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This paper reports correlated product distributions for dissociation of the van der Waals complexes NO-CH4 and NO-N2 on their AÌ state surfaces, providing detailed data sets against which calculations can be benchmarked. NO-CH4 dissociation strongly favors small changes in the CH4 angular momentum, with ΔJ = 0 and 1 providing the bulk of the products. Conversely, the associated NO products show little constraint in terms of the rotational angular momentum transfer, with the full range of energetically accessible angular momentum states populated, although the distributions show minima. The lack of angular momentum transfer to methane accompanied by broad, structured, angular momentum transfer to NO gives the NO-CH4 dissociation some qualitative similarities to NO-Rg complex dissociation. In contrast, for NO-N2, the cluster of highest probability products corresponds to high N2 angular momentum and low NO angular momentum, with a sharp drop in the probability for populating the highest energetically accessible J states. For both the NO and N2 products, there appears to be a constraint limiting angular momentum transfer at the highest energetically accessible rotational states. Both complexes show product distributions that include a component attributed to excitation from warm complexes, which provides insight into their internal energies. Interestingly, for NO-N2, the 44,475 cm-1 photolysis translational energy release distribution for N = 8 extends to energies beyond those accessible from the highest bound XÌ states. This indicates either that there are long-lived (>100 µs) states above the XÌ state binding energy or that there is another mechanism that also contributes to this distribution.
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OBJECTIVE: Patients with kidney stones are counseled to eat a diet low in animal protein, sodium, and oxalate and rich in fruits and vegetables, with a modest amount of calcium, usually from dairy products. Restriction of sodium, potassium, and oxalate may also be recommended in patients with chronic kidney disease. Recently, plant-based diets have gained popularity owing to health, environmental, and animal welfare considerations. Our objective was to compare concentrations of ingredients important for kidney stones and chronic kidney disease in popular brands of milk alternatives. DESIGN AND METHODS: Sodium, calcium, and potassium contents were obtained from nutrition labels. The oxalate content was measured by ion chromatography coupled with mass spectrometry. RESULTS: The calcium content is highest in macadamia followed by soy, almond, rice, and dairy milk; it is lowest in cashew, hazelnut, and coconut milk. Almond milk has the highest oxalate concentration, followed by cashew, hazelnut, and soy. Coconut and flax milk have undetectable oxalate levels; coconut milk also has comparatively low sodium, calcium, and potassium, while flax milk has the most sodium. Overall, oat milk has the most similar parameters to dairy milk (moderate calcium, potassium and sodium with low oxalate). Rice, macadamia, and soy milk also have similar parameters to dairy milk. CONCLUSION: As consumption of plant-based dairy substitutes increases, it is important for healthcare providers and patients with renal conditions to be aware of their nutritional composition. Oat, macadamia, rice, and soy milk compare favorably in terms of kidney stone risk factors with dairy milk, whereas almond and cashew milk have more potential stone risk factors. Coconut milk may be a favorable dairy substitute for patients with chronic kidney disease based on low potassium, sodium, and oxalate. Further study is warranted to determine the effect of plant-based milk alternatives on urine chemistry.
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Cálculos Renais , Insuficiência Renal Crônica , Animais , Cálcio , Cálcio da Dieta , Dieta Hipossódica , Feminino , Humanos , Masculino , Oxalatos , Potássio , Insuficiência Renal Crônica/complicações , Fatores de Risco , SódioRESUMO
Oxalobacter formigenes, a unique anaerobic bacterium that relies solely on oxalate for growth, is a key oxalate-degrading bacterium in the mammalian intestinal tract. Degradation of oxalate in the gut by O. formigenes plays a critical role in preventing renal toxicity in animals that feed on oxalate-rich plants. The role of O. formigenes in reducing the risk of calcium oxalate kidney stone disease and oxalate nephropathy in humans is less clear, in part due to difficulties in culturing this organism and the lack of studies which have utilized diets in which the oxalate content is controlled. Herein, we review the literature on the 40th anniversary of the discovery of O. formigenes, with a focus on its biology, its role in gut oxalate metabolism and calcium oxalate kidney stone disease, and potential areas of future research. Results from ongoing clinical trials utilizing O. formigenes in healthy volunteers and in patients with primary hyperoxaluria type 1 (PH1), a rare but severe form of calcium oxalate kidney stone disease, are also discussed. Information has been consolidated on O. formigenes strains and best practices to culture this bacterium, which should serve as a good resource for researchers.
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Oxalatos/metabolismo , Oxalobacter formigenes , Animais , Microbioma Gastrointestinal , Genômica , Humanos , Inativação Metabólica , Metabolômica , Nefrolitíase , Oxalatos/urina , Oxalobacter formigenes/genética , Oxalobacter formigenes/metabolismo , Oxalobacter formigenes/fisiologiaRESUMO
PURPOSE OF REVIEW: The review of potential therapies in the treatment of hyperoxaluria is timely, given the current excitement with clinical trials and the mounting evidence of the importance of oxalate in both kidney stone and chronic kidney disease. RECENT FINDINGS: Given the significant contribution of both endogenous and dietary oxalate to urinary oxalate excretions, it is not surprising therapeutic targets are being studied in both pathways. This article covers the existing data on endogenous and dietary oxalate and the current targets in these pathways. SUMMARY: In the near future, there will likely be therapies targeting both endogenous and dietary oxalate, especially in subsets of kidney stone formers.
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Hiperoxalúria/metabolismo , Hiperoxalúria/terapia , Oxalatos/efeitos adversos , Oxalatos/metabolismo , Adulto , Animais , Dieta/efeitos adversos , Humanos , Hiperoxalúria/etiologia , Cálculos Renais/química , Cálculos Renais/etiologia , Cálculos Renais/metabolismo , Cálculos Renais/terapia , Camundongos , Ratos , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/terapiaRESUMO
Dietary oxalate is plant-derived and may be a component of vegetables, nuts, fruits, and grains. In normal individuals, approximately half of urinary oxalate is derived from the diet and half from endogenous synthesis. The amount of oxalate excreted in urine plays an important role in calcium oxalate stone formation. Large epidemiological cohort studies have demonstrated that urinary oxalate excretion is a continuous variable when indexed to stone risk. Thus, individuals with oxalate excretions >25 mg/day may benefit from a reduction of urinary oxalate output. The 24-h urine assessment may miss periods of transient surges in urinary oxalate excretion, which may promote stone growth and is a limitation of this analysis. In this review we describe the impact of dietary oxalate and its contribution to stone growth. To limit calcium oxalate stone growth, we advocate that patients maintain appropriate hydration, avoid oxalate-rich foods, and consume an adequate amount of calcium.
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Cálculos Renais/etiologia , Oxalatos , Cálcio/urina , Oxalato de Cálcio , Cálcio da Dieta/urina , Dieta , Humanos , Cálculos Renais/urinaRESUMO
Background Endogenous oxalate synthesis contributes to calcium oxalate stone disease and is markedly increased in the inherited primary hyperoxaluria (PH) disorders. The incomplete knowledge regarding oxalate synthesis complicates discovery of new treatments. Hydroxyproline (Hyp) metabolism results in the formation of oxalate and glycolate. However, the relative contribution of Hyp metabolism to endogenous oxalate and glycolate synthesis is not known.Methods To define this contribution, we performed primed, continuous, intravenous infusions of the stable isotope [15N,13C5]-Hyp in nine healthy subjects and 19 individuals with PH and quantified the levels of urinary 13C2-oxalate and 13C2-glycolate formed using ion chromatography coupled to mass detection.Results The total urinary oxalate-to-creatinine ratio during the infusion was 73.1, 70.8, 47.0, and 10.6 mg oxalate/g creatinine in subjects with PH1, PH2, and PH3 and controls, respectively. Hyp metabolism accounted for 12.8, 32.9, and 14.8 mg oxalate/g creatinine in subjects with PH1, PH2, and PH3, respectively, compared with 1.6 mg oxalate/g creatinine in controls. The contribution of Hyp to urinary oxalate was 15% in controls and 18%, 47%, and 33% in subjects with PH1, PH2, and PH3, respectively. The contribution of Hyp to urinary glycolate was 57% in controls, 30% in subjects with PH1, and <13% in subjects with PH2 or PH3.Conclusions Hyp metabolism differs among PH types and is a major source of oxalate synthesis in individuals with PH2 and PH3. In patients with PH1, who have the highest urinary excretion of oxalate, the major sources of oxalate remain to be identified.
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Glicolatos/urina , Hidroxiprolina/metabolismo , Hiperoxalúria Primária/metabolismo , Ácido Oxálico/urina , Adulto , Creatinina/urina , Feminino , Humanos , Hiperoxalúria Primária/urina , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND AND AIMS: Hyperoxaluria after Roux-en-Y gastric bypass (RYGB) is generally attributed to fat malabsorption. If hyperoxaluria is indeed caused by fat malabsorption, magnitudes of hyperoxaluria and steatorrhea should correlate. Severely obese patients, prior to bypass, ingest excess dietary fat that can produce hyperphagic steatorrhea. The primary objective of the study was to determine whether urine oxalate excretion correlates with elements of fat balance in severely obese patients before and after RYGB. METHODS: Fat balance and urine oxalate excretion were measured simultaneously in 26 severely obese patients before and 1 year after RYGB, while patients consumed their usual diet. At these time points, stool and urine samples were collected. Steatorrhea and hyperoxaluria were defined as fecal fat >7 g/day and urine oxalate >40 mg/day. Differences were evaluated using paired 2-tailed t tests. RESULTS: Prior to RYGB, 12 of 26 patients had mild to moderate steatorrhea. Average urine oxalate excretion was 61 mg/day; there was no correlation between fecal fat and urine oxalate excretion. After RYGB, 24 of 26 patients had steatorrhea and urine oxalate excretion averaged 69 mg/day, with a positive correlation between fecal fat and urine oxalate excretions (r = 0.71, P < .001). For each 10 g/day increase in fecal fat output, fecal water excretion increased only 46 mL/day. CONCLUSIONS: Steatorrhea and hyperoxaluria were common in obese patients before bypass, but hyperoxaluria was not caused by excess unabsorbed fatty acids. Hyperphagia, obesity, or metabolic syndrome could have produced this previously unrecognized hyperoxaluric state by stimulating absorption or endogenous synthesis of oxalate. Hyperoxaluria after RYGB correlated with steatorrhea and was presumably caused by excess fatty acids in the intestinal lumen. Because post-bypass steatorrhea caused little increase in fecal water excretion, most patients with steatorrhea did not consider themselves to have diarrhea. Before and after RYGB, high oxalate intake contributed to the severity of hyperoxaluria.
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Gorduras na Dieta/metabolismo , Derivação Gástrica , Hiperoxalúria/metabolismo , Hiperfagia/metabolismo , Obesidade/metabolismo , Esteatorreia/metabolismo , Adulto , Idoso , Fezes/química , Feminino , Humanos , Hiperoxalúria/epidemiologia , Masculino , Pessoa de Meia-Idade , Obesidade/epidemiologia , Obesidade/cirurgia , Oxalatos/urina , Índice de Gravidade de Doença , Esteatorreia/epidemiologiaRESUMO
Primary hyperoxaluria type 1 (PH1), an inherited rare disease of glyoxylate metabolism, arises from mutations in the enzyme alanine-glyoxylate aminotransferase. The resulting deficiency in this enzyme leads to abnormally high oxalate production resulting in calcium oxalate crystal formation and deposition in the kidney and many other tissues, with systemic oxalosis and ESRD being a common outcome. Although a small subset of patients manages the disease with vitamin B6 treatments, the only effective treatment for most is a combined liver-kidney transplant, which requires life-long immune suppression and carries significant mortality risk. In this report, we discuss the development of ALN-GO1, an investigational RNA interference (RNAi) therapeutic targeting glycolate oxidase, to deplete the substrate for oxalate synthesis. Subcutaneous administration of ALN-GO1 resulted in potent, dose-dependent, and durable silencing of the mRNA encoding glycolate oxidase and increased serum glycolate concentrations in wild-type mice, rats, and nonhuman primates. ALN-GO1 also increased urinary glycolate concentrations in normal nonhuman primates and in a genetic mouse model of PH1. Notably, ALN-GO1 reduced urinary oxalate concentration up to 50% after a single dose in the genetic mouse model of PH1, and up to 98% after multiple doses in a rat model of hyperoxaluria. These data demonstrate the ability of ALN-GO1 to reduce oxalate production in preclinical models of PH1 across multiple species and provide a clear rationale for clinical trials with this compound.
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Oxirredutases do Álcool , Hiperoxalúria Primária/enzimologia , Hiperoxalúria Primária/terapia , Oxalatos/metabolismo , Terapêutica com RNAi , Oxirredutases do Álcool/genética , Animais , Modelos Animais de Doenças , Inativação Gênica , Fígado/enzimologia , Masculino , Camundongos , Primatas , RNA Mensageiro , RatosRESUMO
The hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) is caused by a functional deficiency of the liver-specific, peroxisomal, pyridoxal-phosphate-dependent enzyme, alanine:glyoxylate aminotransferase (AGT). One third of PH1 patients, particularly those expressing the p.[(Pro11Leu; Gly170Arg; Ile340Met)] mutant allele, respond clinically to pharmacological doses of pyridoxine. To gain further insight into the metabolic effects of AGT dysfunction in PH1 and the effect of pyridoxine, we established an "indirect" glycolate cytotoxicity assay using CHO cells expressing glycolate oxidase (GO) and various normal and mutant forms of AGT. In cells expressing GO the great majority of glycolate was converted to oxalate and glyoxylate, with the latter causing the greater decrease in cell survival. Co-expression of normal AGTs and some, but not all, mutant AGT variants partially counteracted this cytotoxicity and led to decreased synthesis of oxalate and glyoxylate. Increasing the extracellular pyridoxine up to 0.3µM led to an increased metabolic effectiveness of normal AGTs and the AGT-Gly170Arg variant. The increased survival seen with AGT-Gly170Arg was paralleled by a 40% decrease in oxalate and glyoxylate levels. These data support the suggestion that the effectiveness of pharmacological doses of pyridoxine results from an improved metabolic effectiveness of AGT; that is the increased rate of transamination of glyoxylate to glycine. The indirect glycolate toxicity assay used in the present study has potential to be used in cell-based drug screening protocols to identify chemotherapeutics that might enhance or decrease the activity and metabolic effectiveness of AGT and GO, respectively, and be useful in the treatment of PH1.
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Hiperoxalúria Primária/metabolismo , Oxalatos/metabolismo , Piridoxina/metabolismo , Transaminases/metabolismo , Animais , Células CHO , Sobrevivência Celular , Cricetulus , Glicolatos/metabolismo , Humanos , Hiperoxalúria Primária/genética , Mutação , Espécies Reativas de Oxigênio/metabolismo , Transaminases/genéticaRESUMO
Excessive endogenous oxalate synthesis can result in calcium oxalate kidney stone formation and renal failure. Hydroxyproline catabolism in the liver and kidney contributes to endogenous oxalate production in mammals. To quantify this contribution we have infused Wt mice, Agxt KO mice deficient in liver alanine:glyoxylate aminotransferase, and Grhpr KO mice deficient in glyoxylate reductase, with (13)C5-hydroxyproline. The contribution of hydroxyproline metabolism to urinary oxalate excretion in Wt mice was 22±2%, 42±8% in Agxt KO mice, and 36%±9% in Grhpr KO mice. To determine if blocking steps in hydroxyproline and glycolate metabolism would decrease urinary oxalate excretion, mice were injected with siRNA targeting the liver enzymes glycolate oxidase and hydroxyproline dehydrogenase. These siRNAs decreased the expression of both enzymes and reduced urinary oxalate excretion in Agxt KO mice, when compared to mice infused with a luciferase control preparation. These results suggest that siRNA approaches could be useful for decreasing the oxalate burden on the kidney in individuals with Primary Hyperoxaluria.
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Oxirredutases do Álcool/genética , Hidroxiprolina/metabolismo , Hiperoxalúria Primária/genética , Hiperoxalúria Primária/terapia , Prolina Oxidase/metabolismo , Terapêutica com RNAi , Oxirredutases do Álcool/metabolismo , Animais , Modelos Animais de Doenças , Hiperoxalúria Primária/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxalatos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Terapêutica com RNAi/métodosRESUMO
TRPV5 is a Ca2+-selective channel that plays a key role in the reabsorption of Ca2+ ions in the kidney. Recently, a rare L530R variation (rs757494578) of TRPV5 was found to be associated with recurrent kidney stones in a founder population. However, it was unclear to what extent this variation alters the structure and function of TRPV5. To evaluate the function and expression of the TRPV5 variant, Ca2+ uptake in Xenopus oocytes and western blot analysis were performed. The L530R variation abolished the Ca2+ uptake activity of TRPV5 in Xenopus oocytes. The variant protein was expressed with drastic reduction in complex glycosylation. To assess the structural effects of this L530R variation, TRPV5 was modeled based on the crystal structure of TRPV6 and molecular dynamics simulations were carried out. Simulation results showed that the L530R variation disrupts the hydrophobic interaction between L530 and L502, damaging the secondary structure of transmembrane domain 5. The variation also alters its interaction with membrane lipid molecules. Compared to the electroneutral L530, the positively charged R530 residue shifts the surface electrostatic potential towards positive. R530 is attracted to the negatively charged phosphate group rather than the hydrophobic carbon atoms of membrane lipids. This shifts the pore helix where R530 is located and the D542 residue in the Ca2+-selective filter towards the surface of the membrane. These alterations may lead to misfolding of TRPV5, reduction in translocation of the channel to the plasma membrane and/or impaired Ca2+ transport function of the channel, and ultimately disrupt TRPV5-mediated Ca2+ reabsorption.
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Variação Genética/genética , Cálculos Renais/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Humanos , Modelos Moleculares , Oócitos/metabolismo , Canais de Cátion TRPV/química , XenopusRESUMO
Transient receptor potential cation channels, vanilloid subfamily, member 5 (TRPV5) plays a key role in active Ca(2+) reabsorption in the kidney. Variations in TRPV5 occur at high frequency in African populations and may contribute to their higher efficiency of Ca(2+) reabsorption. One of the African specific variations, A563T, exhibits increased Ca(2+) transport ability. However, it is unclear how this variation influences the channel pore. On the basis of the structure of TRPV1, a TRPV5 model was generated to simulate the structural and dynamical changes induced by the A563T variation. On the basis of this model, amino acid residue 563 interacts with V540, which is one residue away from the key residue, D542, involved in Ca(2+) selectivity and Mg(2+) blockade. The A563T variation increases secondary structure stability and reduces dynamical motion of D542. In addition, the A563T variation alters the electrostatic potential of the outer surface of the pore. Differences in contact between selective filter residues and residue 563 and in electrostatic potential between the two TRPV5 variants were also observed in another model derived from an alternative alignment in the selective filters between TRPV5 and TRPV1. These findings indicate that the A563T variation induces structural, dynamical, and electrostatic changes in the TRPV5 pore, providing structural insight into the functional alterations associated with the A563T variation.
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Simulação de Dinâmica Molecular , Mutação Puntual , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética , Animais , População Negra/genética , Cálcio/metabolismo , Humanos , Conformação Proteica , Ratos , Eletricidade Estática , Canais de Cátion TRPV/metabolismoRESUMO
Primary Hyperoxaluria Type 3 is a recently discovered form of this autosomal recessive disease that results from mutations in the gene coding for 4-hydroxy-2-oxoglutarate aldolase (HOGA1). This enzyme is one of the 2 unique enzymes in the hydroxyproline catabolism pathway. Affected individuals have increased urinary excretions of oxalate, 4-hydroxy-L-glutamate (4-OH-Glu), 4-hydroxy-2-oxoglutarate (HOG), and 2,4-dihydroxyglutarate (DHG). While 4-OH-Glu and HOG are precursor substrates of HOGA1 and increases in their concentrations are expected, how DHG is formed and how HOG to oxalate are unclear. To resolve these important questions and to provide insight into possible therapeutic avenues for treating this disease, an animal model of the disease would be invaluable. We have developed a mouse model of this disease which has null mutations in the Hoga1 gene and have characterized its phenotype. It shares many characteristics of the human disease, particularly when challenged by the inclusion of hydroxyproline in the diet. An increased oxalate excretion is not observed in the KO mice which may be consistent with the recent recognition that only a small fraction of the individuals with the genotype for HOGA deficiency develop PH.
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We present the results of an investigation into the rotational and angular distributions of the NO à state fragment following photodissociation of the NO-He, NO-Ne, and NO-Ar van der Waals complexes excited via the à â XÌ transition. For each complex, the dissociation is probed for several values of Ea, the available energy above the dissociation threshold. For NO-He, the Ea values probed were 59, 172, and 273 cm(-1); for NO-Ne they were 50 and 166 cm(-1); and for NO-Ar they were 44, 94, 194, and 423 cm(-1). The NO à state rotational distributions arising from NO-He are cold, with most products in low angular momentum states. NO-Ne leads to broader NO rotational distributions but they do not extend to the maximum possible given the energy available. In the case of NO-Ar, the distributions extend to the maximum allowed at that energy and show the unusual shapes associated with the rotational rainbow effect reported in previous studies. This is the only complex for which a rotational rainbow effect is observed at the chosen Ea values. Product angular distributions have also been measured for the NO à photodissociation product for the three complexes. NO-He produces nearly isotropic fragments, but the anisotropy parameter, ß, for NO-Ne and NO-Ar photofragments shows a surprising change in sign from negative to positive as Ea increases within the unstructured excitation profile. Franck-Condon selection of a broader distribution of geometries including more linear geometries at lower excitation energies and more T-shaped geometries at higher energies can account for the changing recoil anisotropy. Two-dimensional wavepacket calculations are reported to model the rotational state distributions and the bound-continuum absorption spectra.
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The primary hyperoxalurias (PH), types 1-3, are disorders of glyoxylate metabolism that result in increased oxalate production and calcium oxalate stone formation. The breakdown of trans-4-hydroxy-L-proline (Hyp) from endogenous and dietary sources of collagen makes a significant contribution to the cellular glyoxylate pool. Proline dehydrogenase 2 (PRODH2), historically known as hydroxyproline oxidase, is the first step in the hydroxyproline catabolic pathway and represents a drug target to reduce the glyoxylate and oxalate burden of PH patients. This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C-terminal truncation variants indicated that residues 157-515 contain the catalytic core with one FAD molecule. The 12-fold higher k(cat)/K(m) value of 0.93 M⻹·s⻹ for Hyp over Pro demonstrates the preference for Hyp as substrate. Moreover, an anaerobic titration determined a K(d) value of 125 µM for Hyp, a value ~1600-fold lower than the K(m) value. A survey of ubiquinone analogues revealed that menadione, duroquinone, and CoQ1 reacted more efficiently than oxygen as the terminal electron acceptor during catalysis. Taken together, these data and the slow reactivity with sodium sulfite support that PRODH2 functions as a dehydrogenase and most likely utilizes CoQ10 as the terminal electron acceptor in vivo. Thus, we propose that the name of PRODH2 be changed to hydroxyproline dehydrogenase (HYPDH). Three Hyp analogues were also identified to inhibit the activity of HYPDH, representing the first steps toward the development of a novel approach to treat all forms of PH.
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Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/metabolismo , Hidroxiprolina/metabolismo , Hiperoxalúria Primária/enzimologia , Modelos Moleculares , Prolina Oxidase/metabolismo , Ubiquinona/análogos & derivados , Biocatálise , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Flavina-Adenina Dinucleotídeo/química , Flavoproteínas/química , Flavoproteínas/genética , Flavoproteínas/isolamento & purificação , Furanos/farmacologia , Furanos/uso terapêutico , Humanos , Hidroxiprolina/química , Hiperoxalúria Primária/tratamento farmacológico , Ligantes , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Prolina/metabolismo , Prolina Oxidase/química , Prolina Oxidase/genética , Prolina Oxidase/isolamento & purificação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Terminologia como Assunto , Ubiquinona/química , Ubiquinona/metabolismoRESUMO
Mutations in the gene encoding for 4-hydroxy-2-oxoglutarate aldolase (HOGA) are associated with an excessive production of oxalate in Primary Hyperoxaluria type 3 (PH3). This enzyme is the final step of the hydroxyproline degradation pathway within the mitochondria and catalyzes the cleavage of 4-hydroxy-2-oxoglutarate (HOG) to pyruvate and glyoxylate. No analyses have been performed to assess the consequences of the mutations identified, particularly for those variants that produce either full-length or nearly full-length proteins. In this study, the expression, stability, and activity of nine PH3 human HOGA variants were examined. Using recombinant protein produced in Escherichia coli as well as transfected Chinese hamster ovary (CHO) cells, it was found that all nine PH3 variants are quite unstable, have a tendency to aggregate, and retain no measurable activity. A buildup of HOG was confirmed in the urine, sera and liver samples from PH3 patients. To determine how HOG is cleaved in the absence of HOGA activity, the ability of N-acetylneuraminate aldolase (NAL) to cleave HOG was evaluated. NAL showed minimal activity towards HOG. Whether the expected buildup of HOG in mitochondria could inhibit glyoxylate reductase (GR), the enzyme mutated in PH2, was also evaluated. GR was inhibited by HOG but not by 2-hydroxyglutarate or 2-oxoglutarate. Thus, one hypothetical component of the molecular basis for the excessive oxalate production in PH3 appears to be the inhibition of GR by HOG, resulting in a phenotype similar to PH2.
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Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/metabolismo , Hiperoxalúria Primária/enzimologia , Oxo-Ácido-Liases/antagonistas & inibidores , Oxo-Ácido-Liases/metabolismo , Oxirredutases do Álcool/genética , Animais , Células CHO , Células Cultivadas , Cricetinae , Glutaratos/metabolismo , Humanos , Hidroxiprolina/metabolismo , Hiperoxalúria Primária/genética , Ácidos Cetoglutáricos/metabolismo , Fígado/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Oxalatos/metabolismo , Oxo-Ácido-Liases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção/métodosRESUMO
INTRODUCTION: Certain dietary modifications limit the risk of stone recurrence. Compliance is an important component of dietary therapy for stone prevention, and self-efficacy is an important ingredient of compliance. We developed an internet program to facilitate dietary compliance for stone prevention and performed a pilot study to assess its effectiveness. MATERIALS AND METHODS: The internet program provides information regarding dietary modifications including increased fluid consumption, limited animal protein, sodium, and oxalate intake, and adequate calcium consumption. Participants record their daily food and fluid intake and receive immediate feedback as to whether they were compliant or not. Five adult calcium stone formers collected three 24 hour urine specimens on self-selected diets, three 24 hour urine specimens while on a stone preventive metabolic diet, and three 24 hour urine specimens after utilizing the internet program for 1 month. Urinary stone risk parameters were measured, and data were analyzed using repeated measures ANOVA and Student's t test. RESULTS: All participants recorded their meals and snacks for each day and found the program easy to navigate. The mean time in hours from food consumption to log in was 35.25 +/- 70.8 hours. There were no statistically significant differences in stone risk factors between the controlled and internet dietary phases. Oxalate excretion was significantly higher during the self-selected dietary intake (p = 0.03). CONCLUSIONS: This pilot study demonstrates that subjects appear to be compliant with utilization of an interactive internet program for stone prevention with dietary modifications. In addition, improvement in certain stone risk parameters occurred.
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Comportamento Alimentar , Internet , Cálculos Renais/prevenção & controle , Cooperação do Paciente , Software , Adulto , Registros de Dieta , Estudos de Viabilidade , Feminino , Humanos , Cálculos Renais/epidemiologia , Cálculos Renais/urina , Masculino , Pessoa de Meia-Idade , Oxalatos/urina , Educação de Pacientes como Assunto , Projetos Piloto , Fatores de Risco , AutocuidadoRESUMO
Primary hyperoxaluria type 1 (PH1) and type 2 (PH2) are rare genetic diseases that result from deficiencies in glyoxylate metabolism. The increased oxalate synthesis that occurs can lead to kidney stone formation, deposition of calcium oxalate in the kidney and other tissues, and renal failure. Hydroxyproline (Hyp) catabolism, which occurs mainly in the liver and kidney, is a prominent source of glyoxylate and could account for a significant portion of the oxalate produced in PH. To determine the sensitivity of mouse models of PH1 and PH2 to Hyp-derived oxalate, animals were fed diets containing 1% Hyp. Urinary excretions of glycolate and oxalate were used to monitor Hyp catabolism and the kidneys were examined to assess pathological changes. Both strains of knockout (KO) mice excreted more oxalate than wild-type (WT) animals with Hyp feeding. After 4 wk of Hyp feeding, all mice deficient in glyoxylate reductase/hydroxypyruvate reductase (GRHPR KO) developed severe nephrocalcinosis in contrast to animals deficient in alanine-glyoxylate aminotransferase (AGXT KO) where nephrocalcinosis was milder and with a lower frequency. Plasma cystatin C measurements over 4-wk Hyp feeding indicated no significant loss of renal function in WT and AGXT KO animals, and significant and severe loss of renal function in GRHPR KO animals after 2 and 4 wk, respectively. These data suggest that GRHPR activity may be vital in the kidney for limiting the conversion of Hyp-derived glyoxylate to oxalate. As Hyp catabolism may make a major contribution to the oxalate produced in PH patients, Hyp feeding in these mouse models should be useful in understanding the mechanisms associated with calcium oxalate deposition in the kidney.
Assuntos
Hidroxiprolina/metabolismo , Hiperoxalúria/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Ração Animal/análise , Animais , Dieta , Masculino , Camundongos , Camundongos Knockout , Nefrocalcinose/metabolismo , Oxalatos/metabolismo , Transaminases/genética , Transaminases/metabolismoRESUMO
Hydroxyproline (Hyp) metabolism is a key source of glyoxylate production in the body and may be a major contributor to excessive oxalate production in the primary hyperoxalurias where glyoxylate metabolism is impaired. Important gaps in our knowledge include identification of the tissues with the capacity to degrade Hyp and the development of model systems to study this metabolism and how to suppress it. The expression of mRNA for enzymes in the pathway was examined in 15 different human tissues. Expression of the complete pathway was identified in liver, kidney, pancreas, and small intestine. HepG2 cells also expressed these mRNAs and enzymes and were shown to metabolize Hyp in the culture medium to glycolate, glycine, and oxalate. [(18)O]- and [(13)C(5)]Hyp were synthesized and evaluated for their use with in vitro and in vivo models. [(18)O]Hyp was not suitable because of an apparent tautomerism of [(18)O]glyoxylate between enol and hydrated forms, which resulted in a loss of isotope. [(13)C(5)]Hyp, however, was metabolized to [(13)C(2)]glycolate, [(13)C(2)]glycine, and [(13)C(2)]oxalate in vitro in HepG2 cells and in vivo in mice infused with [(13)C(5)]Hyp. These model systems should be valuable tools for exploring various aspects of Hyp metabolism and will be useful in determining whether blocking Hyp catabolism is an effective therapy in the treatment of primary hyperoxaluria.
Assuntos
Hidroxiprolina/metabolismo , Hiperoxalúria/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Isótopos de Carbono , Regulação da Expressão Gênica/fisiologia , Células Hep G2 , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Glycolate and 2-phosphoglycolate (PG) are 2-carbon monocarboxylic acids with ill-defined metabolic roles. Their concentrations have not yet been described in tissues apart from body fluids and erythrocytes. We describe the use of ion chromatography coupled with mass spectrometry (IC-MS) to quantify levels of glycolate and PG in tissue. Sample preparation and analysis can be performed within an hour. Low concentrations of glycolate (12-48 nmol/g) and PG (4-17 nmol/g) were detected in all tissues. The availability of this IC-MS assay will facilitate investigations of the origin, function, and metabolism of glycolate and PG in tissues.