Concentrations Of Glutaric Acid Research Articles

Enhancing the photostability and mechanical properties of poly‐lactic acid (PLA) composites with glutaric acid functionalized titanium dioxide (TiO2)

AbstractThe performance of Poly(lactic acid) (PLA) composites reinforced with functionalized titanium dioxide (TiO2) nanoparticles in rutile and anatase crystalline phases were systematically investigated. The results underline that the incorporation of TiO2, regardless of its crystalline structure, significantly enhances PLA's crystallinity by 85%. Notably, rutile TiO2, when functionalized with higher glutaric acid concentrations, was an effective crystallinity booster by 95%, passing from 20.39% to 39.58% crystallinity for the PLA/TiO2 R20 + 10% system. Furthermore, this research elucidates that the selection between anatase and rutile phases plays a vital role in the photostabilization. Functionalized Rutile TiO2 exhibited exceptional UV resistance, effectively inhibiting photodegradation; for example, after 15 days of exposure, the functionalized systems exhibited a 497.6% improvement for PLA/TiO2 R10 + 5% (22.1 ± 2.5 MPa), while the PLA/TiO2 R10 + 5% showed an 85.1% increase in composite stiffness (35.5 ± 3.5 MPa) after 7 days of degradation, in comparison with pristine PLA (19.2 ± 2.9 MPa). Anatase, conversely, displayed varying effects depending on concentration, acting as a stabilizer at lower levels and a promoter of degradation at higher concentrations. This knowledge enables the precise modifying of PLA composites for specific applications, balancing crystallinity, mechanical properties, and UV resistance for a diverse array of applications.Highlights TiO2 enhances photostability in PLA confirming its potential as a UV stabilizer. Comparing anatase and rutile phase: Unveiling PLA composite dynamics. Glutaric acid on TiO2 enhances PLA composite strength significantly. Optimal acid concentration key to composite performance. Tailored PLA composites balance crystallinity, mechanics, and UV resistance.

Modeling Glutaric Aciduria Type I in human neuroblastoma cells recapitulates neuronal damage that can be rescued by gene replacement.

Glutaric Aciduria type I (GA1) is a rare neurometabolic disorder caused by mutations in the GDCH gene encoding for glutaryl-CoA dehydrogenase (GCDH) in the catabolic pathway of lysine, hydroxylysine and tryptophan. GCDH deficiency leads to increased concentrations of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body fluids and tissues. These metabolites are the main triggers of brain damage. Mechanistic studies supporting neurotoxicity in mouse models have been conducted. However, the different vulnerability to some stressors between mouse and human brain cells reveals the need to have a reliable human neuronal model to study GA1 pathogenesis. In the present work we generated a GCDH knockout (KO) in the human neuroblastoma cell line SH-SY5Y by CRISPR/Cas9 technology. SH-SY5Y-GCDH KO cells accumulate GA, 3-OHGA, and glutarylcarnitine when exposed to lysine overload. GA or lysine treatment triggered neuronal damage in GCDH deficient cells. SH-SY5Y-GCDH KO cells also displayed features of GA1 pathogenesis such as increased oxidative stress vulnerability. Restoration of the GCDH activity by gene replacement rescued neuronal alterations. Thus, our findings provide a human neuronal cellular model of GA1 to study this disease and show the potential of gene therapy to rescue GCDH deficiency.

Glutaric Acidemia Type 1: Diagnosis, Clinical features, and Outcome in a Portuguese Cohort.

Glutaric acidemia type 1 (GA1) is a rare autosomal recessive disorder characterized by a deficiency of glutaryl-CoA dehydrogenase, resulting in the accumulation of glutaric acid (GA), 3-hydroxyglutaric acid, and glutarylcarnitine, especially in the brain. GA1-affected children are clinically characterized by macrocephaly. Neurological abnormalities usually appear between 6 and 18 months of age, often triggered by a catabolic event. On the other hand, several biochemically affected individuals may remain asymptomatic or experience an insidious onset of mild neurological abnormalities. Retrospective study of GA1 patients followed at a Portuguese Hereditary Metabolic Disease Center, to characterize the phenotypic and genotypic variations associated with GA1. Therefore, we analyzed the clinical, neuroradiological, biochemical, and genetic information from 14 patients. 14 patients (four months-27 years old) were identified in the last 26 years, 9 were male, 1 was from a consanguineous family. 11 were diagnosed by newborn screening (NBS), and 3 identified following clinical symptoms (later diagnosed, LD). There were 3 phenotypic presentations: 6 asymptomatic, 3 with a motor disability after encephalopathic crisis (EC), and 5 with insidious onset. Acute EC occurred in 1/3 of the LD patients and in 2/11 NBS-identified patients. About urinary GA concentrations: 5 were low excretors (LE), 9 were high excretors (HE). All LE showed symptoms, and 2 had EC. Concerning HE, 3 showed symptoms and 1 had EC. GCDH analysis showed: 6 compound heterozygotes and 8 homozygotes. most frequent variant was c.1204C>T (p.R402W). All of them received appropriate therapy from the time of diagnosis, with a mean age of 23.3 months in LD patients and 13.3 days in NBS-identified patients. The outcomes were different between the two groups: all the LD patients presented motor dysfunction however in the NBS-identified patients only 5 developed this symptom. Patients identified by NBS had better outcomes showing that NBS enables an early diagnosis, and treatment, and consequently improves the clinical outcomes for these patients. No correlation was observed with clinical phenotype between LE and HE, as both groups can suffer the most severe neurological manifestations. These conclusions are in agreement with previous cohorts described in the literature.

Development of glutaric acid production consortium system with α-ketoglutaric acid regeneration by glutamate oxidase in Escherichia coli

Glutaric acid is a C5 dicarboxylic acid that can be used as a building block for bioplastics. Although high concentrations of glutaric acid can be produced by fermentation or bioconversion, a large amount of α-ketoglutaric acid (α-KG) is necessary to accept the amine group from 5-aminovaleric acid. To decrease the demand for α-KG, we introduced l-glutamate oxidase (GOX) from Streptomyces mobaraensis in our previous system for cofactor regeneration in combination with a glutaric acid production system from 5-aminovaleric acid. To enhance glutaric acid production, critical factors were optimized such as the expression vector, pH, temperature, and cell ratio. As a result, the demand for α-KG was decreased by more than 6-fold under optimized conditions. Additionally, the effect of catalase was also demonstrated by blocking the degradation of α-KG to succinic acid because of the hydrogen peroxide. Finally, 468.5 mM glutaric acid was produced from 800 mM 5-aminovaleric acid using only 120 mM α-KG. Moreover, this system containing davBA, gabTD-nox, and gox can be applied to produce glutaric acid from L-lysine by reusing α-KG with GOX. This improved cofactor regeneration system has a potential to apply much larger production of glutaric acid.

Glutaric Acid Affects Pericyte Contractility and Migration: Possible Implications for GA-I Pathogenesis.

Glutaric acidemia I (GA-I) is an inherited neurometabolic childhood disease characterized by bilateral striatal neurodegeneration upon brain accumulation of millimolar concentrations of glutaric acid (GA) and related metabolites. Vascular dysfunction, including abnormal cerebral blood flow and blood-brain barrier damage, is an early pathological feature in GA-I, although the affected cellular targets and underlying mechanisms remain unknown. In the present study, we have assessed the effects of GA on capillary pericyte contractility in cerebral cortical slices and pericyte cultures, as well as on the survival, proliferation, and migration of cultured pericytes. GA induced a significant reduction in capillary diameter at distances up to ~ 10μm from the center of pericyte somata. However, GA did not affect the contractility of cultured pericytes, suggesting that the response elicited in slices may involve GA evoking pericyte contraction by acting on other cellular components of the neurovascular unit. Moreover, GA indirectly inhibited migration of cultured pericytes, an effect that was dependent on soluble glial factors since it was observed upon application of conditioned media from GA-treated astrocytes (CM-GA), but not upon direct GA addition to the medium. Remarkably, CM-GA showed increased expression of cytokines and growth factors that might mediate the effects of increased GA levels not only on pericyte migration but also on vascular permeability and angiogenesis. These data suggest that some effects elicited by GA might be produced by altering astrocyte-pericyte communication, rather than directly acting on pericytes. Importantly, GA-evoked alteration of capillary pericyte contractility may account for the reduced cerebral blood flow observed in GA-I patients.

Ammonium accumulation and chemokine decrease in culture media of Gcdh−/− 3D reaggregated brain cell cultures

Glutaric Aciduria type I (GA-I) is caused by mutations in the GCDH gene. Its deficiency results in accumulation of the key metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body tissues and fluids. Present knowledge on the neuropathogenesis of GA-I suggests that GA and 3-OHGA have toxic properties on the developing brain.We analyzed morphological and biochemical features of 3D brain cell aggregates issued from Gcdh−/− mice at two different developmental stages, day-in-vitro (DIV) 8 and 14, corresponding to the neonatal period and early childhood. We also induced a metabolic stress by exposing the aggregates to 10 mM l-lysine (Lys).Significant amounts of GA and 3-OHGA were detected in Gcdh−/− aggregates and their culture media. Ammonium was significantly increased in culture media of Gcdh−/− aggregates at the early developmental stage. Concentrations of GA, 3-OHGA and ammonium increased significantly after exposure to Lys. Gcdh−/− aggregates manifested morphological alterations of all brain cell types at DIV 8 while at DIV 14 they were only visible after exposure to Lys. Several chemokine levels were significantly decreased in culture media of Gcdh−/− aggregates at DIV 14 and after exposure to Lys at DIV 8.This new in vitro model for brain damage in GA-I mimics well in vivo conditions. As seen previously in WT aggregates exposed to 3-OHGA, we confirmed a significant ammonium production by immature Gcdh−/− brain cells. We described for the first time a decrease of chemokines in Gcdh−/− culture media which might contribute to brain cell injury in GA-I.

Impairment of astrocytic glutaminolysis in glutaric aciduria type I.

Glutaric aciduria type I is a rare, autosomal recessive, inherited defect of glutaryl-CoA dehydrogenase. Deficiency of this protein in L-lysine degradation leads to the characteristic accumulation of nontoxic glutarylcarnitine and neurotoxic glutaric acid (GA), glutaryl-CoA, and 3-hydroxyglutaric acid. Untreated patients develop bilateral lesions of basal ganglia resulting in a complex movement disorder with predominant dystonia in infancy and early childhood. The current pathomechanistic concept strongly focuses on imbalanced neuronal energy metabolism due to accumulating metabolites, whereas little is known about the pathomechanistic role of astrocytes, which are thought to be in constant metabolic crosstalk with neurons. We found that glutaric acid (GA) causes astrocytic cell death under starvation cell culture conditions, i.e. low glucose, without glutamine and fetal calf serum. Glutamine completely abolished GA-induced toxicity, suggesting involvement of glutaminolysis. Increasing dependence on glutaminolysis by chemical induction of hypoxia signaling-potentiated GA-induced toxicity. We further show that GA disturbs glutamine degradation by specifically inhibiting glutamate dehydrogenase. Summarizing our study shows that pathologically relevant concentrations of GA block an important step in the metabolic crosstalk between neurons and astrocytes, ultimately leading to astrocytic cell death.

Toxic Synergism Between Quinolinic Acid and Glutaric Acid in Neuronal Cells Is Mediated by Oxidative Stress: Insights to a New Toxic Model.

It has been shown that synergistic toxic effects of quinolinic acid (QUIN) and glutaric acid (GA), both in isolated nerve endings and in vivo conditions, suggest the contribution of these metabolites to neurodegeneration. However, this synergism still requires a detailed characterization of the mechanisms involved in cell damage during its occurrence. In this study, the effects of subtoxic concentrations of QUIN and/or GA were tested in neuronal cultures, co-cultures (neuronal cells + astrocytes), and mixed cultures (neuronal cells + astrocytes + microglia) from rat cortex and striatum. The exposure of different cortical and striatal cell cultures to QUIN + GA resulted in cell death and stimulated different markers of oxidative stress, including reactive oxygen species (ROS) formation; changes in the activity of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; and depletion of endogenous antioxidants such as -SH groups and glutathione. The co-incubation of neuronal cultures with QUIN + GA plus the N-methyl-D-aspartate antagonist MK-801 prevented cell death but not ROS formation, whereas the antioxidant melatonin reduced both parameters. Our results demonstrated that QUIN and GA can create synergistic scenarios, inducing toxic effects on some parameters of cell viability via the stimulation of oxidative damage. Therefore, it is likely that oxidative stress may play a major causative role in the synergistic actions exerted by QUIN + GA in a variety of cell culture conditions involving the interaction of different neural types.

Gas chromatography-mass spectrometry profiles of urinary organic acids in healthy captive cheetahs (Acinonyx jubatus).

In captivity, cheetahs (Acinonyx jubatus) frequently suffer from several unusual chronic diseases that rarely occur in their free-ranging counterparts. In order to develop a better understanding of their metabolism and health we documented the urine organic acids of 41 apparently healthy captive cheetahs, in an untargeted metabolomic study, using gas chromatography-mass spectrometry. A total of 339 organic acids were detected and annotated. Phenolic compounds, thought to be produced by the anaerobic fermentation of aromatic amino acids in the distal colon, as well as their corresponding glycine conjugates, were present in high concentrations. The most abundant organic acids in the cheetahs' urine were an as yet unidentified compound and a novel cadaverine metabolite, tentatively identified as N1,N5-dimethylpentane-1,5-diamine. Pantothenic acid and citramalic acid concentrations correlated negatively with age, while glutaric acid concentrations correlated positively with age, suggesting possible dysregulation of coenzyme A metabolism in older cheetahs. This study provides a baseline of urine organic acid reference values in captive cheetahs and suggests important avenues for future research in this species.

Proposed recommendations for diagnosing and managing individuals with glutaric aciduria type I: second revision.

Glutaric aciduria type I (GA-I; synonym, glutaric acidemia type I) is a rare inherited metabolic disease caused by deficiency of glutaryl-CoA dehydrogenase located in the catabolic pathways of L-lysine, L-hydroxylysine, and L-tryptophan. The enzymatic defect results in elevated concentrations of glutaric acid, 3-hydroxyglutaric acid, glutaconic acid, and glutaryl carnitine in body tissues, which can be reliably detected by gas chromatography/mass spectrometry (organic acids) and tandem mass spectrometry (acylcarnitines). Most untreated individuals with GA-I experience acute encephalopathic crises during the first 6 years of life that are triggered by infectious diseases, febrile reaction to vaccinations, and surgery. These crises result in striatal injury and consequent dystonic movement disorder; thus, significant mortality and morbidity results. In some patients, neurologic disease may also develop without clinically apparent crises at any age. Neonatal screening for GA-I us being used in a growing number of countries worldwide and is cost effective. Metabolic treatment, consisting of low lysine diet, carnitine supplementation, and intensified emergency treatment during catabolism, is effective treatment and improves neurologic outcome in those individuals diagnosed early; treatment after symptom onset, however, is less effective. Dietary treatment is relaxed after age 6years and should be supervised by specialized metabolic centers. The major aim of this second revision of proposed recommendations is to re-evaluate the previous recommendations (Kölker et al. J Inherit Metab Dis 30:5-22, 2007b; J Inherit Metab Dis 34:677-694, 2011) and add new research findings, relevant clinical aspects, and the perspective of affected individuals.

White Matter Injury Induced by Perinatal Exposure to Glutaric Acid

Glutaric acid (GA) is a neurotoxic metabolite that accumulates in the CNS of patients with glutaric acidemia-I (GA-I), a neurometabolic disease caused by deficient activity of glutaryl-CoA dehydrogenase. Most GA-I patients display characteristic CNS lesions, mainly in the gray and white matter of basal ganglia and cerebral cortex. Neurons and astrocytes are believed to be vulnerable to millimolar concentrations of GA. However, little is known about the effects of GA on oligodendrocytes (OL) and the myelination process in the postnatal brain. Here, we show that a single intracerebroventricular administration of GA to rat neonatal pups induced a selective and long-lasting myelination failure in the striatum but no deleterious effect in the myelination of the corpus callosum. At 45 days post-GA injection, the myelinated area of striatal axonal bundles was decreased by 35 %, and the expression of myelin basic protein and myelin-associated glycoprotein (MAG) reduced by 25 and 60 %, respectively. This was accompanied by long lasting cytopathology features in MAG and CC-1-expressing OLs, which was confirmed by transmission electron microscopy. Remarkably, GA did not induce acute loss of pre-OLs in the striatum as assessed by NG2 or PDGFRα immunohistochemistry, suggesting an indirect and progressive mechanism for OL damage. In accordance, GA-induced white matter injury was restricted to the striatum and associated to GA-induced astrocytosis and neuronal loss. In conclusion, the current evidence indicates a pathogenic mechanism by which GA can permanently affect myelin status.

Therapeutic modulation of cerebral l-lysine metabolism in a mouse model for glutaric aciduria type I

Glutaric aciduria type I, an inherited deficiency of glutaryl-coenzyme A dehydrogenase localized in the final common catabolic pathway of L-lysine, L-hydroxylysine and L-tryptophan, leads to accumulation of neurotoxic glutaric and 3-hydroxyglutaric acid, as well as non-toxic glutarylcarnitine. Most untreated patients develop irreversible brain damage during infancy that can be prevented in the majority of cases if metabolic treatment with a low L-lysine diet and L-carnitine supplementation is started in the newborn period. The biochemical effect of this treatment remains uncertain, since cerebral concentrations of neurotoxic metabolites can only be determined by invasive techniques. Therefore, we studied the biochemical effect and mechanism of metabolic treatment in glutaryl-coenzyme A dehydrogenase-deficient mice, an animal model with complete loss of glutaryl-coenzyme A dehydrogenase activity, focusing on the tissue-specific changes of neurotoxic metabolites and key enzymes of L-lysine metabolism. Here, we demonstrate that low L-lysine diet, but not L-carnitine supplementation, lowered the concentration of glutaric acid in brain, liver, kidney and serum. L-carnitine supplementation restored the free L-carnitine pool and enhanced the formation of glutarylcarnitine. The effect of low L-lysine diet was amplified by add-on therapy with L-arginine, which we propose to result from competition with L-lysine at system y(+) of the blood-brain barrier and the mitochondrial L-ornithine carriers. L-lysine can be catabolized in the mitochondrial saccharopine or the peroxisomal pipecolate pathway. We detected high activity of mitochondrial 2-aminoadipate semialdehyde synthase, the rate-limiting enzyme of the saccharopine pathway, in the liver, whereas it was absent in the brain. Since we found activity of the subsequent enzymes of L-lysine oxidation, 2-aminoadipate semialdehyde dehydrogenase, 2-aminoadipate aminotransferase and 2-oxoglutarate dehydrogenase complex as well as peroxisomal pipecolic acid oxidase in brain tissue, we postulate that the pipecolate pathway is the major route of L-lysine degradation in the brain and the saccharopine pathway is the major route in the liver. Interestingly, treatment with clofibrate decreased cerebral and hepatic concentrations of glutaric acid in glutaryl-coenzyme A dehydrogenase-deficient mice. This finding opens new therapeutic perspectives such as pharmacological stimulation of alternative L-lysine oxidation in peroxisomes. In conclusion, this study gives insight into the discrepancies between cerebral and hepatic L-lysine metabolism, provides for the first time a biochemical proof of principle for metabolic treatment in glutaric aciduria type I and suggests that further optimization of treatment could be achieved by exploitation of competition between L-lysine and L-arginine at physiological barriers and enhancement of peroxisomal L-lysine oxidation and glutaric acid breakdown.

Induction of Oxidative Stress by Chronic and Acute Glutaric Acid Administration to Rats

: 1. Glutaric acidemia type I (GA I) is a neurometabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase, which leads to tissue accumulation of predominantly glutaric acid (GA) and also 3-hydroxyglutaric acid to a lesser amount. Affected patients usually present progressive cortical atrophy and acute striatal degeneration attributed to the toxic accumulating metabolites.2. In the present study, we determined a number of oxidative stress parameters, namely chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), total antioxidant reactivity (TAR), glutathione (GSH) levels, and the activities of catalase and glutathione peroxidase (GPx), in various tissues from rats chronically exposed to GA or to saline (controls). High GA concentrations, similar to those found in glutaric aciduria type I, were induced in the brain by three daily subcutaneous injections of saline-buffered GA (5 micromol/g body weight) to Wistar rats of 5-22 days of life. The parameters were assessed 12 h after the last GA administration in different brain structures, skeletal muscle, heart, liver, erythrocytes, and plasma. The lipid peroxidation parameters chemiluminescence and/or TBA-RS measurements were found significantly increased in midbrain, liver, and erythrocytes of GA-injected rats. The activity of GPx was significantly reduced in midbrain and markedly increased in liver. TAR measurement was significantly reduced in midbrain and liver. Furthermore, GSH levels were reduced in liver and heart. We also investigated the acute in vivo effect of GA administration on the same oxidative stress parameters in cerebral structures and erythrocytes from 22-day-old rats. We found that TBA-RS values were significantly increased in erythrocytes, TAR levels were markedly decreased in midbrain and cerebellum, and GPx activity mildly reduced in the midbrain.3. These data showing an imbalance between antioxidant defences and oxidative damage, particularly in midbrain, liver, and erythrocytes from GA-injected rats, indicate that oxidative stress might be involved in GA toxicity and that the midbrain, where the striatum is located, is the brain structure more susceptible to GA chronic and acute exposition.

Energy Metabolism is Compromised in Skeletal Muscle of Rats Chronically-Treated with Glutaric Acid

Glutaric acidemia type I (GA I) (GA I, McKusick 23167; OMIM # 231670) is an autosomal recessive metabolic disorder caused by glutaryl-CoA dehydrogenase deficiency (EC 1.3.99.7). Clinically, the disease is characterized by macrocephaly, hypotonia, dystonia and diskinesia. Since the pathophysiology of this disorder is not yet well established, in the present investigation we determined a number of energy metabolism parameters, namely (14)CO(2) production, the activities of the respiratory chain complexes I-IV and of creatine kinase, in tissues of rats chronically exposed to glutaric acid (GA). High tissue GA concentrations (0.6 mM in the brain, 4 mM in skeletal muscle and 6 mM in plasma) were induced by three daily subcutaneous injections of saline-buffered GA (5 micromol x g(-1) body weight) to Wistar rats from the 5th to the 21st day of life. The parameters were assessed 12 h after the last GA injection in cerebral cortex and middle brain, as well as in skeletal muscle homogenates of GA-treated rats. GA administration significantly inhibited the activities of the respiratory chain complexes I-III and II and induced a significant increase of complex IV activity in skeletal muscle of rats. Furthermore, creatine kinase activity was also inhibited by GA treatment in skeletal muscle. In contrast, these measurements were not altered by GA administration in the brain structures studied. Taken together, it was demonstrated that chronic GA administration induced an impairment of energy metabolism in rat skeletal muscle probably due to a higher tissue concentration of this organic acid that may be possibly associated to the muscle weakness occurring in glutaric acidemic patients.

Intracerebral accumulation of glutaric and 3‐hydroxyglutaric acids secondary to limited flux across the blood–brain barrier constitute a biochemical risk factor for neurodegeneration in glutaryl‐CoA dehydrogenase deficiency

Glutaric acid (GA) and 3-hydroxyglutaric acids (3-OH-GA) are key metabolites in glutaryl co-enzyme A dehydrogenase (GCDH) deficiency and are both considered to be potential neurotoxins. As cerebral concentrations of GA and 3-OH-GA have not yet been studied systematically, we investigated the tissue-specific distribution of these organic acids and glutarylcarnitine in brain, liver, skeletal and heart muscle of Gcdh-deficient mice as well as in hepatic Gcdh-/- mice and in C57Bl/6 mice following intraperitoneal loading. Furthermore, we determined the flux of GA and 3-OH-GA across the blood-brain barrier (BBB) using porcine brain microvessel endothelial cells. Concentrations of GA, 3-OH-GA and glutarylcarnitine were significantly elevated in all tissues of Gcdh-/- mice. Strikingly, cerebral concentrations of GA and 3-OH-GA were unexpectedly high, reaching similar concentrations as those found in liver. In contrast, cerebral concentrations of these organic acids remained low in hepatic Gcdh-/- mice and after intraperitoneal injection of GA and 3-OH-GA. These results suggest limited flux of GA and 3-OH-GA across the BBB, which was supported in cultured porcine brain capillary endothelial cells. In conclusion, we propose that an intracerebral de novo synthesis and subsequent trapping of GA and 3-OH-GA should be considered as a biochemical risk factor for neurodegeneration in GCDH deficiency.

Glutaric Acid Administration Impairs Energy Metabolism in Midbrain and Skeletal Muscle of Young Rats

A genetic mice model of glutaric acidemia type I (GAI) has recently been developed, however affected animals do not develop the striatal damage characteristic of patients with this disorder. Therefore, the initial aim of the present work was to induce high glutaric acid (GA) concentrations in rat brain similar to those found in GAI patients through subcutaneous injection of GA. High brain GA concentrations (up to 0.60 micromol/g congruent with 0.60mM) were achieved by a single subcutaneous injection of saline-buffered GA (5 micromol/g body weight) to Wistar rats of 7-22 days of life. GA brain levels were about 10-fold lower than in plasma and 5-fold lower than in skeletal and cardiac muscles, indicating that the permeability of the blood brain barrier to GA is low. We also aimed to use this model to investigate neurochemical parameters in the animals. Thus, we evaluated the effect of this model on energy metabolism parameters in midbrain, in which the striatum is localized, as well as in peripheral tissues (skeletal and cardiac muscles) of 22-day-old rats. Control rats were treated with saline in the same volumes. We verified that CO2 production from glucose was not altered in midbrain of rats treated with GA, indicating a normal functioning of the tricarboxylic acid cycle. Creatine kinase activity was also not changed in midbrain, skeletal and cardiac muscles. In contrast, complex I-III activity of the respiratory chain was inhibited in midbrain (25%), while complexes I-III (25%) and II-III (15%) activities were reduced in skeletal muscle, with no alterations found in cardiac muscle. These data indicate that GA administration moderately impairs cellular energy metabolism in midbrain and skeletal muscle of young rats.

Glutaric Acid and Its Metabolites Cause Apoptosis in Immature Oligodendrocytes: A Novel Mechanism of White Matter Degeneration in Glutaryl-CoA Dehydrogenase Deficiency

Glutaryl-CoA dehydrogenase deficiency is an inherited metabolic disease characterized by elevated concentrations of glutaric acid (GA) and its metabolites glutaconic acid (GC) and 3-hydroxy-glutaric acid (3-OH-GA). Its hallmarks are striatal and cortical degeneration, which have been linked to excitotoxic neuronal cell death. However, magnetic resonance imaging studies have also revealed widespread white matter disease. Correspondingly, we decided to investigate the effects of GA, GC, and 3-OH-GA on the rat immature oligodendroglia cell line, OLN-93. For comparison, we also exposed the neuroblastoma line SH-SY5Y and the microglia line BV-2 to GA, GC, and 3-OH-GA. Cell viability was measured by metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium. Flow cytometry was used to assess apoptosis via annexin-V, anti-active caspase-3 antibody, and propidium iodide staining. GA, GC, and 3-OH-GA reduced OLN-93 oligodendroglia cell viability in a dose-dependent manner. Toxicity of GA, GC, and 3-OH-GA was abrogated by preincubation with the pan-caspase inhibitor z-VAD-fmk. Apoptosis but not necrosis was detected at various stages (early: annexin-V; effector: caspase-3) after 24-48 h of incubation with GA, GC, or 3-OH-GA in OLN-93 but not in neuroblastoma or microglia cells. OLN-93 lacked expression of N-methyl-d-aspartate receptors, making classical glutamatergic excitotoxicity an unlikely explanation for the selective toxicity of GA, GC, and 3-OH-GA for OLN-93 cells. GA, GC, and 3-OH-GA directly initiate the apoptotic cascade in oligodendroglia cells. This mechanism may contribute to the white matter damage observed in glutaryl-CoA dehydrogenase deficiency.

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [ 3 H ]glutamate by synaptosomes, Na +-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na +-dependent glutamate uptake into astrocytes, respectively. GA (1–100 nM) significantly stimulated [ 3 H ]glutamate binding to brain plasma membranes (40–70%) in the absence of extracellular Na + concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [ 3 H ]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na +-dependent [ 3 H ]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 μM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.

Glutaryl-CoA Dehydrogenase Deficiency: Region-Specific Analysis of Organic Acids and Acylcarnitines inpost mortemBrain Predicts Vulnerability of the Putamen

The neurometabolic disorder glutaryl-CoA dehydrogenase (GCDH) deficiency is biochemically characterised by an accumulation of the marker metabolites 3-hydroxyglutaric acid, glutaric acid, and glutarylcarnitine. If untreated, the disease is complicated by acute encephalopathic crises, resulting in neurodegeneration of vulnerable brain regions, in particular the putamen. 3-hydroxyglutaric acid is considered the major neurotoxin in this disease. There are only preliminary data concerning glutaric acid concentrations in the brains of affected children and the distribution of 3-hydroxyglutaric acid and glutarylcarnitine has not been described. In the present study, we investigated post mortem the distribution of 3-hydroxyglutaric and glutaric acids as well as glutarylcarnitine in 14 different brain regions, internal organs, and body fluids (urine, plasma, cerebrospinal fluid) in a 14-year-old boy. 3-Hydroxyglutaric acid showed the highest concentration (62 nmol/g protein) in the putamen among all brain areas investigated. The glutarylcarnitine concentration was also highest in the putamen (7.1 nmol/g protein). We suggest that the regional-specific differences in the relative concentrations of 3-hydroxyglutaric acid contribute to the pattern of neuronal damage in this disease. These results provide an explanatory basis for the high vulnerability of the putamen in this disease, adding to the strong corticostriatal glutamatergic input into the putamen and the high excitotoxic susceptibility of neostriatal medium spiny neurons.

IGF-1 and bFGF reduce glutaric acid and 3-hydroxyglutaric acid toxicity in striatal cultures.

Glutaric acid (GA) and 3-hydroxyglutaric acid (3GA) are thought to contribute to the degeneration of the caudate and putamen that is seen in some children with glutaric acidaemia type I, a metabolic disorder caused by a glutaryl-CoA dehydrogenase deficiency. This study assessed the neurotoxicity of GA and 3GA (0-50 mmol/L) compared to quinolinic acid (QUIN) in striatal and cortical cultures. All three acids were neurotoxic in a dose-dependent manner; however, GA and 3GA were both more toxic than QUIN. The neurotoxic effects of low concentrations of GA or 3GA were additive to QUIN toxicity. A series of hormones and growth factors were tested for protection against GA and 3GA toxicity. Insulin (5-500 microU /ml), basic fibroblast growth factor (bFGF; 10 ng/ml), insulin-like growth factor (IGF-1; 50 ng/ml), brain-derived neurotrophic factor (BDNF; 10 ng/ml), glial-derived neurotrophic factor (GDNF; 10 ng/ml), and two glutamate antagonists were evaluated in brain cultures to which 7 mmol/L GA or 3GA were added. GA and 3GA neurotoxicities were prevented by bFGF. Attenuation of 3GA-induced neurotoxicity was seen with insulin (5 microU/ml) and IGF-1. BDNF and GDNF had no effects on neuronal survival. Glutamate antagonists MK801 (10 micromol/L) and NBQX (10 micromol/L) failed to prevent GA or 3GA neurotoxicity. We conclude that GA and 3GA are neurotoxic in cultures of embryonic rat striatum and cortex. Striatal neurons were rescued from death by bFGF and IGF-1 but not by glutamate antagonist, suggesting that toxicity in this embryonic system is not necessarily mediated by glutamate receptors.