Extreme metabolic phenotypes present unique opportunities to understand the participation of different organs in specific metabolite pathways. One such condition is the inherited metabolic disorder alkaptonuria (AKU), caused by mutations in the gene encoding the homogentisate 1,2-dioxygenase (HGD) enzyme. HGD is expressed in liver and kidney. In AKU, lack of functional HGD results in incomplete breakdown of the amino acid tyrosine and accumulation of homogentisic acid (HGA), the indicative metabolite in AKU. Here, we aimed to delineate the role of the kidney in production and metabolism of HGA. We generated for the first time a mouse with specific deletion of Hgd in the kidney but not in the liver, using Cre recombinase driven by Six2 (Six2GCiP), a transcription factor expressed early in kidney development. With intact liver HGD in this mouse, plasma HGA remained equivalent to wild-type concentrations. Minimal circulating HGA combined with no apparent renal HGD activity enabled us to unmask the portion of HGA produced locally within the kidney. Urine HGA (mean ± SEM) was > 100-fold lower in kidney-specific Hgd knockout mice (717 ± 129 μmol/L) compared to AKU mice (118 364 ± 8494 μmol/L) with complete liver and kidney knockout, but higher than wild-type controls (2.3 ± 0.1 μmol/L). Profiling of tyrosine pathway metabolic enzymes showed human and mouse kidney lack detectable tyrosine aminotransferase, a key enzyme involved in tyrosine-HGA metabolism. We demonstrate both tyrosine metabolism and HGA production are minimal in kidney compared to liver. The kidney is therefore not a viable target for HGA-lowering therapies aiming to restore HGD activity.

Abstract Background Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by deficiency of homogentisate 1,2-dioxygenase (HGD), resulting in systemic accumulation of homogentisic acid (HGA), ochronosis, and progressive multisystem disease. Although nitisinone (NTBC) lowers HGA levels, it does not correct the underlying genetic defect and induces hypertyrosinemia, highlighting the need for curative treatment approaches. We evaluated liver-directed adeno-associated virus (AAV)-mediated HGD gene therapy as a potential treatment for AKU. Methods Hgd-deficient ( Hgd -/- ) mice received liver-directed AAV2/8 vectors expressing codon-optimized human HGD under a liver-specific promoter. Reporter vectors were first used to assess hepatic biodistribution and transduction efficiency. Therapeutic efficacy was subsequently evaluated following AAV2/8-HGD administration (1 x 10 12 vg/mouse). HGD expression was assessed by DNAscope, Western blotting, and RT-qPCR. Metabolic correction was determined using targeted LC-MS/MS and untargeted LC-HRMS metabolomics and compared with NTBC-treated Hgd -/- mice. Results Reporter studies demonstrated liver-predominant transduction, with dose-dependent hepatocyte transduction reaching 89-93% at the highest dose. AAV2/8-HGD treatment produced robust hepatic HGD expression, with codon-optimized human HGD transcript levels approximately 33-fold higher than endogenous murine Hgd expression. Twelve weeks after treatment, plasma and urinary HGA levels were significantly reduced, with plasma HGA restored to near wild-type concentrations. Untargeted metabolomics further demonstrated marked reductions in HGA-derived phase I and II metabolites and revealed significant modulation of tricarboxylic acid cycle metabolism, consistent with partial restoration of metabolic homeostasis. Compared with NTBC-treated mice, AAV2/8-HGD achieved comparable plasma HGA reduction without elevation of upstream tyrosine pathway metabolites. Conclusions Liver-directed AAV2/8-HGD gene therapy achieved substantial biochemical correction in Hgd -/- mice and restored metabolic flux without inducing hypertyrosinemia. These findings provide proof-of-concept supporting AAV-mediated HGD replacement as a promising long-term therapeutic strategy for AKU.

Bone resorption involves dissolution of minerals and enzymatic degradation of bone matrix. The primary enzyme is cathepsin K but other proteases including matrix metalloproteinases are involved. Some cathepsin K cleavage products have been partially identified, including cross-linked telopeptides of type I collagen. Here, we aimed to characterize the entire complement of bone breakdown products resulting from osteoclast action under controlled conditions in vitro. We analyzed extracellular media from human osteoclasts cultured on dentin substrates, using untargeted liquid chromatography mass spectrometry. We discovered 22 breakdown products resulting from osteoclastic action. These products were peptide fragment sequences that mapped to various collagen proteins present in bone and dentin. Nine peptide fragments mapped exclusively to collagen I alpha-1 chain (COL1A1), the most abundant protein in bone. We subsequently detected 21 of the fragment products, initially observed in vitro, in human serum and/or urine. Consistent positive correlations were observed between the COL1A1-specific peptide fragments and established bone biochemical markers in serum and urine. Ten urine fragments and two serum fragments markedly increased (p < .05) following total hip arthroplasty, capturing the transient local peri-prosthetic osteolysis observed in these patients (serum, n = 86 patients; urine, n = 83 patients). Among these candidate osteolytic markers, four (two COL1A1-specific products) showed decreases from baseline (p < .05) in patients on denosumab (n = 10 patients). Additionally, two fragment peptides were higher (p < .05, fold change >2) in urine from patients with bone metastasis (24 out of 112) among a lung cancer cohort. The range of collagen peptide fragments we discovered as a direct result of osteoclast activity indicates a complexity of bone resorption pathways not previously known, extending beyond the known proteolytic cleavage events in bone collagen proteins. Monitoring biofluid concentrations of these novel bone markers has the potential to capture multiple pathways of bone resorption activity beyond the existing assays based on cathepsin K.

Abstract Background Accurately recognizing that a person may be dying is central to improving their experience of care at the end-of-life. However, predicting dying is frequently inaccurate and often occurs only hours or a few days before death. Methods We performed urinary metabolomics analysis on patients with lung cancer to create a metabolite model to predict dying over the last 30 days of life. Results Here we show a model, using only 7 metabolites, has excellent accuracy in the Training cohort n = 112 (AUC = 0·85, 0·85, 0·88 and 0·86 on days 5, 10, 20 and 30) and Validation cohort n = 49 (AUC = 0·86, 0·83, 0·90, 0·86 on days 5, 10, 20 and 30). These results are more accurate than existing validated prognostic tools, and uniquely give accurate predictions over a range of time points in the last 30 days of life. Additionally, we present changes in 125 metabolites during the final four weeks of life, with the majority exhibiting statistically significant changes within the last week before death. Conclusions These metabolites identified offer insights into previously undocumented pathways involved in or affected by the dying process. They not only imply cancer’s influence on the body but also illustrate the dying process. Given the similar dying trajectory observed in individuals with cancer, our findings likely apply to other cancer types. Prognostic tests, based on the metabolites we identified, could aid clinicians in the early recognition of people who may be dying and thereby influence clinical practice and improve the care of dying patients.

Introduction: Protein nutrition disorder in alkaptonuria (AKU), resulting in increased homogentisic acid (HGA) before nitisinone therapy and increased tyrosine (TYR) during nitisinone therapy, may benefit from dietetic intervention. The aim of this study was to characterise the diet and their effects prospectively in those who received formal dietetic intervention in the nitisinone-receiving National Alkaptonuria Centre (NAC) patients with those who did not in no-nitisinone Suitability of Nitisinone in Alkaptonuria 2 (SN2 N-) and nitisinone-treated SN2 (SN2 N+) randomised study groups. Patients and methods: A total of 63, 69, and 69 AKU patients from the NAC, SN2 N-, and SN2 N+ were studied for anthropometric (weight, BMI), body composition (including muscle mass, %body fat, hand grip strength), chemical characteristics (serum TYR, serum phenylalanine, urine urea or uUREA, and urine creatinine or uCREAT), and corneal keratopathy. Nitisinone 2 mg and 10 mg were employed in the NAC and SN2 N+ groups, respectively. Dieticians managed protein intake in the NAC, while the SN2 N- and SN2 N+ groups only received advice on self-directed protein restriction during four years of study duration. Results: uUREA decreased in the NAC, SN2 N-, and SN2 N+ groups, showing that protein restriction was achieved in these groups. Body weight and BMI increased in the NAC and SN2 N+ groups. uCREAT decreased significantly in SN2 N- and SN2 N+ compared with the NAC over four years of study. Corneal keratopathy was less frequent in the NAC than in the SN2 N+ group. Active dietetic intervention in NAC stabilised lean body mass (muscle mass, hand grip strength) despite a decrease in uUREA and uCREAT, as well as sTYR. Conclusion: Ongoing dietetic intervention prevented loss of lean body mass despite protein restriction and moderated serum tyrosine increase, leading to less prevalent corneal keratopathy. Protein restriction risks fat mass gain.

BackgroundIncreased homogentisic acid (HGA) in alkaptonuria (AKU) causes severe arthritis. Nitisinone reduces the production of HGA, but whether it also decreases arthroplasty was examined in 237 AKU patients.Patients and methodsPatients attending the United Kingdom National Alkaptonuria Centre (NAC) and the Suitability of Nitisinone in Alkaptonuria 2 (SONIA 2) study were studied. Assessments included questionnaires eliciting details of arthroplasty. Nitisinone was administered from baseline, 2 mg in the NAC and 10 mg in SONIA 2. In SONIA 2, subgroups consisted of those with baseline arthroplasty on and not on nitisinone (BR + N+, BR + N-), as well as those without baseline arthroplasty on and not on nitisinone (BR-N+, BR-N-).ResultsIn the SONIA2 subgroups, new joint replacement (JR) probabilities after baseline were significantly different (BR + N+, BR + N-, BR-N+, BR-N-) (χ2 = 23.3, p < 0.001); mean (SD) was 3.8 (0.1) years in BR-N-, 3.7 (0.1) years in BR-N+, 3.4 (0.3) years in BR + N-, and 3.0 (0.3) years in BR + N+. Further, the BR + N- showed more JR than the BR-N- subgroup (p < 0.01), while BR + N+ similarly showed more JR than the BR-N+ subgroup (p < 0.001).In the NAC, the BR- group had a mean age of 51.6 (7.0) years at baseline but 57.7 (8.7) years at final follow up during nitisinone therapy and showed only 7 incident JR. The BR+ group had an age at baseline of 57.4 (8.5) years and had undergone 94 JRs at baseline.ConclusionThe incidence of arthroplasty was earlier and more frequent after the first JR and was not affected by nitisinone.

Bone resorption involves dissolution of mineral and enzymatic degradation of bone matrix. The primary enzyme is cathepsin K but other proteases including matrix metalloproteinases are involved. Some cleavage products of cathepsin K have been partially identified, including crossed-linked telopeptides of type I collagen. However, the pathway of type I bone collagen degradation has not been fully elucidated. The aim of this study was to comprehensively characterise the entire complement of bone breakdown products resulting from osteoclast action under controlled conditions in vitro . Complete characterisation of these breakdown products will advance understanding of osteoclast biology and has the potential to reveal new biomarkers of bone resorption. We analysed extracellular media from osteoclasts cultured on dentine substrates, using untargeted liquid chromatography mass spectrometry. We discovered 22 breakdown products resulting from osteoclastic action. These products were peptide fragment sequences that mapped to various collagen proteins present in bone and dentine matrix. Nine peptide fragments mapped exclusively to collagen I alpha-1 chain (COL1A1), the most abundant protein in bone. Analysis of the reported cleavage sites in the COL1A1 protein sequence indicated 7/9 COL1A1-specific fragments not explained by known proteolytic events. We subsequently showed that 14 of the fragment products were present in human serum and/or urine from metabolomic datasets obtained from patients with the inherited metabolic disease alkaptonuria (serum) and lung cancer (urine). Two products were at higher concentration (P <0.05, fold change >2) in urine from patients with bone metastasis (29/112) from the lung cancer cohort. The range of collagen peptide fragments we discovered as a direct result of osteoclast activity indicates a complexity of bone resorption pathways not previously known. Monitoring the concentrations of these novel bone markers in biofluids has the potential to capture multiple pathways of bone resorption activity beyond the existing assays based on Cathepsin K. Lay summary Breakdown of bone tissue is performed by specialised bone cells called osteoclasts in a process known as bone resorption. Knowledge of specific molecules produced from osteoclasts acting on bone is important for a) understanding bone resorption in health and disease, and b) clinical tests of bone resorption from measurement of these breakdown products. Here we aimed to characterise the entire complement of bone breakdown products resulting from osteoclast action under controlled conditions in the laboratory. We found a total of 22 breakdown products produced from osteoclasts cultured on wafers of dentine, a tissue with almost identical composition to bone. Analysis of the structures of these products revealed fragments of varying size produced from digestion of specific proteins present in bone. We then showed that 14/22 bone protein fragments observed in our cell culture experiments were also present in human blood and/or urine. In our analysis of urine from patients with lung cancer, we found that two of the protein fragments we identified were higher in a subset (29/112) of patients with bone metastasis. Our findings provide new insights into the mechanisms of bone resorption and show the potential for monitoring the range of urine bone protein fragments in bone disorders. ### Competing Interest Statement The authors have declared no competing interest.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean [±SD] = 1003[±410] μmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] μmol/L). Biliary tyrosine, 3(4-hydroxyphenyl)pyruvic acid (HPPA) and 3(4-hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model.

MicroRNAs (miRNAs) are a family of small, single-stranded, and non-protein coding RNAs about 19 to 22 nucleotides in length, that have been reported to have important roles in the control of bone development. MiRNAs have a strong influence on osteoblast differentiation through stages of lineage commitment and maturation, as well as via controlling the activities of osteogenic signal transduction pathways. Generally, miRNAs may modulate cell stemness, proliferation, differentiation, and apoptosis by binding the 3′-untranslated regions (3′-UTRs) of the target genes, which then can subsequently undergo messenger RNA (mRNA) degradation or protein translational repression. MiRNAs manage the gene expression in osteogenic differentiation by regulating multiple signalling cascades and essential transcription factors, including the transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP), Wingless/Int-1(Wnt)/β-catenin, Notch, and Hedgehog signalling pathways; the Runt-related transcription factor 2 (RUNX2); and osterix (Osx). This shows that miRNAs are essential in regulating diverse osteoblast cell functions. TGF-βs and BMPs transduce signals and exert diverse functions in osteoblastogenesis, skeletal development and bone formation, bone homeostasis, and diseases. Herein, we highlighted the current state of in vitro and in vivo research describing miRNA regulation on the canonical TGF-β/BMP signalling, their effects on osteoblast linage, and understand their mechanism of action for the development of possible therapeutics. In this review, particular attention and comprehensive database searches are focused on related works published between the years 2000 to 2022, using the resources from PubMed, Google Scholar, Scopus, and Web of Science.

Alkaptonuria (AKU) is an ultra-rare inherited inborn error of metabolism that afflicts the tyrosine metabolic pathway, resulting in the accumulation of homogentisic acid (HGA) in the circulation, and significant excretion in urine. Clinical manifestations, typically observed from the third decade of life, are lifelong and significantly affect the quality of life. This review provides a comprehensive overview of the natural history of AKU, including clinical, biochemical and genetic perspectives. An update on the major advances on studies in murine models and human subjects, providing mechanistic insight into the molecular and biochemical processes that underlie pathophysiology and its response to treatment are presented. The impact of treatment with nitisinone is also presented with a specific emphasis on hypertyrosinemia, as uncertainty on this topic remains. Future perspectives are explored, such as novel approaches to treat hypertyrosinemia including the use of binding agents and amino acid transporter inhibitors, as well as advanced potentially curative gene and cell therapy initiatives.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of the metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type-1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in the bile in AKU (mean [±SD] = 1003[±410] μmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] μmol/L). Biliary tyrosine, HPPA and HPLA are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion and possible metabolism of HGA by the gut microbiome. These data provide new insights into the pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism.Take-home message This paper presents the first observation of elevated hepatobiliary circulation of metabolites associated with disease in alkaptonuria, including homogentisic acid in addition to tyrosine and the tyrosine metabolites 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid on nitisinone treatment.### Competing Interest StatementLRR received fees for lectures and consultations from Swedish Orphan Biovitrum. All other authors declare no competing interests.

Background: Accurately recognising that a person may be dying is central for improving their experience of care. Yet recognising dying is difficult and predicting dying frequently inaccurate.Methods: Urine samples from patients (n=112 and 49, training and validation cohorts, respectively)) with lung cancer were analysed using high resolution mass spectrometry. Cox lasso regression was engaged to develop a multivariable model predicting the probability of survival within the last 30 days of life. ANOVA and volcano plot analysis demonstrated metabolites that changed in the last weeks of life. Further analysis identified potential biological pathways affected.Findings: A model predicting time to death using 7 metabolites had excellent accuracy in the training cohort (AUC = 0×85, 0×85, 0×88 and 0×86 on days 5, 10, 20 and 30) and validation cohort (AUC = 0×86, 0×83, 0×90, 0×86 on days 5, 10, 20 and 30). The model enabled classification of patients at low, medium and high risk of dying on a Kaplan-Meier survival curve. 124 metabolites changed. ANOVA analysis identified 93 metabolites and volcano plot analysis 85 metabolites. 53 metabolites changed using both approaches. Pathways altered in the last weeks included those associated with decreased oral intake, muscle loss, decreased RNA and protein synthesis, mitochondrial dysfunction, disrupted β-oxidation and one carbon metabolism. Epinephrine and cortisol increased in the last 2 weeks and week respectively.Interpretation: Metabolomic analysis identified metabolites and their associated pathways that change in the last days of life in patients with lung cancer. Prognostic tests, based on the metabolites identified in this study, could aid clinicians in the early recognition of people who may be dying, and have the potential to influence clinical practice and improve the care of dying patients.Funding: This research received a Wellcome Trust Seed award for Science (202022/Z/16/Z), North West Cancer Research award (SI2018.11), University of Liverpool Enterprise Investment Fund award and Novo Nordisk Foundation (grant NNF20CC0035580).Declaration of Interest: None.Ethical Approval: Ethical approval was provided by North Wales (West) Research Ethics Committee (REC reference 15/WA/0464).

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.

Hereditary tyrosinemia type 1(HT1)is a life-threatening disease caused by the patient's inability to break down tyrosine due to loss-of-function mutations in the fumar-ylacetoacetate hydrolase(FAH)enzyme(Fig.S1).Currently,the only available life-saving treatment is niti-sinone(NTBC).However,nitisinone therapy comes with debilitating side effects and requires a strict drug regime combined with a tyrosine-and phenylalanine-restricted diet.Consequently,therapy adherence is often experi-enced as an additional burden.In this study,transcriptional profiling was conducted parallel to high-resolution metab-olomics on,respectively,liver tissue and serum samples of Fah-deficient mice.The experimental workflow of our study is shown in Figure 1A.

Despite urgent warnings about the spread of multidrug-resistant bacteria, the antibiotic development pipeline has remained sparsely populated. Naturally occurring antibacterial compounds may provide novel chemical starting points for antibiotic development programs and should be actively sought out. Evaluation of homogentisic acid (HGA), an intermediate in the tyrosine degradation pathway, showed that the compound had innate activity against Gram-positive and Gram-negative bacteria, which was lost following conversion into the degradation product benzoquinone acetic acid (BQA). Anti-staphylococcal activity of HGA can be attributed to effects on bacterial membranes. Despite an absence of haemolytic activity, the compound was cytotoxic to human HepG2 cells. We conclude that the antibacterial activity and in vitro safety profile of HGA render it more suitable for use as a topical agent or for inclusion in a small-molecule medicinal chemistry program.

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.

BACKGROUND Adaptations within the phenylalanine (PHE)/tyrosine (TYR) pathway during nitisinone (NIT) are not fully understood. OBJECTIVE To characterise the temporal changes in metabolic features in NIT-treated patients with alkaptonuria. PATIENTS AND METHODS Serum (s) and 24-urine (u) homogentisic acid (sHGA, uHGA24), TYR (sTYR, uTYR24), PHE (sPHE, uPHE24), hydroxyphenylpyruvate (sHPPA, uHPPA24), hydroxyphenyllactate (sHPLA, uHPLA24) and sNIT were measured at baseline (V1) and until month 48 (V6) in 69 NIT-treated patients, recommended to reduce protein intake. The 24-h urine urea (uUREA24), creatinine (uCREAT24) and body weight were also measured. Amounts of tyrosine metabolites in total body water (TBW) were derived by multiplying the serum concentrations by 60% body weight, and sum of TBW and urine metabolites resulted in combined values (c). RESULTS uUREA24 and uCREAT24 decreased between V1 and V6 during NIT, whereas body weight and sNIT increased. Linear regression coefficient between uUREA24 and uCREAT24 was extremely strong (R = 0.84). sPHE, TBWPHE and cPHE24 increased gradually from V1 to V6. A decrease in cTYR24/cPHE24, sTYR/sPHE and TBWTYR/TBWPHE was seen from V2 to V6. Serum, 24-urine and combined TYR, HPPA and HPLA either remained stable or decreased from V2 to V6. DISCUSSION The gradual increase in PHE suggests adaptation to increasing TYR during NIT therapy. The decrease in protein intake resulted in decreased muscle mass and increased weight gain. CONCLUSION Progressive adaptation by decreasing PHE conversion to TYR occurs over time during NIT therapy. A low protein diet results in loss of muscle mass but also weight gain suggesting an increase in fat mass.

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.

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.

Background: Although changes in the tyrosine pathway during nitisinone therapy are known, a complete characterization of the induced tyrosinaemia is lacking to improve disease management. Patients and methods: Our research aims were addressed by 24-h blood sampling. 40 patients with alkaptonuria (AKU), treated with 0, 1, 2, 4 and 8 mg nitisinone daily (n = 8), were studied over four weeks. Serum homogentisic acid (sHGA), tyrosine (sTYR), phenylalanine (sPHE), hydroxyphenylpyruvate (sHPPA), hydroxyphenyllactate (sHPLA) and nitisinone (sNIT) were measured at baseline and after four weeks. Results: sNIT showed a clear dose-proportional response. sTYR increased markedly but with less clear-cut dose responses after nitisinone. Fasting and average 24-h (C-av) sTYR responses were similar. Individual patient sTYR 24-h profiles showed significant fluctuations during nitisinone therapy. At week 4, sTYR, sHPPA and sHPPL all showed dose-related increases compared to VO, with the greatest difference between 1 and 8 mg nitisinone seen for HPLA, while there was no change from VO in sPHE. sHGA decreased to values around the lower limit of quantitation. Discussion: There was sustained tyrosinaemia after four weeks of nitisinone therapy with significant fluctuations over the day in individual patients. Diet and degree of conversion of HPPA to HPLA may determine extent of nitisinone-induced tyrosinaemia. Conclusion: A fasting blood sample is recommended to monitor sTYR during nitisinone therapy Adaptations in HPPA metabolites as well as the inhibition of tyrosine aminotransferase could be contributing factors generating tyrosinaemia during nitisinone therapy.