details and outcomes of the trial are published (1). Serum and urine samples were collected from participants at baseline (pre-treatment) then at 3 months and 1, 2, 3 and 4 years on 10 mg oral daily nitisinone (Orfadin®) treatment or no treatment. The serum and urine samples from visit 1 (baseline; pre-treatment), visit 4 (2 years), visit 6 (4 years) underwent metabolomic analysis. In this study, only the data from patients with samples available for these three time points were included; 53 and 50 patients in treated and untreated groups respectively for urine, and 47 and 45 patients in treated and untreated groups respectively for serum. Reference: (1) Ranganath, L.R.; Psarelli, E.E.; Arnoux, J.B.; Braconi, D.; Briggs, M.; Bröijersén, A.; Loftus, N.; Bygott, H.; Cox, T.F.; Davison, A.S.; et al. Efficacy and Safety of Once-Daily Nitisinone for Patients with Alkaptonuria (SONIA 2): An International, Multicentre, Open-Label, Randomised Controlled Trial. Lancet Diabetes Endocrinol. 2020, 8, 762–772, doi:10.1016/S2213-8587(20)30228-X. |
| Treatment Protocol ID: | Clinical trial ID: EudraCT no. 2013-001633-41 |
| Treatment Compound: | Nitisinone (NTBC) |
| Treatment Dose: | 10 mg daily |
| Treatment Doseduration: | 48 months total |
Sample Preparation:
| Sampleprep ID: | SP002389 |
| Sampleprep Summary: | Prior to analysis, urine samples were thawed at room temperature before vortexing and centrifugation at 1500 ×g for 5 minutes. 150 µL of each urine sample was aliquoted into a 1 mL 96-well plate (Waters Corporation, Wilmslow, UK) and diluted with 450 µL of deionised water. Samples were mixed on a plate shaker (MTS 2/4m IKA) at 600 rpm for 10 min and sub-aliquoted into multiple replicate 96-well plates before storage at -80 °C ready for analysis. Prior to analysis, sample plates were thawed then agitated on a plate shaker (MTS 2/4m IKA) at 600 rpm for 10 min. |
Combined analysis:
| Analysis ID | AN003752 | AN003753 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Agilent 1290 Infinity II | Agilent 1290 Infinity II |
| Column | Waters Atlantis dC18 (100 x 3mm,3um) | Waters Atlantis dC18 (100 x 3mm,3um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Agilent 6550 QTOF | Agilent 6550 QTOF |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | peak area (pareto-scaled, log2-transformed, 24h creatinine normalised) | peak area (pareto-scaled, log2-transformed, 24h creatinine normalised) |
Chromatography:
| Chromatography ID: | CH002777 |
| Chromatography Summary: | Analysis of serum and urine samples was performed using a published LC-QTOF-MS acquisition method, which employed a 1290 Infinity II HPLC coupled to a 6550 QTOF-MS equipped with dual AJS electrospray ionisation source (Agilent, Cheadle, UK). Data acquisition parameters are detailed in brief below and in full in Supplementary Materials. Reversed-phase LC was performed on an Atlantis dC18 column (3×100 mm, 3 μm, Waters, Manchester, UK) maintained at 60 °C. Mobile phase composition was (A) water and (B) methanol, both with 5 mmol/L ammonium formate and 0.1 % formic acid. The elution gradient began at 5 % B 0–1 min and increased linearly to 100 % B by 12 min, held at 100 % B until 14 min, then at 5 % B for a further 5 min. MS data acquisition was performed in positive and negative ionisation polarity with mass range 50–1700 in 2 GHz mode with acquisition rate at 3 spectra/second. Sample injection volume was 1 and 2 µL in negative and positive polarities, respectively. |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Waters Atlantis dC18 (100 x 3mm,3um) |
| Column Temperature: | 60 |
| Flow Gradient: | The elution gradient began at 5 % B 0–1 min and increased linearly to 100 % B by 12 min, held at 100 % B until 14 min, then at 5 % B for a further 5 min. |
| Solvent A: | 100% water; 0.1% formic acid; 5mM ammonium formate |
| Solvent B: | 100% methanol; 0.1% formic acid; 5mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003496 |
| Analysis ID: | AN003752 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | MS acquisition conditions detailed in attached Supplementary Materials. Raw data were mined using the targeted feature extraction function in Masshunter Profinder (build 10.00, Agilent) with mass targets based on chemical formulae of known/predicted phe-tyr pathway metabolites from the customised compound databases described below. A combined compound database was compiled using PCDL Manager (Agilent, build 08.00). Accurate mass retention time (AMRT) matched metabolites were present in our published AMRT database, which was generated from chemical standards using the same LC-QTOF-MS methodology employed here: phenylalanine, phenylethylamine, tyrosine, N-acetyl-tyrosine, tyramine, HPPA, HPLA and HGA. Other established phenylalanine metabolites added to the database for mining by accurate mass alone were hydroxyphenylacetic acid, phenylacetaldehyde, phenylacetamide, phenylacetic acid, phenylacetylglutamine, phenylethylamine, phenyllactic acid and phenylpyruvic acid. The remaining formulae were from non-established but theoretically possible phase 1 and 2 biotransformation products derived from phenylalanine (n=74), tyrosine (n=74), HPPA (n=67) and HPLA (n=67) predicted using the Biotransformation Mass Defects tool (Agilent), in addition to the HGA biotransformation products (n=7) previously established by our group. Feature extraction parameters were accurate mass match window ±5 ppm with addition of matched retention time (RT; window ±0.3 min) for AMRT database metabolites. Allowed ion species were: H+, Na+, and NH4+ in positive polarity, and H− and CHO2- in negative polarity. Charge state range was 1–2, and dimers were allowed. ‘Find by formula’ filters were: score >60 in at least 60 % of samples in at least one sample group. Where compounds were detected in both positive and negative ionisation, the polarity with the clearest signal was selected for further analysis. Extracted peak area intensity data were exported in .csv file format and imported into Mass Profiler Professional (MPP; build 15.1, Agilent), in which all statistical analyses were performed unless stated otherwise. In MPP, all data were log2 transformed and pareto scaled. Urine data were normalised to 24-h creatinine values. QC was performed based on compound signal intensity data from the pooled samples interspersed throughout each analytical sequence. Compounds were retained for subsequent statistical analyses if a) observed in 100 % of replicate injections for at least one sample group pool, and b) peak area coefficient of variation (CV) remained <30% across replicate injections for each sample group pool across batches 1 and 2 combined. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003497 |
| Analysis ID: | AN003753 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | MS acquisition conditions detailed in attached Supplementary Materials. Raw data were mined using the targeted feature extraction function in Masshunter Profinder (build 10.00, Agilent) with mass targets based on chemical formulae of known/predicted phe-tyr pathway metabolites from the customised compound databases described below. A combined compound database was compiled using PCDL Manager (Agilent, build 08.00). Accurate mass retention time (AMRT) matched metabolites were present in our published AMRT database, which was generated from chemical standards using the same LC-QTOF-MS methodology employed here: phenylalanine, phenylethylamine, tyrosine, N-acetyl-tyrosine, tyramine, HPPA, HPLA and HGA. Other established phenylalanine metabolites added to the database for mining by accurate mass alone were hydroxyphenylacetic acid, phenylacetaldehyde, phenylacetamide, phenylacetic acid, phenylacetylglutamine, phenylethylamine, phenyllactic acid and phenylpyruvic acid. The remaining formulae were from non-established but theoretically possible phase 1 and 2 biotransformation products derived from phenylalanine (n=74), tyrosine (n=74), HPPA (n=67) and HPLA (n=67) predicted using the Biotransformation Mass Defects tool (Agilent), in addition to the HGA biotransformation products (n=7) previously established by our group. Feature extraction parameters were accurate mass match window ±5 ppm with addition of matched retention time (RT; window ±0.3 min) for AMRT database metabolites. Allowed ion species were: H+, Na+, and NH4+ in positive polarity, and H− and CHO2- in negative polarity. Charge state range was 1–2, and dimers were allowed. ‘Find by formula’ filters were: score >60 in at least 60 % of samples in at least one sample group. Where compounds were detected in both positive and negative ionisation, the polarity with the clearest signal was selected for further analysis. Extracted peak area intensity data were exported in .csv file format and imported into Mass Profiler Professional (MPP; build 15.1, Agilent), in which all statistical analyses were performed unless stated otherwise. In MPP, all data were log2 transformed and pareto scaled. Urine data were normalised to 24-h creatinine values. QC was performed based on compound signal intensity data from the pooled samples interspersed throughout each analytical sequence. Compounds were retained for subsequent statistical analyses if a) observed in 100 % of replicate injections for at least one sample group pool, and b) peak area coefficient of variation (CV) remained <30% across replicate injections for each sample group pool across batches 1 and 2 combined. |
| Ion Mode: | POSITIVE |
