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.

Disuse-induced muscle atrophy commonly occurs following illness, injury, or falls and becomes increasingly frequent with ageing. Whether skeletal muscle retains a "memory" of repeated disuse remains unknown. We investigated repeated lower-limb immobilization in young adults and a refined aged rat model, integrating physiological, multi-omic, immunohistochemical, biochemical, and primary human muscle stem cell (MuSC) analyses. To enable robust age comparisons, we integrated previously published young rat data with newly generated aged rat data. In young human muscle, repeated disuse elicited attenuated transcriptional perturbations in oxidative and mitochondrial pathways, suggestive of a protective molecular memory, despite similar atrophy to initial disuse. In contrast, aged muscle exhibited a detrimental memory, characterized by greater atrophy, exaggerated suppression of aerobic metabolism genes despite recovery after initial disuse, NAD+ and mitochondrial DNA depletion, and activation of proteasomal, extracellular-matrix, and DNA-damage pathways. Whereas young rats recovered muscle mass after initial disuse, aged rats failed to do so. Across species, repeated disuse induced DNA hypermethylation and downregulation of aerobic metabolism and mitochondrial gene networks. NR4A1 and NR4A3 were among the strongest disuse-suppressed genes; NR4A1 acquired recovery-phase hypermethylation that maintained its transcriptional repression, while NR4A3 was the most downregulated gene after initial atrophy and remained persistently suppressed into recovery. Acetylcholine receptor subunit genes (CHRNA1, CHRND) were epigenetically primed, demonstrating hypomethylation and strong upregulation after disuse, and further amplification after repeated atrophy, while CHRNG was selectively induced after repeated atrophy only. NMRK2, an NAD+ biosynthesis gene, was the most downregulated gene across both atrophy periods, and supplementation with its substrate, nicotinamide riboside (NR), improved myotube size in MuSCs derived post-atrophy. Overall, repeated disuse atrophy imprints a molecular memory in skeletal muscle shaping transcriptional resilience in young adults and exaggerated susceptibility in aged muscle.

Resistance training (RT) promotes muscle protein accretion and myofiber hypertrophy, driven by dynamic processes of protein synthesis and degradation. While molecular studies have focused on acute signalling or long-term hypertrophy and strength gains, a critical gap remains in understanding the intermediate processes of muscle adaptation. Acute signalling does not always correlate directly with long-term outcomes, highlighting the need for a time-course analysis of protein abundance and turnover rates. To address this, we utilised deuterium oxide labelling and peptide mass spectrometry to quantify absolute protein content and synthesis rates in skeletal muscle. A daily programmed resistance training regimen was applied to the rat tibialis anterior (TA) via electrical stimulation of the left hind limb for 10, 20, and 30 days (5 sets of 10 repetitions daily). Muscle samples from stimulated (Stim) and contralateral control (Ctrl) limbs were analysed, quantifying 658 protein abundances and 215 protein synthesis rates. Unsupervised temporal clustering of protein responses revealed distinct phases of muscle adaptation, with early (0-10 days) and mid (10-20 days) responses driven by differential protein accretion rates in ribosomal and mitochondrial networks, respectively. These findings suggest that subsets of proteins exhibit distinct adaptation timelines due to variations in translation and/or degradation rates. A deeper understanding of these temporal shifts could improve strategies for optimising muscle growth and functional adaptation to resistance training. ### Competing Interest Statement The authors have declared no competing interest.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor whose dysfunction is linked to developmental dysplasia of the hip, osteoporosis and osteoarthritis. Our work addresses the critical question of how these skeletal pathologies emerge. Here, we show the abundant expression of LRP1 in skeletal progenitor cells at mouse embryonic stage E10.5 and onwards, especially in the perichondrium, the stem cell layer surrounding developing limbs essential for bone formation. Lrp1 deficiency in these stem cells causes joint fusion, malformation of cartilage/bone template and markedly delayed or lack of primary ossification. These abnormalities, which resemble phenotypes associated with Wnt signalling pathways, result in severe and persistent skeletal defects including a severe deficit in hip joint and patella, and markedly deformed and low-density long bones leading to dwarfism and impaired mobility. Mechanistically, we show that LRP1 regulates core non-canonical Wnt/planar cell polarity (PCP) components that may explain the malformation of long bones. LRP1 directly binds to Wnt5a, facilitates its cell-association and endocytic degradation and recycling. In the developing limbs, LRP1 partially colocalises with Wnt5a and its deficiency alters abundance and distribution of Wnt5a and Vangl2. Finally, using Xenopus as a model system, we show the regulatory role for LRP1 in Wnt/PCP signalling. We propose that in skeletal progenitors, LRP1 plays a critical role in formation and maturity of multiple bones and joints by regulating Wnt signalling, providing novel insights into the fundamental processes of morphogenesis and the emergence of skeletal pathologies.

Resistance training promotes muscle protein accretion and myofiber hypertrophy, driven by dynamic processes of protein synthesis and degradation. Muscle adaptations to ongoing resistance training occur over weeks, but most molecular knowledge on the process of adaptation is derived from static measurements at specific time points, which do not capture the dynamics of the adaptation process. To address this, we utilized deuterium oxide labeling and peptide mass spectrometry to quantify absolute protein content (grams) and synthesis rates (grams/day) in skeletal muscle during a time series experimental design. A daily programmed resistance training regimen was applied to male rat tibialis anterior via electrical stimulation of the left hindlimb for 10, 20, and 30 days (5 sets of 10 repetitions daily). Muscle samples from stimulated and contralateral control limbs were analyzed, quantifying 658 protein abundances and 215 protein synthesis rates. Unsupervised temporal clustering of protein responses revealed distinct phases of muscle adaptation. The early (0-10 days) response was driven by greater rates of ribosomal protein accretion and the mid (10-20 days) response by expansion of mitochondrial networks. These findings highlight that subsets of proteins exhibit distinct adaptation timelines due to variations in translation and/or degradation rates. The new understanding of temporal patterns highlighted by our dynamic proteomic data helps interpret static data from studies at isolated time points and could improve the development of strategies for optimizing muscle growth and functional adaptation to resistance training.NEW & NOTEWORTHY We used stable isotope labeling and proteomic analyses to quantify absolute changes in the synthesis and abundance of muscle proteins during programmed resistance training in rat in vivo. This novel time-resolved approach revealed distinct phases of adaptation, characterized by early ribosomal and later mitochondrial protein accretion. Strikingly, we observed substantial "oversynthesis" of muscle proteins; that is, the net gain in muscle protein content was much less than the amount of newly synthesized protein.

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.

Resistance training increases muscle size but little is known about the dynamic processes that underpin the gains in muscle mass. We investigated rat muscle during 30 days of unilateral programmed resistance training (PRT) using the stable isotope (deuterium oxide; D2O) in vivo and proteomics. Three-month-old, male Wistar rats (body weight 348 ± 20 g) were assigned to four groups (n = 4, in each), including a control group and experimental groups that received D2O for 10, 20 or 30 days. Under ethically approved procedures, deuterium was administered, and a stimulating device was surgically implanted to activate the left peroneal nerve and cause maximal contraction of the dorsiflexors (inc. tibialis anterior; TA) and partial contraction of the plantar flexors. PRT consisted of 1 bout per day of 5 sets of 10 repetitions (1 repetition consisted of 2 s stimulation at 100 Hz with 2 s rest between repetitions and 2.5 min between sets). One hour after the final training bout (i.e. day 10, 20, or 30) animals were killed and the left (stimulated) and right (contralateral control) TA were extracted and weighed. Muscles were fractionated into myofibrillar and sarcoplasmic components. Protein content was calculated via Bradford assay and proteomic analysis of myofibrillar proteins was conducted via liquid chromatography-tandem mass spectrometry of peptide digests. Statistical analysis was conducted using R (v4.3.2.) and functional annotation was performed using the STRING database. Two-way mixed ANOVA was used to investigate differences between condition (stimulated vs contralateral control) and time (0, 10, 20, or 30 days). The protein content of stimulated TA increased (P < 0.05) from 0.579 ± 0.05 mg at day zero to 1.096 ± 0.15 mg after 30 days of PRT, whereas the protein content of control TA (0.618 ± 0.04 mg) was consistent across the 30-day experimental period. Proteomic analysis encompassed 244 proteins and the abundance of 45 proteins exhibited a significant (P < 0.05) interaction between condition and time. Stimulated muscle was enriched in mitochondrial enzymes. For example, the beta-subunit of ATPase synthase (ATPB) increased from 14.6 ± 4.5 to 74 ± 15.6 μg in stimulated TA and was 17 ± 5 in control TA. PRT also increased the abundance of neonatal myosin heavy chain (MYH8), four-and-a-half LIM domains protein 1 (FHL1) and muscular LMNA interacting protein (MLIP). These findings suggest the energy requirements of daily PRT are met by mitochondrial metabolism and PRT may be associated with activation of the muscle developmental programme.

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.

The layer of stem cells surrounding developing limbs is essential for bone formation and regeneration. Our work addresses the critical question of how these stem cells and bone template communicate to ensure that limbs form correctly. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor whose mutations are linked to bone and joint pathologies. Here, we show the abundant expression of LRP1 in skeletal progenitor cells, especially in the perichondrium – the dense layer of fibrous connective tissue enveloping the cartilage of the developing limb bud. Our mouse models reveal that LRP1 deficiency in these stem cells ( Lrp1 flox/flox / Prrx1 Cre ) but not in chondrocytes ( Lrp1 flox/flox / Acan CreERT2 ) causes disrupted articulation and cavitation starting at as early as embryonic stage 16.5. LRP1 deficiency is also associated with aberrant accumulation of LRP1 ligands including tissue-inhibitor of metalloproteinase 3 and CCN2. These early abnormalities result in severe defects in multiple joints, plus markedly deformed and low-density long bones leading to dwarfism and impaired mobility. Our in vitro exploration shows unique regulation of non-canonical WNT components by LRP1 that may explain the malformation of long bones. Mechanistically, we found that LRP1 facilitates cell-association, endocytic recycling but not degradation, and graded distribution of Wnt5a in the developing limbs. We propose that LRP1-mediated endocytic regulation of availability and distribution of extracellular signalling molecules play a critical role in limb development. This provides a novel mechanism for crosstalk among skeletal elements.

Mice are often used in gain or loss of function studies to understand how genes regulate metabolism and adaptation to exercise in skeletal muscle. Once-daily resistance training with electrical nerve stimulation produces hypertrophy of the dorsiflexors in rat, but not in mouse. Using implantable pulse generators, we assessed the acute transcriptional response (1-hour post exercise) after 2, 10 and 20 days of training in free living mice and rats using identical nerve stimulation paradigms. RNA-sequencing revealed strong concordance in the timecourse of many transcriptional responses in the tibialis anterior muscles of both species including responses related to ‘stress responses/immediate-early genes’, and ‘collagen homeostasis’, ‘ribosomal subunits’, ‘autophagy’ and ‘focal adhesion’. However, pathways associated with energy metabolism including ‘carbon metabolism’, ‘oxidative phosphorylation’, ‘mitochondrial translation’, ‘propanoate metabolism’ and ‘valine, leucine and isoleucine degradation’ were oppositely regulated between species. These pathways were suppressed in the rat but upregulated in the mouse. Our transcriptional analysis suggests that although many pathways associated with growth show remarkable similarities between species, absence of an actual growth response in the mouse may be because the mouse prioritises energy metabolism, specifically replenishment of fuel stores and intermediate metabolites.

We present the time course of change in the muscle transcriptome 1 h after the last exercise bout of a daily resistance training program lasting 2, 10, 20, or 30 days. Daily exercise in rat tibialis anterior muscles (5 sets of 10 repetitions over 20 min) induced progressive muscle growth that approached a new stable state after 30 days. The acute transcriptional response changed along with progressive adaptation of the muscle phenotype. For example, expression of type 2B myosin was silenced. Time courses recently synthesized from human exercise studies do not demonstrate so clearly the interplay between the acute exercise response and the longer-term consequences of repeated exercise. We highlight classes of transcripts and transcription factors whose expression increases during the growth phase and declines again as the muscle adapts to a new daily pattern of activity and reduces its rate of growth. Myc appears to play a central role.

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.

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: 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 nitisinone—4 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.

Muscle adaptations to exercise are underpinned by alterations to the abundance of individual proteins, which may occur through a change either to the synthesis or degradation of each protein. We used deuterium oxide ((H2O)-H-2) labeling and chronic low-frequency stimulation (CLFS)in vivo to investigate the synthesis, abundance, and degradation of individual proteins during exercise-induced muscle adaptation. Independent groups of rats received CLFS (10 Hz, 24 h/d) and(2)H(2)O for 0, 10, 20, or 30 days. The extensor digitorum longus (EDL) was isolated from stimulated (Stim) and contralateral non-stimulated (Ctrl) legs. Proteomic analysis encompassed 38 myofibrillar and 46 soluble proteins and the rates of change in abundance, synthesis, and degradation were reported in absolute (ng/d) units. Overall, synthesis and degradation made equal contributions to the adaptation of the proteome, including instances where a decrease in protein-specific degradation primarily accounted for the increase in abundance of the protein.

Background and Purpose alkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of the enzyme homogentisate 1,2-dioxygenase (HGD). The primary biochemical consequence of HGD-deficiency is increased circulating homogentisic acid (HGA), which is central to AKU disease pathology. The aim of this study was to investigate the wider metabolic consequences of targeted Hgd disruption. Experimental Approach the first metabolomic analysis of the Hgd −/− AKU mouse model was performed. Urinary metabolites altered in Hgd −/− were further validated by showing that the HGA-lowering drug nitisinone reversed their direction of alteration in AKU Key Results comparison of Hgd −/− (AKU) versus Hgd +/− (heterozygous control) urine revealed increases in HGA and a group of 8 previously unreported HGA-derived transformation products from phase I and II metabolism. HGA biotransformation products HGA-sulfate, HGA-glucuronide, HGA-hydrate and hydroxymethyl-HGA were also decreased in urine from both mice and patients with AKU on the HGA-lowering agent nitisinone. Hgd knockout also revealed a host of previously unrecognised associations between tyrosine, purine and TCA cycle metabolic pathways. Conclusion and Implications AKU is rare, but our findings further what is currently understood about tyrosine metabolism more generally, and show for the first time that phase I and II detoxification is recruited to prevent accumulation of endogenously-produced metabolites in inborn errors of metabolism. The data highlight the misconception that phase I and II metabolic biotransformations are reserved solely for drug clearance; these are ancient mechanisms, which represent new potential treatment targets in inherited metabolic diseases. ![Figure][1] Bullet point summary What is already known What this study adds Clinical significance ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes