5-oxoproline Concentrations Research Articles

Severe Hemolytic Anemia and Metabolic Acidosis at Birth with Glutathione Synthetase Deficiency and Progressive Neurological Symptoms on Follow-Up.

BACKGROUND Glutathione synthetase deficiency (GSD) is a rare autosomal recessive disorder caused by glutathione synthetase (GSS) gene variants that occur in 1 in 1 million individuals. The severe form of GSD is characterized by hemolytic anemia, metabolic acidosis with 5-oxoprolinuria, progressive neurological symptoms, and recurrent bacterial infections. This case report presents a male Japanese infant with severe hemolytic anemia and metabolic acidosis at birth caused by GSD, who developed progressive neurological symptoms on follow-up. CASE REPORT A Japanese male term infant developed severe hemolytic anemia and metabolic acidosis in the early neonatal period. We suspected GSD based on his symptoms and a high 5-oxoproline urine concentration. We began correcting his metabolic acidosis and administering vitamins C and E supplements. The patient required blood transfusion twice during the acute phase for hemolytic anemia. After age 1 month, he maintained good control of metabolic acidosis and hemolytic anemia. A definitive diagnosis of GSD was made based on high concentrations of 5-oxoproline in urine, low concentrations of glutathione and GSS activity in erythrocytes, and genetic testing. Several episodes of febrile convulsions were started at age 11 months, but none occurred after 2 years. At the last follow-up at age 25 months, metabolic acidosis and hemolytic anemia were well controlled, but he had mild neurodevelopmental delay. CONCLUSIONS This case report shows that GSD can present with severe hemolytic anemia and metabolic acidosis at birth, and manifest with subsequent neurological impairment despite early diagnosis and treatment. Therefore, a careful long-term follow-up that includes neurological evaluation is essential for patients with GSD.

Lactiplantibacillus plantarum P9 improved gut microbial metabolites and alleviated inflammatory response in pesticide exposure cohorts

SummaryMultiple pesticide residue accumulations increase the probability of chronic metabolic diseases in humans. Thus, we applied multi-omics techniques to reveal how the gut microbiome responded to pesticide exposure. Then, we explored how probiotic Lactiplantibacillus plantarum P9 (P9) consumption impacted the gut microbiota and immune factors after high pesticide exposure. Multi-omics results indicated frequent exposure to pesticides did not alter the composition of the intestinal microbiota, but it did increase the abundance of Lipopolysaccharide in the gut, which might contribute to chronic inflammation. Supplementation with P9 maintained the homeostasis of the gut microbiota and reduced the abundance of pathogens in the high pesticide-exposed subjects. By detecting metabolites, we observed uridine and 5-oxoproline concentrations increased significantly after P9 consumption. Furthermore, P9 alleviated immune factors disorder and promoted pesticide residue excretion. Our findings provide new insights into the application of probiotics for pesticide detoxification, and suggest probiotics as daily supplements for pesticide exposure prevention.

Glycine, a Dispensable Amino Acid, Is Conditionally Indispensable in Late Stages of Human Pregnancy

Recently, we showed that there are higher protein, lysine, and phenylalanine requirements in late stages of pregnancy compared with early stages. Animal studies have suggested an increased dietary need for specific dispensable amino acids in pregnancy; whether such a need exists in human pregnancies is unknown. The objective of the current study was to examine whether healthy pregnant women at midgestation (20-29 wk) and late gestation (30-40 wk) have a dietary demand for glycine, a dispensable amino acid, using the indicator amino acid oxidation method and measurement of plasma 5-oxoproline concentrations. Seventeen healthy women (aged 26-36 y) randomly received different test glycine intakes (range: 5-100 mg·kg-1·d-1) during each study day in midgestation (∼26 wk, n=17 observations in 9 women) and late gestation (∼35 wk, n=19 observations in 8 women). Diets were isocaloric with energy at 1.7 × resting energy expenditure. Protein was given as a crystalline amino acid mixture based on egg protein composition at current estimated average requirement (EAR; 0.88 g·kg-1·d-1). Breath samples were collected at baseline and isotopic steady state to measure oxidation of L-[1-13C]phenylalanine to 13CO2 (F13CO2). Plasma was collected at the sixth hour of the study day. Linear regression crossover analysis and simple linear regression were used to assess responses in F13CO2 and plasma 5-oxoproline concentrations to different glycine intakes. No statistically significant responses were observed in midgestation. However, in late gestation, lower glycine intakes resulted in higher rates of F13CO2 (suggesting low protein synthesis) with a breakpoint for phenylalanine oxidation at >37mgglycine·kg-1·d-1 and higher plasma 5-oxoproline (suggesting low glycine availability) with a breakpoint >27mg glycine·kg-1·d-1. The findings suggest that glycine should be considered a "conditionally" indispensable amino acid during late gestation, especially when protein intakes are at 0.88 g·kg-1·d-1, the current EAR. This trial was registered at clinicaltrials.gov as NCT02149953.

5-Oxoproline concentrations in acute acetaminophen overdose

Introduction: Several reports describe high anion gap metabolic acidosis with 5-oxoproline (5-OP) after acetaminophen exposure, including therapeutic use of acetaminophen. The mechanism may involve disordered glutathione metabolism. It is unknown whether acute acetaminophen overdose consistently causes elevations in 5-oxoproline concentration.Methods: We enrolled 23 consecutive adult and adolescent patients with measureable plasma APAP after acute APAP overdose. We used plasma left over in the laboratory after blood tests obtained in clinical care of the patients. We measured plasma [5-OP] by GC/MS. We compared the [5-OP] to laboratory results obtained in the care of these patients to search for correlations. The study had IRB approval.Results: Eighteen patients had non-detectable or normal (<100 μmol/L) 5-oxoproline concentrations. Six more patients had concentrations between 100 μmol/L and 300 μmol/L. There was no significant correlation of 5-OP with APAP, AST, ALT, creatinine, anion gap, INR, or total bilirubin.Discussion: Limitations of the study include small sample size and treatment with IV N-acetylcysteine for all patients with APAP concentrations above the 150 line of the Rumack Matthew nomogram or with hepatotoxicity. We believe that inherited enzyme deficiencies more likely explain cases of 5-oxoprolinemia.Conclusion: Acetaminophen overdose generally results in normal 5-oxoproline concentrations with some patients having slightly elevated 5-oxoproline concentrations.

5-0xoproline (pyroglutamic acid) acidosis and acetaminophen- a differential diagnosis in high anion gap metabolic acidosis

Rare cases of high anion gap metabolic acidosis during long-term paracetamol administration in therapeutic doses with causative 5-oxoproline (pyroglutamic acid} accumulation have been reported. Other concomitant risk factors such as malnutrition, alcohol abuse, renal or hepatic dysfunction, comedication with flue/oxacillin, vigabatrin, netilmicin or sepsis have been described. The etiology seems to be a drug-induced reversible inhibition of glutathione synthetase or 5-oxoprolinase leading to elevated serum and urine levels of 5-oxoproline. Other more frequent differential diagnoses, such as intoxications, ketoacidosis or lactic acidosis should be excluded. Causative substances should be stopped. 5-oxoproline concentrations in urine can be quantified to establish the diagnosis. Adverse drug reactions, which are not listed or insufficiently described in the respective Swiss product information, should be reported to the regional pharmacovigilance centres for early signal detection. 5-0 xoproline acidosis will be integrated as a potential adverse drug reaction in the Swiss product information for paracetamol.

Hemolytic Anemia and Metabolic Acidosis: Think about Glutathione Synthetase Deficiency

Glutathione synthetase deficiency (GSSD) is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with hemolytic anemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low glutathione synthetase activity in erythrocytes or cultured skin fibroblasts. The prognosis seems to depend on early diagnosis and treatment. We report a 4 months old Tunisian male infant who presented with severe metabolic acidosis with high anion gap and hemolytic anemia. High level of 5-oxoproline was detected in her urine and diagnosis of GSSD was made. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. He died of severe metabolic acidosis and sepsis at the age of 15 months.

Multiscale modelling approach combining a kinetic model of glutathione metabolism with PBPK models of paracetamol and the potential glutathione-depletion biomarkers ophthalmic acid and 5-oxoproline in humans and rats

A key role of the antioxidant glutathione is detoxification of chemically reactive electrophilic drug metabolites within the liver. Therefore glutathione depletion can have severe toxic consequences. Ophthalmic acid and 5-oxoproline are metabolites involved in glutathione metabolism, which can be measured readily in the blood and urine and have been proposed as candidate biomarkers of hepatic glutathione content. However, currently it is unclear whether their concentrations in plasma exhibit a robust correlation with hepatic glutathione content. To explore this important question, we have developed a novel approach which combines a physiologically based pharmacokinetic (PBPK) model of metabolism and disposition of paracetamol (acetaminophen) with a previously developed mathematical systems model of hepatic glutathione homeostasis. Paracetamol is metabolised to reactive intermediates which deplete glutathione and cause toxicity when given at high doses. Our model correctly predicted that hepatic glutathione depletion following paracetamol administration resulted in elevated concentrations of 5-oxoproline and ophthalmic acid in blood and of 5-oxoproline in urine. However, we also found from the model that concentrations of both of the compounds were likely to be influenced by prolonged administration of paracetamol and by the concentrations of intracellular metabolites such as methionine. We conclude that care must be taken when extrapolating from concentrations of these biomarkers to hepatic glutathione status.

5‐Oxoprolinuria as a cause of high anion gap metabolic acidosis

5‐Oxoprolinuria as a cause of high anion gap metabolic acidosis

A mathematical modelling approach to assessing the reliability of biomarkers of glutathione metabolism

One of the main pathways for the detoxification of reactive metabolites in the liver involves glutathione conjugation. Metabolic profiling studies have shown paradoxical responses in glutathione-related biochemical pathways. One of these is the increase in 5-oxoproline and ophthalmic acid concentrations with increased dosage of paracetamol. Experimental studies have thus far failed to resolve these paradoxes and the robustness of how these proposed biomarkers correlate with liver glutathione levels has been questioned. To better understand how these biomarkers behave in the glutathione system a kinetic model of this pathway was made. By using metabolic control analysis and by simulating biomarker levels under a variety of conditions, we found that 5-oxoproline and ophthalmic acid concentrations may not only depend on the glutathione but also on the methionine status of the cell. We show that neither of the two potential biomarkers are reliable on their own since they need additional information about the methionine status of the system to relate them uniquely to intracellular glutathione concentration. However, when both biomarkers are measured simultaneously a direct inference of the glutathione concentration can be made, irrespective of the methionine concentration in the system.

Diagnosis of glutathione synthetase deficiency in newborn screening

Glutathione synthetase (GSS) deficiency is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with haemolytic anaemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low GSS activity in erythrocytes or cultured skin fibroblasts. Diagnosis can be confirmed by mutational analysis. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. The most important determinants for outcome and survival in patients with GSS deficiency are early diagnosis and early initiation of treatment. The case of a newborn with GSS deficiency diagnosed by tandem mass spectrometry (MS/MS)-based newborn screening is described. After onset of clinical symptoms on the 2nd day of life, expanded newborn screening revealed normal results for all disorders included in the German screening programme; however, selective MS/MS screening revealed a >10-fold elevation of 5-oxoproline in dried blood, leading to the presumptive diagnosis of GSS deficiency by the 5th day of life. Diagnosis was later confirmed by detection of markedly reduced glutathione concentration in erythrocytes and mutational analysis of the GSS gene. Presently, GSS deficiency is not included in newborn screening programmes in Europe. As outcome depends significantly on early start of treatment, routine inclusion of this disorder in newborn screening panels should be considered.

Relationship of Dimethylglycine, Choline, and Betaine with Oxoproline in Plasma of Pregnant Women and Their Newborn Infants ,

Choline and glycine are inter-related through their roles in methyl metabolism. Choline is metabolized to betaine, which donates a methyl group to homocysteine to form methionine, also generating dimethylglycine, which is further metabolized to glycine. Choline is transported across the placenta and is higher in fetal than maternal plasma. Placental glycine transfer, however, is limited and poor glycine status has been suggested in preterm infants. Insufficient glycine for glutathione (GSH) synthesis results in increased metabolism of γ-glutamyl cysteine to 5-oxoproline. We measured plasma 5-oxoproline as a metabolic indicator to address whether choline, via dimethylglycine, contributes physiologically relevant amounts of glycine in pregnancy. Blood was collected from healthy term pregnant women and their newborn infants at delivery (n = 46) and nonpregnant healthy women (n = 19) as a reference group. Plasma choline, betaine, dimethylglycine, homocysteine, methionine, and 5-oxoproline were quantified by HPLC-tandem MS. Plasma choline was 45% higher, but betaine was 63% lower and dimethylglycine was 28% lower in pregnant than nonpregnant women (P < 0.01). Higher white blood cell choline dehydrogenase messenger RNA levels in a random subset of pregnant (n = 8) than nonpregnant women (n = 7) (P < 0.01) suggest increased betaine and dimethylglycine turnover rather than decreased synthesis. Plasma choline, betaine, and dimethylglycine were higher (P < 0.001) in fetal plasma (36.4 ± 13, 29.4 ± 1.0, and 2.44 ± 0.12 μmol/L, respectively) than maternal plasma (15.3 ± 0.42, 14.1 ± 0.6 and 1.81 ± 0.12 μmol/L, respectively). Concentrations of 5-oxoproline and dimethylglycine were inversely (P < 0.05) correlated in maternal (Spearman rho = −0.35) and fetal plasma (Spearman rho = −0.32), suggesting that choline, via dimethylglycine, contributes glycine for GSH synthesis in human development.

Gamma-Glutamylglutamine identified in plasma and cerebrospinal fluid from hyperammonaemic patients

gamma-Glutamylglutamine has been identified in plasma and cerebrospinal fluid. Preparative high voltage electrophoresis was used to isolate the compound from two to three ml of sample. Analysis of archival material from eight patients with urea cycle disorders showed that plasma γ-glutamylglutamine was 20–214 μmol/l when the plasma glutamine was greater than 1000 μmol/l and that the relationship was strongly positive ( P < 0.0005). Plasma γ-glutamylglutamine concentration was also raised in one patient with secondary hyperammonaemia, but not in three other cases, one of whom had a plasma glutamine of 10000 μmol/l. Cerebrospinal fluid from the last patient contained 97 μmol/l of γ-glutamylglutamine. Samples of cerebrospinal fluid from two patients with urea cycle disorders were available and the γ-glutamylglutamine levels were 203 and 313 μmol/l. gamma-Glutamylglutamine and 5-oxoproline concentrations were not significantly increased in urine from any of these patients.

A therapeutic trial withN‐acetylcysteine in subjects with hereditary glutathione synthetase deficiency (5‐oxoprolinuria)

In a therapeutic trial, the effect of short-term low-dosage N-acetylcysteine supplementation on glutathione metabolism was investigated in two patients with hereditary glutathione deficiency (5-oxoprolinuria). Clinical and neurophysiological examinations of the patients indicated progressive neurological damage. The pretreatment concentrations of total and free glutathione in leukocytes were 15-20% of normal, whereas the corresponding gamma-glutamylcysteine levels were increased. In plasma, the glutathione concentrations were similarly decreased, but no gamma-glutamylcysteine was detected. Total glutathione in erythrocytes was markedly decreased. Low urinary excretion of cysteinylglycine, cyst(e)ine, taurine, N-acetylcysteine, mercaptolactate and mercaptoacetate and reduced leukocyte taurine levels constituted additional evidence of decreased intracellular availability of cysteine, i.e. glutathione. Oral supplementation with N-acetylcysteine (5 mg/kg x 3/day) had no effect on acid-base balance, erythrocyte glutathione levels or 5-oxoproline concentrations in plasma and urine. In leukocytes, the glutathione concentrations were increased by 20-30%, whereas the gamma-glutamylcysteine levels were essentially unaltered. In parallel, the urinary excretion of cysteinylglycine was increased and the leukocyte levels and urinary outputs of sulphur amino acids were restored. No side-effects of the treatment were noted. The results indicate that N-acetylcysteine may be of value in increasing the low intracellular glutathione concentrations and cysteine availability in patients with hereditary glutathione synthetase deficiency.

Red cell glutathione deficiency: clinical and biochemical investigations using sheep as an experimental model system.

The biochemical properties of red cells from normal sheep and sheep with three types of red cell glutathione (GSH)-deficiency were compared. One deficiency was due to an impaired transport system for amino acids (lesion 1), one was the result of a diminished activity of gamma-glutamyl cysteine synthetase (GC-S) (lesion 2) and the third was a combined deficiency produced by selective breeding to give animals with both lesions 1 and 2. Under normal husbandry conditions no clinical symptoms were apparent in sheep with lesion 2, but red cells from sheep with lesion 1 and lesions 1 + 2 showed an increased osmotic fragility, a greater tendency to form Heinz bodies and a shorter potential life span than normal. These deficiencies were not found in tissues other than blood. Normal and GSH-deficient red cells had the expected low concentrations of 5-oxoproline. The effects of the toxic agents phenylhydrazine, s-methylcysteine sulphoxide and nitrite in vivo were measured in sheep of the different types. GSH-deficient sheep responded earlier and more dramatically than normal sheep, showing greater methaemoglobin formation, and for phenylhydrazine and s-methylcysteine sulphoxide, more severe anaemia. Sheep with the combined lesions were in general the most susceptible, but even they had the ability to recover from moderately severe oxidative challenge.