Alkaptonuria and Ochronosis – Experience From Slovakia

Alkaptonuria is a rare autosomal recessive condition resulting from inability to breakdown homogentisic acid (HGA), an intermediate in tyrosine degradation. The condition has a triad of clinical features, the most damaging of which is ochronotic osteoarthropathy. HGA is elevated from birth, but pigmentation takes many years. We hypothesise that interleukins play a role in initiation and progression of ochronotic osteoarthropathy. C20/A4 cells were cultured and maintained in 9-cm petri dishes containing either HGA at 0.33mM, a single interleukin (IL-1β, IL-6 or IL-10) at 1ng/ml or a combination of HGA and a single interleukin. Statistical analysis of pigment deposits and cell viability was performed using analysis of variance with Newman-Keuls post-test. All cultures containing HGA showed a significant increase in pigment deposition compared to control and IL cultures alone. The cultures containing HGA and IL-6 showed a significant increase in pigment deposits compared to HGA alone. The cell viability counts across all cultures on day 10 demonstrated a significant decrease in cultures containing HGA compared to those which did not. There was no significant difference between cultures containing just HGA or those combined with an interleukin. This work demonstrates a role for cytokines present in the joint(s) in the pigmentation process, particularly IL-6, and that the presence of HGA in joint tissues appears more detrimental to chondrocytes than the presence of any of the interleukins found in response to joint injury, trauma and osteoarthritis (OA). This further supports the evidence that the arthropathy in alkaptonuria is much more severe and rapidly progressing.

Inherited Metabolic Disorders: Errors of Phenylalanine and Tyrosine Metabolism

Clinicians require simple quantitative tools for the detection of homogentisic acid in alkaptonuria patients, a rare inherited disorder of amino acid metabolism. In this study, we report a whole-cell biosensor for homogentisic acid to detect alkaptonuria disease through the expression of green fluorescence protein. The assay system utilizes a promoter sequence (hmgA) isolated from the Pseudomonas aeruginosa genome. To increase the sensitivity, the sensor module harboring phmgA::GFP was further transformed into various transposon mutants debilitated in steps involved in the metabolism of phenylalanine and tyrosine via homogentisic acid as a central intermediate. The proposed biosensor was further checked for analytical features such as sensitivity, selectivity, linearity, and precision for the quantification of homogentisic acid in spiked urine samples. The limit of detection for the developed biosensor was calculated to be 3.9 μM, which is comparable to that of the various analytical techniques currently in use. The sensor construct showed no interference from all of the amino acids and its homolog molecules. The accuracy and precision of the proposed biosensor were validated using high-performance liquid chromatography (HPLC) with satisfactory results.

Abnormal tyrosine and phenylalanine metabolism in patients with tyrosyluria and phenylketonuria; gas-liquid chromatographic analysis of urinary metabolites

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis

High-throughput metabolomics using UPLC/Q-TOF-MS coupled with multivariate data analysis reveals the effect and mechanism of syringin against ovariectomized osteoporosis

Disc-coloration of an ochronotic cervical intervertebral disc in a patient with alkaptonuria: Case report and review of the literature

Alkaptonuria (AKU) is a rare genetic autosomal recessive disorder characterized by elevated serum levels of homogentisic acid (HGA). In this disease, tyrosine metabolism is interrupted because of the alterations in homogentisate dioxygenase (HGD) gene. The patient suffers from ochronosis, fractures, and tendon ruptures. To date, no medicine has been approved for the treatment of AKU. However, physiotherapy and strong painkillers are administered to help mitigate the condition. Recently, nitisinone, an FDA-approved drug for type 1 tyrosinemia, has been given to AKU patients in some countries and has shown encouraging results in reducing the disease progression. However, this drug is not the targeted treatment for AKU, and causes keratopathy. Therefore, the foremost aim of this study is the identification of potent and druggable inhibitors of AKU with no or minimal side effects by targeting 4-hydroxyphenylpyruvate dioxygenase. To achieve our goal, we have performed computational modelling using BioSolveIT suit. The library of ligands for molecular docking was acquired by fragment replacement of reference molecules by ReCore. Subsequently, the hits were screened on the basis of estimated affinities, and their pharmacokinetic properties were evaluated using SwissADME. Afterward, the interactions between target and ligands were investigated using Discovery Studio. Ultimately, compounds c and f were identified as potent inhibitors of 4-hydroxyphenylpyruvate dioxygenase.

Metabolomics is an emerging and robust discipline and involves the comprehensive evaluation of small molecule endogenous metabolites and enables the exploration of the pathogenesis of diseases. For example, endometriosis – a common disease which mostly occurs in women of childbearing age. A cure for endometriosis of cold coagulation and blood stasis (ECB) is highly sought after. This study was conducted to discover the potential biomarkers of ECB and the effective mechanism undertaken by Guizhi Fuling Wan (GFW) in treating ECB in rats. Urinary metabolomics were performed by using UPLC-Q-TOF-MS with pattern recognition methods to evaluate the changes in metabolic profiles and to identify biomarkers for elucidating the mechanism of the treatment of ECB with GFW. The results showed that urinary metabolism in the two groups were distinctly separated on the 28th day, and a total of 20 differential biomarkers (16 in the positive mode, 4 in the negative mode) were confirmed involving several key metabolic pathways which included phenylalanine, tyrosine and tryptophan biosynthesis, valine, leucine and isoleucine biosynthesis, glyoxylate and dicarboxylate metabolism, tyrosine metabolism and the citrate cycle. Following the oral administration of GFW, certain pathways were affected; these included the following: phenylalanine, tyrosine and tryptophan biosynthesis, valine, leucine and isoleucine biosynthesis, glyoxylate and dicarboxylate metabolism, tyrosine metabolism, citrate cycle, steroid hormone biosynthesis, tryptophan metabolism, phenylalanine metabolism, primary bile acid biosynthesis, and aminoacyl-tRNA biosynthesis. This study also demonstrated that the administration of GFW affected the levels of urine endogenous metabolites, thereby laying a foundation for further study of the pharmacodynamical mechanism of GFW.

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly called non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Although MASLD has been widely studied in persons with Type 2 diabetes (T2D), far less in known about the pathogenesis and severity of MASLD in Type 1 diabetes (T1D). Determine metabolic perturbations associated with MASLD in persons with T1D. We conducted a cross-sectional study of 30 participants with T1D. Based on the results of a FibroScan, participants were stratified as cases (MASLD) or controls. Metabolomic analyses were performed on plasma obtained from all participants after an overnight (after midnight) fast. 17 of 30 participants were classified as cases (MASLD) and 13 as controls. Cases had higher BMI (p=<0.001) and were taking higher daily insulin doses than controls (p=0.003). Metabolomic analyses revealed that those with MASLD had elevated levels of gluconeogenic substrates pyruvate (p=0.001) and lactate (p=0.043), gluconeogenic amino acids alanine (p<0.001) and glutamate (p=0.004), phenylalanine (p=0.003), and anthranilic acid (p=0.015). Lipidomics revealed, elevated ceramides (P=0.02), diacylglycerols (p=0.0009) and triacylglycerols (P=0.0004) in MASLD group. In those with MASLD, the acylcarnitines, isovalerylcarnitine (CAR.5.0) (P=0.002) and L-Palmitoylcarnitine (CAR.16.0) (P=0.048), were elevated. Pathway analyses using MetaboAnalyst 5.0 Software revealed that, pathways including phenylalanine and tyrosine metabolism, tryptophan metabolism, glucose-alanine cycle, glutamate metabolism, and glutathione metabolism were significantly enriched in those with MASLD. Participants with T1D and MASLD manifest features of insulin resistance and metabolite perturbations suggesting enhanced gluconeogenesis, dysfunctional fat synthesis, and perturbed TCA cycle activity.

Identification of non-invasive biomarkers of disease progression in multiple sclerosis (MS) is critically needed for monitoring the disease progression and for effective therapeutic interventions. Urine is an attractive source for non-invasive biomarkers because it is easily obtained in the clinic. In search of a urine metabolite signature of progression in chronic experimental autoimmune encephalomyelitis (EAE), we profiled urine at the chronic stage of the disease (day 45 post immunization) by global untargeted metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found 105 metabolites (P < 0.05) significantly altered at the chronic stage, indicating a robust alteration in the urine metabolite profile during disease. Assessment of altered metabolites against the Kyoto Encyclopedia of Genes and Genomes revealed distinct non-overlapping metabolic pathways and revealed phenylalanine-tyrosine and associated metabolism being the most impacted. Combined with previously performed plasma profiling, eight common metabolites were significantly altered in both of the biofluids. Metaboanalyst analysis of these common metabolites revealed that phenylalanine metabolism and Valine, leucine, and isoleucine biosynthetic pathways are central metabolic pathways in both bio-fluids and could be analyzed further, either for the discovery of therapeutics or biomarker development. Overall, our study suggests that urine and plasma metabolomics may contribute to the identification of a distinct metabolic fingerprint of EAE disease discriminating from the healthy control which may aid in the development of an objective non-invasive monitoring method for progressive autoimmune diseases like MS. Graphical Abstract Untargeted urinary metabolomics of a chronic mouse model of multiple sclerosis identified Phenylalanine, tyrosine & tryptophan metabolism as the significantly altered metabolic pathway. Eight common metabolites were identified when we combined urinary and plasma metabolic signature, which revealed a perturbation of Phenylalanine metabolism and valine, leucine & isoleucine metabolic pathways, involved in CNS dysfunction during diseases. The identified eight metabolic signature of urine and plasma may be of clinical relevance as potential biomarkers and guide towards the identification of specific metabolic pathways as novel drug targets.

Alkaptonuria (AKU) is a rare autosomal recessive condition caused by deficiency of the enzyme homogentisate 1,2 dioxygenase, resulting in widespread deposition of oxidised homogentisic acid (HGA) polymer, primarily in joint...

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products in connective tissues up to the deposition of melanin-like pigments (ochronosis). Since little is known on the effects of HGA and its metabolites on articular cells, we carried out a proteomic and redox-proteomic analysis to investigate how HGA and ascorbic acid (ASC) affect the human chondrocytic protein repertoire. We settled up an in vitro model using a human chondrocytic cell line to evaluate the effects of 0.33 mM HGA, alone or combined with ASC. We found that HGA and ASC significantly affect the levels of proteins with specific functions in protein folding, cell organization and, notably, stress response and cell defense. Increased protein carbonyls levels were found either in HGA or ASC treated cells, and evidences produced in this paper support the hypothesis that HGA-induced stress might be mediated by protein oxidation. Our finding can lay the basis towards the settling up of more sophisticated models to study AKU and ochronosis.

Introduction:Alkaptonuria is an autosomal extremely rare recessive metabolic disorder with incidence reported to occur as 1:100 000–1:250 000 live births worldwide. This rare metabolic disorder is characterized by the accumulation of homogentisic acid due to a deficiency in homogentisic acid 1,2 dioxygenase. Homogentisic acid subsequently oxidizes and accumulates in the connective tissue. The knee is the most significant peripheral joint to be affected by the disorder. The authors present the first case of ochronotic arthropathy in Syria.Case presentation:A 46-year-old male presented with bilateral pain in the knees. the pain was affecting his day-to-day activities, and not responding to conservative management. Anteroposterior standing radiographs demonstrated extensive degenerative disease. Intraoperatively, the diagnosis was done after noticing that the quadriceps tendon and the articular cartilage of the femur, tibia, and patella were blackened during cemented total knee replacement of the knee.Conclusion:Ochronotic arthropathy should be kept in mind in middle age patients with severe osteoarthritis to not be surprised by the rare alkaptonuria diagnosis if arthroplasty was indicated.

Plants produce various l-tyrosine (Tyr)-derived compounds that are critical for plant adaptation and have pharmaceutical or nutritional importance for human health. Tyrosine aminotransferases (TATs) catalyze the reversible reaction between Tyr and 4-hydroxyphenylpyruvate (HPP), representing the entry point in plants for both biosynthesis of various natural products and Tyr degradation in the recycling of energy and nutrients. To better understand the roles of TATs and how Tyr is metabolized in planta, here we characterized single and double loss-of-function mutants of TAT1 (At5g53970) and TAT2 (At5g36160) in the model plant Arabidopsis thaliana As reported previously, tat1 mutants exhibited elevated and decreased levels of Tyr and tocopherols, respectively. The tat2 mutation alone had no impact on Tyr and tocopherol levels, but a tat1 tat2 double mutant had increased Tyr accumulation and decreased tocopherol levels under high-light stress compared with the tat1 mutant. Relative to WT and the tat2 mutant, the tat1 mutant displayed increased vulnerability to continuous dark treatment, associated with an early drop in respiratory activity and sucrose depletion. During isotope-labeled Tyr feeding in the dark, we observed that the tat1 mutant exhibits much slower 13C incorporation into tocopherols, fumarate, and other tricarboxylic acid (TCA) cycle intermediates than WT and the tat2 mutant. These results indicate that TAT1 and TAT2 function together in tocopherol biosynthesis, with TAT2 having a lesser role, and that TAT1 plays the major role in Tyr degradation in planta Our study also highlights the importance of Tyr degradation under carbon starvation conditions during dark-induced senescence in plants.