Multidisciplinary approach combining food metabolomics and epidemiology identifies meglutol as an important bioactive metabolite in tempe, an Indonesian fermented food.

This study introduces a multidisciplinary approach to investigate bioactive food metabolites often overlooked due to their low concentrations. We integrated an in-house food metabolite library (n = 494), a human metabolite library (n = 891) from epidemiological studies, and metabolite pharmacological databases to screen for food metabolites with potential bioactivity. We identified six potential metabolites, including meglutol (3-hydroxy-3-methylglutarate), an understudied low-density lipoprotein (LDL)-lowering compound. We further focused on meglutol as a case study to showcase the range of characterizations achievable with this approach. Green pea tempe was identified to contain the highest meglutol concentration (21.8 ± 4.6 mg/100 g). Furthermore, we identified a significant cross-sectional association between plasma meglutol (per 1-standard deviation) and lower LDL cholesterol in two Hispanic adult cohorts (n = 1,628) (ß [standard error]: -5.5 (1.6) mg/dl, P = 0.0005). These findings highlight how multidisciplinary metabolomics can serve as a systematic tool for discovering and enhancing bioactive metabolites in food, such as meglutol, with potential applications in personalized dietary approaches for disease prevention.

[Clinical and genetic analysis of two children with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency].

OBJECTIVE: To explore the clinical characteristics and genetic variants of two children with 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMGCLD). METHODS: Two children with HMGCLD diagnosed at Henan Provincial Children's Hospital respectively in December 2019 and June 2022 were selected as the study subjects. Clinical data and results of laboratory testing were analyzed retrospectively. RESULTS: Both children had manifested with repeated convulsions, severe hypoglycemia, metabolic acidosis and liver dysfunction. Blood amino acids and acylcarnitine analysis showed increased 3-hydroxy-isovalyl carnitine (C5OH) and 3-hydroxy-isovalyl carnitine/capryloyl carnitine ratio (C5OH/C8), and urinary organic acid analysis showed increased 3-hydroxyl-3-methyl glutaric acid, 3-methyl glutaric acid, 3-methyl glutaconic acid, 3-hydroxyisoglycine and 3-methylprotarylglycine. Child 1 was found to harbor homozygous c.722C>T variants of the HMGCL gene, which was rated as uncertain significance (PM2_Supporting+PP3). Child 2 was found to harbor homozygous c.121C>T variants of the HMGCL gene, which was rated as pathogenic variant (PVS1+PM2_Supporting+PP4). CONCLUSION: Acute episode of HMGCLD is usually characterized by metabolic disorders such as hypoglycemia and metabolic acidosis, and elevated organic acids in urine may facilitate the differential diagnosis, though definite diagnosis will rely on genetic testing.

Improvement of the functional value of green soybean (edamame) using germination and tempe fermentation: A comparative metabolomics study.

Green soybean, also known as edamame, is a legume with high nutritional and functional value. Despite its growing popularity and potential health benefits, the functionality of green soybean has not been thoroughly studied. Previous research on the functionality of green soybean has largely focused on a limited number of specific, well-studied, bioactive metabolites, without comprehensively investigating the metabolome of this legume. Additionally, very few studies have explored the improvement of the functional value of green soybean. This study aimed to investigate the metabolome profile of green soybean, identify bioactive metabolites, and to further explore the potential improvement of the identified bioactive metabolites using germination and tempe fermentation. A total of 80 metabolites were annotated from green soybean using GC-MS and HPLC-PDA-MS. Among them, 16 important bioactive metabolites were identified: soy isoflavones daidzin, glycitin, genistin, malonyl daidzin, malonyl genistin, malonyl glycitin, acetyl daidzin, acetyl genistin, acetyl glycitin, daidzein, glycitein, and genistein, as well as other metabolites including 3,4-dihydroxybenzoic acid, 3-hydroxyanthranillic acid, 3-hydroxy-3-methylglutaric acid (meglutol), and 4-aminobutyric acid (GABA). Germination and tempe fermentation techniques were employed to potentially improve the concentrations of these bioactive metabolites. While showing improvements in amino acid contents, germination process did not improve bioactive metabolites significantly. In contrast, tempe fermentation was found to significantly increase the concentrations of daidzein, genistein, glycitein, acetyl genistin, acetyl daidzin, 3-hydroxyanthranillic acid, and meglutol (>2-fold increase with p < 0.05) while also improving amino acid levels. This study highlights the potentials of germination and fermentation to improve the functionality of legumes, particularly green soybean.

Serum organic acid metabolites can be used as potential biomarkers to identify prostatitis, benign prostatic hyperplasia, and prostate cancer.

Background: Noninvasive methods for the early identify diagnosis of prostatitis, benign prostatic hyperplasia (BPH), and prostate cancer (PCa) are current clinical challenges. Methods: The serum metabolites of 20 healthy individuals and patients with prostatitis, BPH, or PCa were identified using untargeted liquid chromatography-mass spectrometry (LC-MS). In addition, targeted LC-MS was used to verify the organic acid metabolites in the serum of a validation cohort. Results: Organic acid metabolites had good sensitivity and specificity in differentiating prostatitis, BPH, and PCa. Three diagnostic models identified patients with PROSTATITIS: phenyllactic acid (area under the curve [AUC]=0.773), pyroglutamic acid (AUC=0.725), and pantothenic acid (AUC=0.721). Three diagnostic models identified BPH: citric acid (AUC=0.859), malic acid (AUC=0.820), and D-glucuronic acid (AUC=0.810). Four diagnostic models identified PCa: 3-hydroxy-3-methylglutaric acid (AUC=0.804), citric acid (AUC=0.918), malic acid (AUC=0.862), and phenyllactic acid (AUC=0.713). Two diagnostic models distinguished BPH from PCa: phenyllactic acid (AUC=0.769) and pyroglutamic acid (AUC=0.761). Three diagnostic models distinguished benign BPH from PROSTATITIS: citric acid (AUC=0.842), ethylmalonic acid (AUC=0.814), and hippuric acid (AUC=0.733). Six diagnostic models distinguished BPH from prostatitis: citric acid (AUC=0.926), pyroglutamic acid (AUC=0.864), phenyllactic acid (AUC=0.850), ethylmalonic acid (AUC=0.843), 3-hydroxy-3-methylglutaric acid (AUC=0.817), and hippuric acid (AUC=0.791). Three diagnostic models distinguished PCa patients with PROSTATITISA < 4.0 ng/mL from those with PSA > 4.0 ng/mL: 5-hydromethyl-2-furoic acid (AUC=0.749), ethylmalonic acid (AUC=0.750), and pyroglutamic acid (AUC=0.929). Conclusions: These results suggest that serum organic acid metabolites can be used as biomarkers to differentiate prostatitis, BPH, and PCa.

Early pregnancy metabolites predict gestational diabetes mellitus: implications for fetal programming.

BACKGROUND: Aberrant fetal programming in gestational diabetes mellitus seems to increase the risk of obesity, type 2 diabetes, and cardiovascular disease. The inability to accurately identify gestational diabetes mellitus in the first trimester of pregnancy has thwarted ascertaining whether early therapeutic interventions reduce the predisposition to these prevalent medical disorders. OBJECTIVE: A metabolomics study was conducted to determine whether advanced analytical methods could identify accurate predictors of gestational diabetes mellitus in early pregnancy. STUDY DESIGN: This nested observational case-control study was composed of 92 gravidas (46 in the gestational diabetes mellitus group and 46 in the control group) in early pregnancy, who were matched by maternal age, body mass index, and gestational age at urine collection. Gestational diabetes mellitus was diagnosed according to community standards. A comprehensive metabolomics platform measured 626 endogenous metabolites in randomly collected urine. Consensus multivariate criteria or the most important by 1 method identified low-molecular weight metabolites independently associated with gestational diabetes mellitus, and a classification tree selected a subset most predictive of gestational diabetes mellitus. RESULTS: Urine for both groups was collected at a mean gestational age of 12 weeks (range, 6-19 weeks' gestation). Consensus multivariate analysis identified 11 metabolites independently linked to gestational diabetes mellitus. Classification tree analysis selected a 7-metabolite subset that predicted gestational diabetes mellitus with an accuracy of 96.7%, independent of maternal age, body mass index, and time of urine collection. CONCLUSION: Validation of this high-accuracy model by a larger study is now needed to support future studies to determine whether therapeutic interventions in the first trimester of pregnancy for gestational diabetes mellitus reduce short- and long-term morbidity.

In vivo evidence that bezafibrate prevents oxidative stress and mitochondrial dysfunction caused by 3-methylglutaric acid in rat liver.

High urinary excretion and tissue accumulation of 3-methylglutaric acid (MGA) are observed in patients affected by 3-hydroxy-3-methylglutaric (HMGA) and 3-methylglutaconic (MGTA) acidurias. The pathomechanisms underlying the hepatic dysfunction commonly observed in these disorders are not fully elucidated so that we investigated here the effects of intraperitoneal administration of MGA on redox homeostasis, mitochondrial bioenergetics, biogenesis and dynamics in rat liver. The effects of a pre-treatment with the protective compound bezafibrate (BEZ) were also determined. Our data showed that MGA induced lipid peroxidation and altered enzymatic and non-enzymatic antioxidant defenses in liver, indicating redox homeostasis disruption. BEZ prevented most of these alterations induced by MGA. MGA also decreased the activities of the respiratory chain complexes II and IV and increased of II-III, whereas BEZ prevented the alteration in complex II activity. Furthermore, MGA decreased levels of nuclear PGC-1α and Sirt1, and increased levels of AMPKα1 and cytosolic PPARγ, which were blocked by BEZ. MGA augmented the levels of mitofusin-1 and dynamin-related protein 1, suggesting that both fusion and fission mitochondrial processes are enhanced by MGA. BEZ was able to prevent only the changes in mitofusin-1 levels. Collectively, these findings indicate that oxidative stress and mitochondrial dysfunction are mechanisms involved in the hepatic dysfunction found in HMGA and MGTA. It is also presumed that mitochondrial biogenesis stimulation may constitute an attractive approach to reduce MGA toxicity in liver of individuals affected by HMGA and MGTA.

3-Hydroxy-3-Methylglutaric Acid Impairs Redox and Energy Homeostasis, Mitochondrial Dynamics, and Endoplasmic Reticulum-Mitochondria Crosstalk in Rat Brain.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a neurometabolic disorder characterized by predominant accumulation of 3-hydroxy-3-methylglutaric acid (HMG) in tissues and biological fluids. Patients often present in the first year of life with metabolic acidosis, non-ketotic hypoglycemia, hypotonia, lethargy, and coma. Since neurological symptoms may be triggered or worsened during episodes of metabolic decompensation, which are characterized by high urinary excretion of organic acids, this study investigated the effects of HMG intracerebroventricular administration on redox homeostasis, citric acid cycle enzyme activities, dynamics (mitochondrial fusion and fission), and endoplasmic reticulum (ER)-mitochondria crosstalk in the brain of neonatal rats euthanized 1 (short term) or 20 days (long term) after injection. HMG induced lipid peroxidation and decreased the activities of glutathione peroxidase (GPx) and citric acid cycle enzymes, suggesting bioenergetic and redox disruption, 1 day after administration. Levels of VDAC1, Grp75, and mitofusin-1, proteins involved in ER-mitochondria crosstalk and mitochondrial fusion, were increased by HMG. Furthermore, HMG diminished synaptophysin levels and tau phosphorylation, and increased active caspase-3 content, indicative of cell damage. Finally, HMG decreased GPx activity and synaptophysin levels, and changed MAPK phosphorylation 20 days after injection, suggesting that long-term toxicity is further induced by this organic acid. Taken together, these data show that HMG induces oxidative stress and disrupts bioenergetics, dynamics, ER-mitochondria communication, and signaling pathways in the brain of rats soon after birth. It may be presumed that these mechanisms underlie the onset and progression of symptoms during decompensation occurring in HL-deficient patients during the neonatal period.

3-Methylglutaric acid in energy metabolism.

3-methylglutaric (3MG) acid is a conspicuous C6 dicarboxylic organic acid classically associated with two distinct leucine pathway enzyme deficiencies. 3MG acid is excreted in urine of individuals harboring deficiencies in 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HMGCL) or 3-methylglutaconyl CoA hydratase (AUH). Whereas 3MG CoA is not part of the leucine catabolic pathway, it is likely formed via a side reaction involving reduction of the α-ß trans double bond in the leucine pathway intermediate, 3-methylglutaconyl CoA. While the metabolic basis for the accumulation of 3MG acid in subjects with deficiencies in HMGCL or AUH is apparent, the occurrence of 3MG aciduria in a host of unrelated inborn errors of metabolism associated with compromised mitochondrial energy metabolism is less clear. Herein, a novel mitochondrial biosynthetic pathway termed "the acetyl CoA diversion pathway", provides an explanation. The pathway is initiated by defective electron transport chain function which, ultimately, inhibits acetyl CoA entry into the TCA cycle. When this occurs, 3MG acid is synthesized in five steps from acetyl CoA via a novel reaction sequence, providing a metabolic rationale for the connection between 3MG aciduria and compromised mitochondrial energy metabolism.

Prevention by L-carnitine of DNA damage induced by 3-hydroxy-3-methylglutaric and 3-methylglutaric acids and experimental evidence of lipid and DNA damage in patients with 3-hydroxy-3-methylglutaric aciduria.

3-hydroxy-3-methylglutaric aciduria (HMGA) is an inherited disorder of the leucine catabolic pathway in which occurs a deficiency of the 3-hydroxy-3-methylglutaryl-CoA lyase enzyme. Therefore, the organic acids 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA), mainly, accumulate in tissues of affected patients. Lately, much attention has been focused on free radicals as mediators of tissue damage in human diseases, causing lipid peroxidation, protein oxidation and DNA damage. The treatment of this disease is based in a restricted protein ingest and supplementation with l-carnitine (LC), an antioxidant and detoxifying agent. In the present work, we investigated the in vitro oxidative damage to DNA induced by the accumulation of organic acids and oxidative stress parameters in vivo of patients with 3-HMG, as well as the effect of the recommended therapy. The in vitro DNA damage was analyzed by the alkaline comet assay in leukocytes incubated with HMG and MGA (1 mM, 2.5 mM and 5 mM) and co-incubated with LC (90 µM and 150 µM). The in vivo urinary 15-F2t-isoprostane levels and urinary oxidized guanine species were measured by ELISA kits in patient's urine before and after the treatment with LC. HMG and MGA induced a DNA damage index (DI) significantly higher than that of the control group. The DI was significantly reduced in the presence of LC. It was also verified a significant increase of oxidized guanine species and urinary isoprostane levels, biomarker of oxidative DNA damage and lipid peroxidation respectively, in patients before treatment. After the treatment and supplementation with LC, patients presented significantly lower levels of those biomarkers. Analyzing the data together, we can conclude that HMGA patients present oxidative lipid and DNA damage, which is induced by HMG and MGA, and the antioxidant therapy with LC can prevent that kind of injuries.

Bezafibrate In Vivo Administration Prevents 3-Methylglutaric Acid-Induced Impairment of Redox Status, Mitochondrial Biogenesis, and Neural Injury in Brain of Developing Rats.

3-Methylglutaric acid (MGA) is an organic acid that accumulates in 3-methylglutaconic (MGTA) and 3-hydroxy-3-methylglutaric (HMGA) acidurias. Patients affected by these disorders present with neurological dysfunction that usually appears in the first years of life. In order to elucidate the pathomechanisms underlying the brain injury in these disorders, we evaluated the effects of MGA administration on redox homeostasis, mitochondrial respiratory chain activity, and biogenesis in the cerebral cortex of developing rats. Neural damage markers and signaling pathways involved in cell survival, and death were also measured after MGA administration. Furthermore, since the treatment for MGTA and HMGA is still limited, we tested whether a pre-treatment with the pan-peroxisome proliferator-activated receptor (PPAR) agonist bezafibrate could prevent the alterations caused by MGA. MGA provoked lipid peroxidation, increased heme oxygenase-1 content, and altered the activities of antioxidant enzymes, strongly suggestive of oxidative stress. MGA also impaired mitochondrial function and biogenesis by decreasing the activities of succinate dehydrogenase and various respiratory chain complexes, as well as the nuclear levels of PGC-1α and NT-PGC-1α, and cell content of Sirt1. AMPKα1 was further increased by MGA. Neural cell damage was also observed following the MGA administration, as verified by decreased Akt and synaptophysin content and reduced ERK phosphorylation, and by the increase of active caspase-3 and p38 and Tau phosphorylation. Importantly, bezafibrate prevented MGA-elicited toxic effects towards mitochondrial function, redox homeostasis, and neural cell injury, implying that this compound may be potentially used as an adjunct therapy for MGTA and HMGA and other disorders with mitochondrial dysfunction.

New C-2 diastereomers of flavanone glycosides conjugated with 3-hydroxy-3-methylglutaric acid from the pericarp of Citrus grandis (L.) Osbeck.

Two new 3-hydroxy-3-methylglutaryl (HMG) flavanone 7-O-diglycosides, cigranosides A and B (1 and 2), the known naringenin 7-(2''-α-rhamnosyl-6''-(3''''-hydroxy-3''''-methylglutaryl)-glucoside (melitidin, 3), their common biosynthetic precursor flavanone 7-O-diglycoside (naringin, 4), and one known flavone 7-O-diglycoside (rhoifolin, 5) were isolated from the pericarp of Citrus grandis (L.) Osbeck. The structures of these compounds were elucidated by spectroscopic and chemical techniques. The relative ratios and absolute configurations of the C-2 diastereomers of compounds 1, 2 and 4 were determined by online normal-phase HPLC-CD using a Chiralcel column. The absolute configuration of the HMG fragment in compounds 1-3 was assigned to be S through spectroscopic analysis of the mevalonamide obtained by amidation and reduction of the HMG moiety. The NO inhibitory activities of compounds 1-5 were evaluated using lipopolysaccharide-induced RAW264.7 cells. Compounds 1-5 were not cytotoxic to RAW264.7 cells at 10 µM.

CALB-Catalyzed Two-Step Alcoholytic Desymmetrization of 3-Methylglutaric Diazolides in MTBE.

Optically pure 3-substituted glutarates can be prepared from the alcoholic ring-opening of cyclic anhydride derivatives, esterification of 3-substituted glutaric acid, and hydrolysis, alcoholysis, aminolysis, and ammonolysis of the diester derivatives via hydrolases or organocatalysts. Unfortunately, most of them mainly focus on the first-step desymmetrization, leading to the difficulty on producing optically pure enantiomers. As a general trend in lipase-catalyzed desymmetrization of 3-methylglutarates, poorer enantiomeric excesses with lower chemical yields were found, as the methyl substituent is relatively small to induce a high enzyme stereodiscrimination. The two-step desymmetrization for CALB-catalyzed alcoholysis of 3-methylglutaric di-1,2,4-triazolide 1a in anhydrous MTBE is first developed to increase the enzyme activity in each reaction step. The enantioselectivity for the second-step kinetic resolution is furthermore improved by using 3-methylglutaric dipyrazolide 1b as the substrate. The kinetic and thermodynamic analysis is, moreover, addressed for shedding insights into the desymmetrization process.

Selective screening of glutaric acid acidurias by capillary electrophoresis-mass spectrometry.

A sensitive and selective method for the separation and quantification of the three organic acids 3-hydroxy-3-methylglutaric acid, 3-methylglutaric acid, and glutaric acid in human urine samples by CE with mass spectrometry detection has been developed. This methodology is faster, simpler and less time-consuming, than other methodologies previously described, and requires of reduced amounts of reagents as well. Samples are first filtered and then diluted in water. For the electrophoretic separation, a 20mM ammonium acetate and 10% methanol solution at pH 9.1 was selected as the running electrolyte. With 5-s hydrodynamic injection, detection limits ranging from 15.5 to 39.3µM and linear responses ranging from the LOQ calculated for each analyte to more than 400µM were obtained for the analysis of the different organic acids in less than 13min. Remarkable selectivity is achieved by mass spectrometry detection using 0.25% of formic acid in 50% v/v 2-propanol-water solution as sheath liquid, and enough sensitivity without interferences from the matrices was obtained as well. This methodology has revealed as an efficient approach to help the 3-hydroxy-3-methylglutaric aciduria diagnoses in order to discard or confirm the occurrence of the disease as of the presence or absence of the expected increased levels of these analytes in samples of potential patients.

Coupled brain and urine spectroscopy - in vivo metabolomic characterization of HMG-CoA lyase deficiency in 5 patients.

BACKGROUND: 3-Hydroxy-3-Methylglutaryl-Coenzyme A (HMG-CoA) lyase deficiency is a rare inborn error of leucine metabolism and ketogenesis. Despite recurrent hypoglycemia and metabolic decompensations, most patients have a good clinical and neurological outcome contrasting with abnormal brain magnetic resonance imaging (MRI) signals and consistent abnormal brain proton magnetic resonance spectroscopy (1H-MRS) metabolite peaks. Identifying these metabolites could provide surrogate markers of the disease and improve understanding of MRI-clinical discrepancy and follow-up of affected patients. METHODS: Urine samples, brain MRI and 1H-MRS in 5 patients with HMG-CoA lyase deficiency (4 boys and 1 girl aged from 25days to 10years) were, for each patient, obtained on the same day. Brain and urine spectroscopy were performed at the same pH by studying urine at pH 7.4. Due to pH-induced modifications in chemical shifts and because reference 1H NMR spectra are obtained at pH 2.5, spectroscopy of normal urine added with the suspected metabolite was further performed at this pH to validate the correct identification of compounds. RESULTS: Mild to extended abnormal white matter MRI signals were observed in all cases. Brain spectroscopy abnormal peaks at 0.8-1.1ppm, 1.2-1.4ppm and 2.4ppm were also detected by urine spectroscopy at pH 7.4. Taking into account pH-induced changes in chemical shifts, brain abnormal peaks in patients were formally identified to be those of 3-hydroxyisovaleric, 3-methylglutaconic, 3-methylglutaric and 3-hydroxy-3-methylglutaric acids. CONCLUSION: 3-Methylglutaric, 3-hydroxyisovaleric and 3-hydroxy-3-methylglutaric acids identified on urine 1H-NMR spectra of 5 patients with HMG-CoA lyase deficiency are responsible for the cerebral spectroscopy signature seen in these patients, validating their local involvement in brain and putative contribution to brain neuropathology.

3-Hydroxy-3-methylglutaric and 3-methylglutaric acids impair redox status and energy production and transfer in rat heart: relevance for the pathophysiology of cardiac dysfunction in 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase (HL) deficiency is characterized by tissue accumulation of 3-hydroxy-3-methylglutaric (HMG), and 3-methylglutaric (MGA) acids. Affected patients present cardiomyopathy, whose pathomechanisms are not yet established. We investigated the effects of HMG and MGA on energy and redox homeostasis in rat heart using in vivo and in vitro models. In vivo experiments showed that intraperitoneal administration of HMG and MGA decreased the activities of the respiratory chain complex II and creatine kinase (CK), whereas HMG also decreased the activity of complex II-III. Furthermore, HMG and MGA injection increased reactive species production and carbonyl formation, and decreased glutathione concentrations. Regarding the enzymatic antioxidant defenses, HMG and MGA increased glutathione peroxidase (GPx) and glutathione reductase (GR) activities, while only MGA diminished the activities of superoxide dismutase (SOD) and catalase, as well as the protein content of SOD1. Pre-treatment with melatonin (MEL) prevented MGA-induced decrease of CK activity and SOD1 levels. In vitro results demonstrated that HMG and MGA increased reactive species formation, induced lipid peroxidation and decreased glutathione. We also verified that reactive species overproduction and glutathione decrease provoked by HMG and MGA were abrogated by MEL and lipoic acid (LA), while only MEL prevented HMG- and MGA-induced lipoperoxidation. Allopurinol (ALP) also prevented reactive species overproduction caused by both metabolites. Our data provide solid evidence that bioenergetics dysfunction and oxidative stress are induced by HMG and MGA in heart, which may explain the cardiac dysfunction observed in HL deficiency, and also suggest that antioxidant supplementation could be considered as adjuvant therapy for affected patients.

Experimental Evidence that 3-Methylglutaric Acid Disturbs Mitochondrial Function and Induced Oxidative Stress in Rat Brain Synaptosomes: New Converging Mechanisms.

3-Methylglutaric acid (3MGA) is an organic acid that accumulates in various organic acidemias whose patients present neurodegeneration events in children coursing with metabolic acidurias. Limited evidence describes the toxic mechanisms elicited by 3MGA in the brain. Herein, we explored the effects of 3MGA on different toxic endpoints in synaptosomal and mitochondrial-enriched fractions of adult rat brains to provide novel information on early mechanisms evoked by this metabolite. At 1 and 5 mM concentration, 3MGA increased lipid peroxidation, but decreased mitochondrial function only at 5 mM concentration. Despite less intense effects were obtained at 1 mM concentration, its co-administration with the kynurenine pathway (KP) metabolite and N-methyl-D-aspartate receptor (NMDAr) agonist, quinolinic acid (QUIN, 50 and 100 µM), produced toxic synergism on markers of oxidative stress and mitochondrial function. The toxicity of 3MGA per se (5 mM) was prevented by the cannabinoid receptor agonist WIN55,212-2 and the NMDAr antagonist kynurenic acid (KYNA), suggesting cannabinoid and glutamatergic components in the 3MGA pattern of toxicity. The synergic model (3MGA + QUIN) was also sensitive to KYNA and the antioxidant S-allylcysteine, but not to the nitric oxide synthase inhibitor L-nitroarginine methyl ester. These findings suggest various underlying mechanisms involved in the neurotoxicity of 3MGA that may possibly contribute to the neurodegeneration observed in acidemias.

Prenylfuranocoumarin-HMGA-flavonol glucoside conjugates and other constituents of the fruit peels of Citrus hystrix and their anticholinesterase activity.

Sixteen compounds including dihydroxy prenylfuranocoumarins/3-hydroxy-3-methylglutaric acid conjugates and dihydroxy prenylfuranocoumarins/3-hydroxy-3-methylglutaric acid/1-O-flavonyl-ß-d-glucopyranoside conjugates, together with other dihydroxyprenylfuranocoumarins conjugates, were isolated from the ethyl acetate extract of the fruit peels of Citrus hystrix. Some of the isolates were evaluated for their cholinesterase inhibitory activity, but only one compound possessing a 3-O-ß-d-glucopyranosyl-3,5,7,4'-tetrahydroxy-6,8,3'-trimethoxyflavonol nucleus in the prenylfuranocoumarin-HMGA conjugate showed strong activity.

Induction of a Proinflammatory Response in Cortical Astrocytes by the Major Metabolites Accumulating in HMG-CoA Lyase Deficiency: the Role of ERK Signaling Pathway in Cytokine Release.

3-Hydroxy-3-methylglutaric aciduria (HMGA) is an inherited metabolic disorder caused by 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. It is biochemically characterized by predominant tissue accumulation and high urinary excretion of 3-hydroxy-3-methylglutarate (HMG) and 3-methylglutarate (MGA). Affected patients commonly present acute symptoms during metabolic decompensation, including vomiting, seizures, and lethargy/coma accompanied by metabolic acidosis and hypoketotic hypoglycemia. Although neurological manifestations are common, the pathogenesis of brain injury in this disease is poorly known. Astrocytes are important for neuronal protection and are susceptible to damage by neurotoxins. In the present study, we investigated the effects of HMG and MGA on important parameters of redox homeostasis and cytokine production in cortical cultured astrocytes. The role of the metabolites on astrocyte mitochondrial function (thiazolyl blue tetrazolium bromide (MTT) reduction) and viability (propidium iodide incorporation) was also studied. Both organic acids decreased astrocytic mitochondrial function and the concentrations of reduced glutathione without altering cell viability. In contrast, they increased reactive species formation (2'-7'-dichlorofluorescein diacetate (DCFHDA) oxidation), as well as IL-1ß, IL-6, and TNF α release through the ERK signaling pathway. Taken together, the data indicate that the principal compounds accumulating in HMGA induce a proinflammatory response in cultured astrocytes that may possibly be involved in the neuropathology of this disease.

Evidence that 3-hydroxy-3-methylglutaric and 3-methylglutaric acids induce DNA damage in rat striatum.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a rare autosomal recessive disorderaffecting the final step of leucine degradation and ketogenesis and biochemically characterized by the predominant accumulation of 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA) acids in biological fluids and tissues of affected patients. Considering that previous studies reported that HMG and MGA have pro oxidant properties, the present study evaluated the ex vivo and in vitro effects of HMG and MGA on frequency and index of DNA damage in cerebral cortex and striatum of young rats. The ex vivo effects of both organic acids on 8-hydroxy-2'-deoxyguanosine (OHdG) levels and their in vitro effects on 2',7'-dichlorofluorescin (DCFH) oxidation and glutathione (GSH) concentrations in rat striatum were also determined. We also investigated the ex vivo effects of both organic acids on 8-hydroxy-2'-deoxyguanosine (OHdG) levels in rat striatum. In the ex vivo experiments, DNA damage was determined in striatum homogenates prepared 30 min after a single intrastriatal administration of HMG or MGA. On the other hand, the in vitro evaluation was performed after an incubation of rat cerebral cortex or striatum homogenates or slices in the presence of HMG or MGA during 1 h at 37 °C. We observed that the intrastriatal administration of HMG and MGA increased the frequency and the index of DNA damage, as well as OHdG staining in rat striatum. We also verified that MGA, but not HMG, increased DNA damage frequency and index in vitro in striatum of rats. In contrast, no alterations were verified in vitro in cerebral cortex. Finally, we found that HMG and MGA increased DCFH oxidation and decreased GSH concentrations in rat striatum. Therefore, it may be presumed that DNA damage provoked by HMG and MGA possibly via reactive species generation is involved, at least in part, in the pathophysiology of brain injury, particularly in the striatum of HL-deficient patients.

The neuropsychological profile of patients with 3-methylglutaconic aciduria type III, Costeff syndrome.

Costeff syndrome is a rare genetic neuro-ophthalmological syndrome consisting of early-onset bilateral optic atrophy along with a progressive complex motor disorder with elevated levels of urinary 3-methylglutaconic acid and 3-methylglutaric acid. While borderline to mild cognitive deficits have been considered to be common in patients with this syndrome, a comprehensive cognitive assessment has never been performed. The aim of the current study was to explore the cognitive profile associated with Costeff syndrome. Sixteen adult patients diagnosed with Costeff syndrome were administered a neuropsychological test battery that was composed of standardized verbal tests adapted for the blind. General intelligence ranged from average to borderline, with a group mean consistent with intact general cognitive functioning (VIQmean = 85, z = -1) in the low-average range of the general population. The auditory immediate and delayed memory indexes were in the average range and were significantly higher than the general cognitive functioning, whereas the working memory index was significantly lower than the general cognitive functioning. Adult patients with Costeff syndrome have intact global cognition and learning abilities and strong auditory memory performance. © 2015 Wiley Periodicals, Inc.