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1.
Nat Rev Dis Primers ; 10(1): 16, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38453957

RESUMO

Alkaptonuria is a rare inborn error of metabolism caused by the deficiency of homogentisate 1,2-dioxygenase activity. The consequent homogentisic acid (HGA) accumulation in body fluids and tissues leads to a multisystemic and highly debilitating disease whose main features are dark urine, ochronosis (HGA-derived pigment in collagen-rich connective tissues), and a painful and severe form of osteoarthropathy. Other clinical manifestations are extremely variable and include kidney and prostate stones, aortic stenosis, bone fractures, and tendon, ligament and/or muscle ruptures. As an autosomal recessive disorder, alkaptonuria affects men and women equally. Debilitating symptoms appear around the third decade of life, but a proper and timely diagnosis is often delayed due to their non-specific nature and a lack of knowledge among physicians. In later stages, patients' quality of life might be seriously compromised and further complicated by comorbidities. Thus, appropriate management of alkaptonuria requires a multidisciplinary approach, and periodic clinical evaluation is advised to monitor disease progression, complications and/or comorbidities, and to enable prompt intervention. Treatment options are patient-tailored and include a combination of medications, physical therapy and surgery. Current basic and clinical research focuses on improving patient management and developing innovative therapies and implementing precision medicine strategies.


Assuntos
Alcaptonúria , Ocronose , Masculino , Humanos , Feminino , Alcaptonúria/complicações , Alcaptonúria/diagnóstico , Alcaptonúria/terapia , Qualidade de Vida , Ocronose/complicações , Ocronose/diagnóstico , Rim/metabolismo , Ácido Homogentísico/metabolismo
2.
J Clin Lab Anal ; 37(21-22): e24976, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37877521

RESUMO

OBJECTIVE: Homogentisic acid (HGA) is excreted in excessive amounts in the urine of patients with alkaptonuria, which is a hereditary metabolic disorder of phenylalanine and tyrosine. Therefore, the detection of HGA in urine is useful for the diagnosis of alkaptonuria. To evaluate the detection of HGA, we confirmed the color shift of HGA solutions and analyzed them by electrospray ionization mass spectrometry (ESI-MS). METHODS: We observed the color change of the HGA solutions under different pH conditions (pH 6.0, 7.0, and 8.0) and examined the influences of adding potassium hydroxide (KOH) and ascorbic acid (AA) to the HGA solutions. Then, we analyzed the chemical reaction in HGA solutions using ESI-MS. RESULTS: The HGA solution at pH 8.0 became brown after incubation at room temperature for 24 h and became darker brown with the addition of KOH; however, HGA solutions at pH 6.0 and 7.0 showed no color changes. The brown color change of the HGA solution at pH 8.0 was also inhibited by AA. Moreover, all HGA sample solutions showed the deprotonated molecular ion peak at m/z 167.035 in the negative ion mode after incubation at room temperature for 24 h and with the addition of KOH and AA. CONCLUSION: We identified the molecular ion of HGA in all sample solutions by ESI-MS, regardless of different pH conditions, color changes, or the presence of AA. These results suggest that spectral analysis by ESI-MS is suitable for the detection of HGA and the diagnosis of alkaptonuria.


Assuntos
Alcaptonúria , Humanos , Alcaptonúria/diagnóstico , Alcaptonúria/urina , Espectrometria de Massas por Ionização por Electrospray , Ácido Homogentísico/urina , Hidróxidos , Ácido Ascórbico
3.
BMJ Case Rep ; 16(10)2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37880174

RESUMO

Alkaptonuria is a very rare disorder in which homogentisic acid accumulates due to a deficiency in the activity of homogentisic acid 1,2 dioxygenase. This deficiency results in deposition of a yellowish-brown pigment in connective tissue. Such deposition is termed 'ochronosis' and leads to deterioration in the formation and structure of proteoglycans in hyaline cartilage. These actions lead to fragmentation and rapid destructive arthritis. Often, ochronotic arthritis appears at 40-60 years of age, and many patients are treated symptomatically. Here, we report two patients (three ankles) with ochronotic arthritis who were treated with ankle arthrodesis. In all cases, the postoperative clinical score improved, but the time needed for fusion was prolonged and symptomatic subtalar arthropathy developed in the early postoperative period.


Assuntos
Alcaptonúria , Doenças das Cartilagens , Osteoartrite , Humanos , Alcaptonúria/complicações , Alcaptonúria/cirurgia , Tornozelo , Ácido Homogentísico , Artrodese
4.
Sci Rep ; 13(1): 14374, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37658095

RESUMO

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by pathogenic variants in the homogentisate 1,2-dioxygenase (HGD) gene. This leads to a deficient HGD enzyme with the consequent accumulation of homogentisic acid (HGA) in different tissues causing complications in various organs, particularly in joints, heart valves and kidneys. The genetic basis of AKU in Egypt is completely unknown. We evaluated the clinical and genetic spectrum of six pediatric and adolescents AKU patients from four unrelated Egyptian families. All probands had a high level of HGA in urine by qualitative GC/MS before genetic confirmation by Sanger sequencing. Recruited AKU patients were four females and two males (median age 13 years). We identified four different pathogenic missense variants within HGD gene. Detected variants included a novel variant c.1079G > T;p.(Gly360Val) and three recurrent variants; c.1078G > C;p.(Gly360Arg), c.808G > A;p.(Gly270Arg) and c.473C > T;p.(Pro158Leu). All identified variants were properly segregating in the four families consistent with autosomal recessive inheritance. In this study, we reported the phenotypic and genotypic spectrum of alkaptonuria for the first time in Egypt. We further enriched the HGD-variant database with another novel pathogenic variant. The recent availability of nitisinone may promote the need for genetic confirmation at younger ages to start therapy earlier and prevent serious complications.


Assuntos
Alcaptonúria , Dioxigenases , Adolescente , Feminino , Masculino , Humanos , Criança , Alcaptonúria/genética , Egito , Homogentisato 1,2-Dioxigenase/genética , Fenilacetatos , Ácido Homogentísico
5.
Cells ; 12(13)2023 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-37443717

RESUMO

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.


Assuntos
Alcaptonúria , Humanos , Alcaptonúria/tratamento farmacológico , Alcaptonúria/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas , Ácido Homogentísico/metabolismo , Estudos Prospectivos
6.
Adv Clin Chem ; 114: 47-81, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37268334

RESUMO

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.


Assuntos
Alcaptonúria , Tirosinemias , Humanos , Animais , Camundongos , Alcaptonúria/diagnóstico , Alcaptonúria/tratamento farmacológico , Alcaptonúria/metabolismo , Qualidade de Vida , Ácido Homogentísico/metabolismo , Tirosina/metabolismo , Tirosina/urina
7.
Mol Genet Metab ; 139(3): 107628, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37354891

RESUMO

A 6-yr-old female orangutan presented with a history of dark urine that turned brown upon standing since birth. Repeated routine urinalysis and urine culture were unremarkable. Urine organic acid analysis showed elevation in homogentisic acid consistent with alkaptonuria. Sequence analysis identified a homozygous missense variant, c.1081G>A (p.Gly361Arg), of the homogentisate 1,2-dioxygenase (HGD) gene. Familial studies, molecular modeling, and comparison to human variant databases support this variant as the underlying cause of alkaptonuria in this orangutan. This is the first report of molecular confirmation of alkaptonuria in a nonhuman primate.


Assuntos
Alcaptonúria , Pongo abelii , Animais , Humanos , Feminino , Alcaptonúria/diagnóstico , Alcaptonúria/genética , Pongo abelii/genética , Ácido Homogentísico , Mutação de Sentido Incorreto , Homozigoto
8.
Molecules ; 28(6)2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36985595

RESUMO

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.


Assuntos
4-Hidroxifenilpiruvato Dioxigenase , Alcaptonúria , Ocronose , Humanos , Alcaptonúria/tratamento farmacológico , Alcaptonúria/genética , Alcaptonúria/metabolismo , Simulação de Acoplamento Molecular , Ocronose/tratamento farmacológico , Ácido Homogentísico/metabolismo
9.
Endocr Regul ; 57(1): 61-67, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36966367

RESUMO

Alkaptonuria (AKU, OMIM, No. 203500) is a rare, slow-progressing, irreversible, multisystemic disease resulting from a deficiency of the homogentisate 1,2-dioxygenase enzyme, which leads to the accumulation of homogentisic acid (HGA) and subsequent deposition as pigment in connective tissues called ochronosis. As a result, severe arthropathy of large joints and spondyloarthropathy with frequent fractures, ligament ruptures, and osteoporosis develops in AKU patients. Since 2020, the first-time treatment with nitisinone has become available in the European Union. Nitisinone significantly reduces HGA production and arrests ochronosis in AKU patients. However, blocking of the tyrosine metabolic pathway by the drug leads to tyrosine plasma and tissue concentrations increase. The nitisinone-induced hypertyrosinemia can lead to the development of corneal keratopathy, and once it develops, the treatment needs to be interrupted. A decrease in overall protein intake reduces the risk of the keratopathy during nitisinone-induced hypertyrosinemia in AKU patients. The low-protein diet is not only poorly tolerated by patients, but over longer periods, leads to a severe muscle loss and weight gain due to increased energy intake from carbohydrates and fats. Therefore, the development of novel nutritional approaches is required to prevent the adverse events due to nitisinone-induced hypertyrosinemia and the negative impact on skeletal muscle metabolism in AKU patients.


Assuntos
Alcaptonúria , Ocronose , Tirosinemias , Humanos , Alcaptonúria/tratamento farmacológico , Alcaptonúria/metabolismo , Ocronose/tratamento farmacológico , Tirosina/uso terapêutico , Ácido Homogentísico/metabolismo
10.
Curr Protein Pept Sci ; 24(5): 380-392, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36880186

RESUMO

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs, which occurs because the homogentisate 1,2-dioxygenase (HGD) enzyme is not functional due to gene variants. Over time, HGA oxidation and accumulation cause the formation of the ochronotic pigment, a deposit that provokes tissue degeneration and organ malfunction. Here, we report a comprehensive review of the variants so far reported, the structural studies on the molecular consequences of protein stability and interaction, and molecular simulations for pharmacological chaperones as protein rescuers. Moreover, evidence accumulated so far in alkaptonuria research will be re-proposed as the bases for a precision medicine approach in a rare disease.


Assuntos
Alcaptonúria , Homogentisato 1,2-Dioxigenase , Humanos , Alcaptonúria/genética , Alcaptonúria/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Estudos de Associação Genética , Homogentisato 1,2-Dioxigenase/genética , Homogentisato 1,2-Dioxigenase/metabolismo , Ácido Homogentísico/metabolismo , Doenças Raras , Relação Estrutura-Atividade
11.
Gastroenterology ; 164(7): 1165-1179.e13, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36813208

RESUMO

BACKGROUND & AIMS: Aberrant epigenetic events mediated by histone methyltransferases and demethylases contribute to malignant progression of colorectal cancer (CRC). However, the role of the histone demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in CRC remains poorly understood. METHODS: UTX conditional knockout mice and UTX-silenced MC38 cells were used to investigate UTX function in tumorigenesis and development of CRC. We performed time of flight mass cytometry to clarify the functional role of UTX in remodeling immune microenvironment of CRC. To investigate metabolic interaction between myeloid-derived suppressor cells (MDSCs) and CRC, we analyzed metabolomics data to identify metabolites secreted by UTX-deficient cancer cells and taken up by MDSCs. RESULTS: We unraveled a tyrosine-mediated metabolic symbiosis between MDSC and UTX-deficient CRC. Loss of UTX in CRC resulted in methylation of phenylalanine hydroxylase, preventing its degradation and subsequently increasing tyrosine synthesis and secretion. Tyrosine taken up by MDSCs was metabolized to homogentisic acid by hydroxyphenylpyruvate dioxygenase. Homogentisic acid modified protein inhibitor of activated STAT3 via carbonylation of Cys 176, and relieved the inhibitory effect of protein inhibitor of activated STAT3 on signal transducer and activator of transcription 5 transcriptional activity. This in turn, promoted MDSC survival and accumulation, enabling CRC cells to acquire invasive and metastatic traits. CONCLUSIONS: Collectively, these findings highlight hydroxyphenylpyruvate dioxygenase as a metabolic checkpoint to restrict immunosuppressive MDSCs and to counteract malignant progression of UTX-deficient CRC.


Assuntos
Neoplasias Colorretais , Dioxigenases , Animais , Camundongos , Dioxigenases/metabolismo , Ácido Homogentísico , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Metilação , Microambiente Tumoral
12.
Am J Med Sci ; 365(4): 368-374, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36608845

RESUMO

BACKGROUND: The aim of the current study was to determine whether there is an association between alkaptonuria (AKU) and urinary tract infection (UTI) by exploring the bacterial quality of the urinary tract, as most of the patients with AKU present with frequent occurrence of urinary tract symptoms such as incomplete emptying of urinary bladder, dysuria and nocturia. METHODS: Study samples were collected from 22 participants; 9 from patients with AKU, 9 from individuals who were AKU carriers, and 4 people served as control. Confirmation of AKU diagnosis was established by the ferric chloride test and quantitative determination of urinary homogentisic acid (HGA) levels. RESULTS: In the ferric chloride test, the urine samples of AKU patients showed a characteristic black ring upon addition of few drops of ferric chloride solution. During urinary HGA determination, patients with AKU had increased levels of urinary HGA as compared to carriers and controls. The following 10 bacterial species were isolated from the urinary tract of AKU patients, carriers and controls: Sphingomonas paucimobilis, Escherichia coli, Francisella tularensis, Staphylococcus hominis, Staphylococcus haemolyticus, Leuconostoc mesenteroides, Dermacoccus nishinomiyaensis, Kytococcus sedentarius, Serratia fonticola and Granulicatella adiacens. The presence of S. paucimobilis was found in three male patients, and one female each from the carrier and control groups. Almost all study samples were positive for D. nishinomiyaensis and K. sedentarius. S. fonticola and G. adiacens were found only in AKU carrier females. CONCLUSIONS: The results deduced that males show symptoms of arthritis early and more severely than females and by this it appears that there is an association between these symptoms and the percentage of bacterial infection in males that requires more accurate diagnosis and treatment to clarify such relationship. In the current study, males (patients, carriers, and controls) were more likely to have bacterial infections than females (64% vs. 36%). The 16 and 2 bacterial isolates, detected in 7 males and 2 females AKU patients, respectively, revealed that male AKU patients had a 2.3-fold greater rate of bacterial infection than female AKU patients. Therefore, further studies are warranted to investigate if there's any relationship between higher incidence of bacterial infections and development of AKU-related clinical symptoms in the male population.


Assuntos
Alcaptonúria , Artrite , Sistema Urinário , Humanos , Masculino , Feminino , Alcaptonúria/tratamento farmacológico , Alcaptonúria/urina , Ácido Homogentísico/urina
13.
Orphanet J Rare Dis ; 18(1): 1, 2023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-36600285

RESUMO

Alkaptonuria is a rare inherited disorder for which there was no disease-modifying treatment. In order to develop a successful approved therapy of AKU multiple barriers had to be overcome. These included activities before the conduct of the study including deciding on the drug therapy, the dose of the drug to be used, clarify the nature of the disease, develop outcome measures likely to yield a positive outcome, have a strategy to ensure appropriate patient participation through identification, build a consortium of investigators, obtain regulatory approval for proposed investigation plan and secure funding. Significant barriers were overcome during the conduct of the multicentre study to ensure harmonisation. Mechanisms were put in place to recruit and retain patients in the study. Barriers to patient access following completion of the study and regulatory approval were resolved.


Assuntos
Alcaptonúria , Humanos , Alcaptonúria/tratamento farmacológico , Doenças Raras/tratamento farmacológico , Cicloexanonas/uso terapêutico , Nitrobenzoatos/uso terapêutico , Ácido Homogentísico , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Multicêntricos como Assunto
14.
ACS Chem Biol ; 18(4): 711-723, 2023 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-36215670

RESUMO

Opportunistic infections by Burkholderia cenocepacia are life threatening for patients suffering from cystic fibrosis and chronic granulomatous disease. These infections are often associated with variable clinical outcomes, prompting an interest in molecular investigations of phenotypes associated with disease severity. The production of the pyomelanin pigment is one such phenotype, which was recently linked to the ability of clinical strains to carry out biotransformation of the antibiotic trimethoprim. However, this biotransformation product was not identified, and differences in metabolite production associated with pyomelanin pigmentation are poorly understood. Here, we identify several key metabolites produced exclusively by the pyomelanin-producing strains. To provide insight into the structures and biosynthetic origin of these metabolites, we developed a mass spectrometry-based strategy coupling unsupervised in silico substructure prediction with stable isotope labeling referred to as MAS-SILAC (Metabolite Annotation assisted by Substructure discovery and Stable Isotope Labeling by Amino acids in Cell culture). This approach led to discovery of homogentisic acid as a precursor for biosynthesis of several natural products and for biotransformation of trimethoprim, representing a previously unknown mechanism of antibiotic tolerance. This work presents application of computational methods for analysis of untargeted metabolomic data to link the chemotype of pathogenic microorganisms with a specific phenotype. The observations made in this study provide insights into the clinical significance of the melanated phenotype.


Assuntos
Produtos Biológicos , Trimetoprima , Antibacterianos , Produtos Biológicos/metabolismo , Ácido Homogentísico/metabolismo , Metabolômica , Trimetoprima/química , Trimetoprima/metabolismo
15.
Int J Mol Sci ; 23(24)2022 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-36555443

RESUMO

Alkaptonuria (AKU) is an ultra-rare metabolic disease caused by the accumulation of homogentisic acid (HGA), an intermediate product of phenylalanine and tyrosine degradation. AKU patients carry variants within the gene coding for homogentisate-1,2-dioxygenase (HGD), which are responsible for reducing the enzyme catalytic activity and the consequent accumulation of HGA and formation of a dark pigment called the ochronotic pigment. In individuals with alkaptonuria, ochronotic pigmentation of connective tissues occurs, leading to inflammation, degeneration, and eventually osteoarthritis. The molecular mechanisms underlying the multisystemic development of the disease severity are still not fully understood and are mostly limited to the metabolic pathway segment involving HGA. In this view, untargeted metabolomics of biofluids in metabolic diseases allows the direct investigation of molecular species involved in pathways alterations and their interplay. Here, we present the untargeted metabolomics study of AKU through the nuclear magnetic resonance of urine from a cohort of Italian patients; the study aims to unravel molecular species and mechanisms underlying the AKU metabolic disorder. Dysregulation of metabolic pathways other than the HGD route and new potential biomarkers beyond homogentisate are suggested, contributing to a more comprehensive molecular signature definition for AKU and the development of future adjuvant treatment.


Assuntos
Alcaptonúria , Dioxigenases , Humanos , Alcaptonúria/genética , Metabolômica , Ácido Homogentísico/metabolismo , Biomarcadores , Espectroscopia de Ressonância Magnética
16.
Sci Rep ; 12(1): 19452, 2022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376482

RESUMO

Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis, which results on the long term in an early-onset and severe osteoarthropathy. HGD's quaternary structure is known to be easily disrupted by missense mutations, which makes them an interesting target for novel treatment strategies that aim to rescue enzyme activity. However, only prediction models are available providing information on a structural basis. Therefore, an E. coli based whole-cell screening was developed to evaluate HGD missense variants in 96-well microtiter plates. The screening principle is based on HGD's ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity could be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Optimized screening conditions comprised several E. coli expression strains, varied expression temperatures and varied substrate concentrations. In addition, plate uniformity, signal variability and spatial uniformity were investigated and optimized. Finally, eight HGD missense variants were generated via site-directed mutagenesis and evaluated with the developed high-throughput screening (HTS) assay. For the HTS assay, quality parameters passed the minimum acceptance criterion for Z' values > 0.4 and single window values > 2. We found that activity percentages versus wildtype HGD were 70.37 ± 3.08% (for M368V), 68.78 ± 6.40% (for E42A), 58.15 ± 1.16% (for A122V), 69.07 ± 2.26% (for Y62C), 35.26 ± 1.90% (for G161R), 35.86 ± 1.14% (for P230S), 23.43 ± 4.63% (for G115R) and 19.57 ± 11.00% (for G361R). To conclude, a robust, simple, and cost-effective HTS system was developed to reliably evaluate and distinguish human HGD missense variants by their HGA consumption ability. This HGA quantification assay may lay the foundation for the development of novel treatment strategies for missense variants in AKU.


Assuntos
Alcaptonúria , Dioxigenases , Humanos , Alcaptonúria/genética , Homogentisato 1,2-Dioxigenase/genética , Dioxigenases/genética , Polimorfismo de Nucleotídeo Único , Ensaios de Triagem em Larga Escala , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Homogentísico
17.
J Med Case Rep ; 16(1): 351, 2022 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-36183119

RESUMO

BACKGROUND: We present this report of a new ophthalmic finding in a patient with ochronosis. CASE PRESENTATION: An 85-year-old Caucasian male patient with bilateral dark temporal and nasal pigmentation of conjunctiva and sclera was referred to our hospital owing to low visual acuity. On biomicroscopic examination, bilateral horizontal Descemet's membrane folds were observed. Corneal tomography revealed irregular and asymmetric "against-the-rule" astigmatism in both eyes. Anterior segment optical coherence tomography demonstrated numerous central Descemet's without edema or other corneal structure alterations. CONCLUSION: This is the first report of Descemet's membrane folds in ochronosis. These corneal findings suggest that the accumulation of homogentisic acid in the sclera leads to thickening and stiffness of this region. These alterations could remarkably decrease visual acuity owing to topographic corneal curvature alterations, especially in elderly patients.


Assuntos
Lâmina Limitante Posterior , Ocronose , Idoso , Idoso de 80 Anos ou mais , Córnea/diagnóstico por imagem , Lâmina Limitante Posterior/diagnóstico por imagem , Ácido Homogentísico , Humanos , Masculino , Ocronose/complicações , Ocronose/diagnóstico , Acuidade Visual
18.
Sci Rep ; 12(1): 16083, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36167967

RESUMO

Nitisinone (NIT) produces inevitable but varying degree of tyrosinaemia. However, the understanding of the dynamic adaptive relationships within the tyrosine catabolic pathway has not been investigated fully. The objective of the study was to assess the contribution of protein intake, serum NIT (sNIT) and tyrosine pathway metabolites to nitisinone-induced tyrosinaemia in alkaptonuria (AKU). Samples of serum and 24-h urine collected during SONIA 2 (Suitability Of Nitisinone In Alkaptonuria 2) at months 3 (V2), 12 (V3), 24 (V4), 36 (V5) and 48 (V6) were included in these analyses. Homogentisic acid (HGA), tyrosine (TYR), phenylalanine (PHE), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and sNIT were analysed at all time-points in serum and urine. Total body water (TBW) metabolites were derived using 60% body weight. 24-h urine and TBW metabolites were summed to obtain combined values. All statistical analyses were post-hoc. 307 serum and 24-h urine sampling points were analysed. Serum TYR from V2 to V6, ranging from 478 to 1983 µmol/L were stratified (number of sampling points in brackets) into groups < 701 (47), 701-900 (105), 901-1100 (96) and > 1100 (59) µmol/L. The majority of sampling points had values greater than 900 µmol/L. sPHE increased with increasing sTYR (p < 0.001). Tyrosine, HPPA and HPLA in serum and TBW all increased with rising sTYR (p < 0.001), while HPLA/TYR ratio decreased (p < 0.0001). During NIT therapy, adaptive response to minimise TYR formation was demonstrated. Decreased conversion of HPPA to HPLA, relative to TYR, seems to be most influential in determining the degree of tyrosinaemia.


Assuntos
Alcaptonúria , Encefalopatias Metabólicas Congênitas , Tirosinemias , Alcaptonúria/tratamento farmacológico , Cicloexanonas/uso terapêutico , Ácido Homogentísico , Humanos , Nitrobenzoatos/uso terapêutico , Fenilalanina , Fenilpropionatos , Tirosina/metabolismo , Tirosinemias/tratamento farmacológico
19.
Appl Environ Microbiol ; 88(18): e0128922, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36073941

RESUMO

Shewanella oneidensis is the best understood model microorganism for the study of diverse cytochromes (cytos) c that support its unparallel respiratory versatility. Although RNA chaperone Hfq has been implicated in regulation of cyto c production, little is known about the biological pathways that it affects in this bacterium. In this study, from a spontaneous mutant that secretes pyomelanin and has a lowered cyto c content, we identified Hfq to be the regulator that critically associates with both phenotypes in S. oneidensis. We found that expression of the key genes in biosynthesis and degradation of heme is differentially affected by Hfq at under- and overproduced levels, and through modulating heme levels, Hfq influences the cyto c content. Although Hfq in excess results in overproduction of the enzymes responsible for both generation and removal of homogentisic acid (HGA), the precursor of pyomelanin, it is compromised activity of HmgA that leads to excretion and polymerization of HGA to form pyomelanin. We further show that Hfq mediates HmgA activity by lowering intracellular iron content because HmgA is an iron-dependent enzyme. Overall, our work highlights the significance of Hfq-mediated posttranscriptional regulation in the physiology of S. oneidensis, unraveling unexpected mechanisms by which Hfq affects cyto c biosynthesis and pyomelanin production. IMPORTANCE In bacteria, Hfq has been implicated in regulation of diverse biological processes posttranslationally. In S. oneidensis, Hfq affects the content of cytos c that serve as the basis of its respiratory versatility and potential application in bioenergy and bioremediation. In this study, we found that Hfq differentially regulates heme biosynthesis and degradation, leading to altered cyto c contents. Hfq in excess causes a synthetic effect on HmgA, an enzyme responsible for pyomelanin formation. Overall, the data presented manifest that the biological processes in a given bacterium regulated by Hfq are highly complex, amounting to required coordination among multiple physiological aspects to allow cells to respond to environmental changes promptly.


Assuntos
Proteínas HMGA , Shewanella , Citocromos c/metabolismo , Proteínas HMGA/metabolismo , Heme/metabolismo , Ácido Homogentísico/metabolismo , Ferro/metabolismo , Melaninas , RNA/metabolismo , Shewanella/metabolismo
20.
Rheumatol Int ; 42(12): 2277-2282, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36053307

RESUMO

Alkaptonuria is a disease often forgotten because of its rarity. Its pathogenic mechanism is the deficiency of one of the enzymes of the tyrosine degradation pathway-homogentisate-1, 2-dioxygenase, which sequelae is accumulation and deposition of its metabolite homogentisic acid in connective tissues and urine. Alkaptonuria presents as a clinical triad-darkening urine upon prolonged exposure to air, pigmentation of connective tissues and debilitating arthropathy. We present a case report of a 67-year old patient with alkaptonuria who presented with the clinical triad, but was mistakenly diagnosed as having ankylosing spondylitis in the past. Currently there is no treatment for the disease hence the management strategy was focused on symptoms control with analgesics, physical therapy, dietary modification, vitamin C supplementation, and joint arthroplasty. Alkaptonuria's clinical features are extensively described in the literature and despite the fact that it is a rare disease, due to the similar radiographic changes with spondyloarthropathies, it should be included in the differential diagnosis in young patients presenting with severe joint involvement. Early recognition of the disease is necessary since its natural evolution is joint destruction leading to significant reduction in the quality of life. Alkaptonuria's articular features in the spine and peripheral tissues are well described using the classical imaging techniques. Musculoskeletal ultrasonography shows a characteristic set of findings in the soft tissues, including synovium, cartilage, tendons and entheses.


Assuntos
Alcaptonúria , Doenças das Cartilagens , Dioxigenases , Artropatias , Ocronose , Osteoartrite , Espondiloartropatias , Idoso , Alcaptonúria/complicações , Alcaptonúria/diagnóstico , Alcaptonúria/metabolismo , Ácido Ascórbico , Ácido Homogentísico/metabolismo , Humanos , Ocronose/complicações , Ocronose/diagnóstico , Osteoartrite/complicações , Qualidade de Vida , Espondiloartropatias/complicações , Tirosina
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