Biallelic variants in the SLC13A1 sulfate transporter gene cause hyposulfatemia with a mild spondylo-epi-metaphyseal dysplasia.

Sulfate is the fourth most abundant anion in human plasma but is not measured in clinical practice and little is known about the consequences of sulfate deficiency. Nevertheless, sulfation plays an essential role in the modulation of numerous compounds, including proteoglycans and steroids. We report the first patient with a homozygous loss-of-function variant in the SLC13A1 gene, encoding a renal and intestinal sulfate transporter, which is essential for maintaining plasma sulfate levels. The homozygous (Arg12Ter) variant in SLC13A1 was found by exome sequencing performed in a patient with unexplained skeletal dysplasia. The main clinical features were enlargement of joints and spondylo-epi-metaphyseal radiological abnormalities in early childhood, which improved with age. In addition, autistic features were noted. We found profound hyposulfatemia due to complete loss of renal sulfate reabsorption. Cholesterol sulfate was reduced. Intravenous N-acetylcysteine administration temporarily restored plasma sulfate levels. We conclude that loss of the SLC13A1 gene leads to profound hypersulfaturia and hyposulfatemia, which is mainly associated with abnormal skeletal development, possibly predisposing to degenerative bone and joint disease. The diagnosis might be easily missed and more frequent.

Fecal Amino Acid Profiles Exceed Accuracy of Serum Amino Acids in Diagnosing Pediatric Inflammatory Bowel Disease.

In this prospective intention-to-diagnose pilot study, we aimed to assess accuracy of serum and fecal amino-acids to discriminate de novo pediatric inflammatory bowel disease (IBD) and non-IBD children. Patients with suspected IBD were allocated the IBD (n = 11) or non-IBD group (n = 8) following laboratory testing or endoscopy according to the revised Porto-criteria. Fecal calprotectin levels were obtained, an additional blood and fecal sample were collected. Fecal and serum amino-acid profiles were analyzed using high performance-liquid chromatography. Nine fecal amino-acids (alanine [area under the curve 0.94], citrulline [0.94], glutamine [0.89], leucine [0.98], lysine [0.89], phenylalanine [0.99], serine [0.91], tyrosine [0.96], and valine [0.95]) differed significantly between IBD and non-IBD. In serum, no significant differences were observed. This study underlines the potential of fecal amino-acids as novel, adjuvant noninvasive, and low-cost biomarkers in the diagnostic work-up of pediatric IBD detection.

D-2-hydroxyglutaric aciduria Type I: Functional analysis of D2HGDH missense variants.

D-2-hydroxyglutaric aciduria Type I (D-2-HGA Type I), a neurometabolic disorder with a broad clinical spectrum, is caused by recessive variants in the D2HGDH gene encoding D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). We and others detected 42 potentially pathogenic variants in D2HGDH of which 31 were missense. We developed functional studies to investigate the effect of missense variants on D-2-HGDH catalytic activity. Site-directed mutagenesis was used to introduce 31 missense variants in the pCMV5-D2HGDH expression vector. The wild type and missense variants were overexpressed in HEK293 cells. D-2-HGDH enzyme activity was evaluated based on the conversion of [2 H4 ]D-2-HG to [2 H4 ]2-ketoglutarate, which was subsequently converted into [2 H4 ]L-glutamate and the latter quantified by LC-MS/MS. Eighteen variants resulted in almost complete ablation of D-2-HGDH activity and thus, should be considered pathogenic. The remaining 13 variants manifested residual activities ranging between 17% and 94% of control enzymatic activity. Our functional assay evaluating the effect of novel D2HGDH variants will be beneficial for the classification of missense variants and determination of pathogenicity.

Maternal glutamine supplementation in murine succinic semialdehyde dehydrogenase deficiency, a disorder of γ-aminobutyric acid metabolism.

Murine succinic semialdehyde dehydrogenase deficiency (SSADHD) manifests with high concentrations of γ-aminobutyric acid (GABA) and γ-hydroxybutyrate (GHB) and low glutamine in the brain. To understand the pathogenic contribution of central glutamine deficiency, we exposed aldh5a1-/- (SSADHD) mice and their genetic controls (aldh5a1+/+ ) to either a 4% (w/w) glutamine-containing diet or a glutamine-free diet from conception until postnatal day 30. Endpoints included brain, liver and blood amino acids, brain GHB, ataxia scores, and open field testing. Glutamine supplementation did not improve aldh5a1-/- brain glutamine deficiency nor brain GABA and GHB. It decreased brain glutamate but did not change the ratio of excitatory (glutamate) to inhibitory (GABA) neurotransmitters. In contrast, glutamine supplementation significantly increased brain arginine (30% for aldh5a1+/+ and 18% for aldh5a1-/- mice), and leucine (12% and 18%). Glutamine deficiency was confirmed in the liver. The test diet increased hepatic glutamate in both genotypes, decreased glutamine in aldh5a1+/+ but not in aldh5a1-/- , but had no effect on GABA. Dried bloodspot analyses showed significantly elevated GABA in mutants (approximately 800% above controls) and decreased glutamate (approximately 25%), but no glutamine difference with controls. Glutamine supplementation did not impact blood GABA but significantly increased glutamine and glutamate in both genotypes indicating systemic exposure to dietary glutamine. Ataxia and pronounced hyperactivity were observed in aldh5a1-/- mice but remained unchanged by the diet intervention. The study suggests that glutamine supplementation improves peripheral but not central glutamine deficiency in experimental SSADHD. Future studies are needed to fully understand the pathogenic role of brain glutamine deficiency in SSADHD.

Severe infantile epileptic encephalopathy associated with D-glyceric aciduria: report of a novel case and review.

D-glycerate 2 kinase (DGK) is an enzyme that mediates the conversion of D-glycerate, an intermediate metabolite of serine and fructose metabolism, to 2-phosphoglycerate. Deficiency of DGK leads to accumulation of D-glycerate in various tissues and its massive excretion in urine. D-glyceric aciduria (DGA) is an autosomal recessive metabolic disorder caused by mutations in the GLYCTK gene. The clinical spectrum of DGA is highly variable, ranging from severe progressive infantile encephalopathy to a practically asymptomatic condition. We describe a male patient from a consanguineous Arab family with infantile onset of DGA, characterized by profound psychomotor retardation, progressive microcephaly, intractable seizures, cortical blindness and deafness. Consecutive brain MR imaging showed an evolving brain atrophy, thinning of the corpus callosum and diffuse abnormal white matter signals. Whole exome sequencing identified the homozygous missense variant in the GLYCTK gene [c.455 T > C, NM_145262.3], which affected a highly conserved leucine residue located at a domain of yet unknown function of the enzyme [p.Leu152Pro, NP_660305]. In silico analysis of the variant supported its pathogenicity. A review of the 15 previously reported patients, together with the current one, confirms a clear association between DGA and severe neurological impairment. Yet, future studies of additional patients with DGA are required to better understand the clinical phenotype and pathogenesis.

An overview of combined D-2- and L-2-hydroxyglutaric aciduria: functional analysis of CIC variants.

Combined D-2- and L-2-hydroxyglutaric aciduria (D/L-2-HGA) is a devastating neurometabolic disorder, usually lethal in the first years of life. Autosomal recessive mutations in the SLC25A1 gene, which encodes the mitochondrial citrate carrier (CIC), were previously detected in patients affected with combined D/L-2-HGA. We showed that transfection of deficient fibroblasts with wild-type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels, confirming that deficient CIC is the cause of D/L-2-HGA. We developed and implemented a functional assay and applied it to all 17 missense variants detected in a total of 26 CIC-deficient patients, including eight novel cases, showing reduced activities of varying degrees. In addition, we analyzed the importance of residues affected by these missense variants using our existing scoring system. This allowed not only a clinical and biochemical overview of the D/L-2-HGA patients but also phenotype-genotype correlation studies.

A small molecule inhibitor of mutant IDH2 rescues cardiomyopathy in a D-2-hydroxyglutaric aciduria type II mouse model.

D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.

Plants Possess a Cyclic Mitochondrial Metabolic Pathway similar to the Mammalian Metabolic Repair Mechanism Involving Malate Dehydrogenase and l-2-Hydroxyglutarate Dehydrogenase.

Enzymatic side reactions can give rise to the formation of wasteful and toxic products that are removed by metabolite repair pathways. In this work, we identify and characterize a mitochondrial metabolic repair mechanism in Arabidopsis thaliana involving malate dehydrogenase (mMDH) and l-2-hydroxyglutarate dehydrogenase (l-2HGDH). We analyze the kinetic properties of both A. thaliana mMDH isoforms, and show that they produce l-2-hydroxyglutarate (l-2HG) from 2-ketoglutarate (2-KG) at low rates in side reactions. We identify A. thaliana l-2HGDH as a mitochondrial FAD-containing oxidase that converts l-2HG back to 2-KG. Using loss-of-function mutants, we show that the electrons produced in the l-2HGDH reaction are transferred to the mitochondrial electron transport chain through the electron transfer protein (ETF). Thus, plants possess the biochemical components of an l-2HG metabolic repair system identical to that found in mammals. While deficiencies in the metabolism of l-2HG result in fatal disorders in mammals, accumulation of l-2HG in plants does not adversely affect their development under a range of tested conditions. However, orthologs of l-2HGDH are found in all examined genomes of viridiplantae, indicating that the repair reaction we identified makes an essential contribution to plant fitness in as yet unidentified conditions in the wild.

Human pyrroline-5-carboxylate reductase (PYCR1) acts on Δ(1)-piperideine-6-carboxylate generating L-pipecolic acid.

We have conducted biochemical studies with commercial available pyrroline-5-carboxylate (P5C) reductase (PYCR1) to investigate whether this enzyme plays a role in L-lysine degradation. Our recent studies with antiquitin/ALDH7A1 deficient fibroblasts revealed an alternative genesis of L-pipecolic acid, and we then hypothesized that PYCR1 was responsible for the conversion of Δ(1)-piperideine-6-carboxylate (P6C) into pipecolic acid. We here present evidence that PYCR1 is indeed able to produce L-pipecolic acid from P6C preparations, and the observed K m for this conversion is of the same magnitude as the K m described for the conversion of P5C to L-proline by PYCR1. Urine samples from antiquitin deficient individuals, who accumulate P6C, were also incubated with PYCR1 which resulted in a marked decrease of P6C and a huge increase of L-pipecolic acid as measured by LC-MS/MS, confirming that indeed PYCR1 generates L-pipecolic acid from P6C.

Plant D-2-hydroxyglutarate dehydrogenase participates in the catabolism of lysine especially during senescence.

D-2-Hydroxyglutarate dehydrogenase (D-2HGDH) catalyzes the specific and efficient oxidation of D-2-hydroxyglutarate (D-2HG) to 2-oxoglutarate using FAD as a cofactor. In this work, we demonstrate that D-2HGDH localizes to plant mitochondria and that its expression increases gradually during developmental and dark-induced senescence in Arabidopsis thaliana, indicating an enhanced demand of respiration of alternative substrates through this enzymatic system under these conditions. Using loss-of-function mutants in D-2HGDH (d2hgdh1) and stable isotope dilution LC-MS/MS, we found that the D-isomer of 2HG accumulated in leaves of d2hgdh1 during both forms of carbon starvation. In addition to this, d2hgdh1 presented enhanced levels of most TCA cycle intermediates and free amino acids. In contrast to the deleterious effects caused by a deficiency in D-2HGDH in humans, d2hgdh1 and overexpressing lines of D-2HGDH showed normal developmental and senescence phenotypes, indicating a mild role of D-2HGDH in the tested conditions. Moreover, metabolic fingerprinting of leaves of plants grown in media supplemented with putative precursors indicated that D-2HG most probably originates during the catabolism of lysine. Finally, the L-isomer of 2HG was also detected in leaf extracts, indicating that both chiral forms of 2HG participate in plant metabolism.

IDH1 R132H decreases proliferation of glioma cell lines in vitro and in vivo.

OBJECTIVE: A high percentage of grade II and III gliomas have mutations in the gene encoding isocitrate dehydrogenase (IDH1). This mutation is always a heterozygous point mutation that affects the amino acid arginine at position 132 and results in loss of its native enzymatic activity and gain of alternative enzymatic activity (producing D-2-hydroxyglutarate). The objective of this study was to investigate the cellular effects of R132H mutations in IDH1. METHODS: Functional consequences of IDH1(R132H) mutations were examined among others using fluorescence-activated cell sorting, kinome and expression arrays, biochemical assays, and intracranial injections on 3 different (glioma) cell lines with stable overexpression of IDH1(R132H) . RESULTS: IDH1(R132H) overexpression in established glioma cell lines in vitro resulted in a marked decrease in proliferation, decreased Akt phosphorylation, altered morphology, and a more contact-dependent cell migration. The reduced proliferation is related to accumulation of D-2-hydroxyglutarate that is produced by IDH1(R132H) . Mice injected with IDH1(R132H) U87 cells have prolonged survival compared to mice injected with IDH1(wt) or green fluorescent protein-expressing U87 cells. INTERPRETATION: Our results demonstrate that IDH1(R132H) dominantly reduces aggressiveness of established glioma cell lines in vitro and in vivo. In addition, the IDH1(R132H) -IDH1(wt) heterodimer has higher enzymatic activity than the IDH1(R132H) -IDH1(R132H) homodimer. Our observations in model systems of glioma might lead to a better understanding of the biology of IDH1 mutant gliomas, which are typically low grade and often slow growing.

Fecal Amino Acid Analysis in Newly Diagnosed Pediatric Inflammatory Bowel Disease: A Multicenter Case-Control Study.

BACKGROUND: Fecal metabolomic profiles differ between pediatric inflammatory bowel disease (IBD) patients and controls and may provide new insights in the pathophysiology of IBD. The role of amino acids, however, is not fully elucidated. We aimed to assess fecal amino acid profiles in pediatric IBD. METHODS: In this case-control study, treatment-naïve, newly diagnosed pediatric IBD patients and a non-IBD control group, matched based on sex and age, were included in 2 tertiary centres. Fecal amino acid profiles were assessed using a targeted high-performance liquid chromatography technique. A random forest classifier method was used to develop a prediction model differentiating IBD from controls and predicting IBD phenotype. The association between IBD localization and amino acid concentrations was tested with ordinal regression models. RESULTS: We included 78 newly diagnosed IBD patients (40 Crohn's disease [CD], 38 ulcerative colitis [UC]) and 105 controls. Patients with IBD could be differentiated from controls with an accuracy of 82% (sensitivity 63%, specificity 97%). Twenty-nine out of the 42 measured unique amino acids were included in the prediction model. Increased levels of tryptophan, taurine, alanine, ornithine, valine, histidine, and leucine were the most differentiating features. Children with CD and UC could be differentiated from the controls with an accuracy of 80% and 90%, respectively. Inflammatory bowel disease phenotype could not be predicted. Tryptophan, valine, and histidine levels were positively associated with more extended disease in UC patients (P < .05). CONCLUSIONS: Fecal amino acids may enhance understanding of the role of host-microbial interactions in the pathophysiology of IBD and may evolve into biomarkers for pediatric IBD diagnostic and personalized medicine.

Fecal amino acid analysis could differentiate newly diagnosed children with IBD from a non-IBD control group with an accuracy of 82%. Increased levels of tryptophan, taurine, alanine, ornithine, and valine were the most differentiating features. This may enhance understanding of IBD pathophysiology.

Is impaired energy production a novel insight into the pathogenesis of pyridoxine-dependent epilepsy due to biallelic variants in ALDH7A1?

BACKGROUND: Pyridoxine-dependent epilepsy (PDE) is due to biallelic variants in ALDH7A1 (PDE-ALDH7A1). ALDH7A1 encodes α-aminoadipic semialdehyde dehydrogenase in lysine catabolism. We investigated the gamma aminobutyric acid (GABA) metabolism and energy production pathways in human PDE-ALDH7A1 and its knock-out aldh7a1 zebrafish model. METHODS: We measured GABA pathway, and tricarboxylic acid cycle metabolites and electron transport chain activities in patients with PDE-ALDH7A1 and in knock-out aldh7a1 zebrafish. RESULTS: We report results of three patients with PDE-ALDH7A1: low paired complex I+II and complex II+III and individual complex IV activities in muscle biopsy in patient 1 (likely more severe phenotype); significantly elevated CSF glutamate in the GABA pathway and elevated CSF citrate, succinate, isocitrate and α-ketoglutarate in the TCA cycle in patient 3 (likely more severe phenotype); and normal CSF GABA pathway and TCA cycle metabolites on long-term pyridoxine therapy in patient 2 (likely milder phenotype). All GABA pathway metabolites (γ-hydroxybutyrate, glutamine, glutamate, total GABA, succinic semialdehyde) and TCA cycle metabolites (citrate, malate, fumarate, isocitrate, lactate) were significantly low in the homozygous knock-out aldh7a1 zebrafish compared to the wildtype zebrafish. Homozygous knock-out aldh7a1 zebrafish had decreased electron transport chain enzyme activities compared to wildtype zebrafish. DISCUSSION: We report impaired electron transport chain function, accumulation of glutamate in the central nervous system and TCA cycle dysfunction in human PDE-ALDH7A1 and abnormal GABA pathway, TCA cycle and electron transport chain in knock-out aldh7a1 zebrafish. Central nervous system glutamate toxicity and impaired energy production may play important roles in the disease neuropathogenesis and severity in human PDE-ALDH7A1.

Preferential accumulation of the active S-(+) isomer in murine retina highlights novel mechanisms of vigabatrin-associated retinal toxicity.

((S)-(+)/(R)-(-)) vigabatrin (SabrilR; γ-vinyl GABA), an antiepileptic irreversibly inactivating GABA-transaminase, was administered to male C57Bl6 J mice via continuous infusion (0, 40, 80 mg/kg/d) for 12 days. Our study design pooled retina, eye (minus retina), whole brain and plasma from n = 24 animals for each dose to provide n = 8 triplicates per treatment group. Hypothesizing that (S)-(+) VGB (active isomer) would preferentially accumulate in retina, we determined VGB isomers, comprehensive amino acids, and pharmacokinetic parameters. In brain, eye and plasma, the ((S)-(+)/(R)-(-)) ratio varied from 0.73 to 1.29 and 13.3 in retina, accompanied by a partition coefficient (tissue/plasma, ((S)-(+);(R)-(-))) of 5.8;0.34, 0.63;0.49, and 0.51;0.34 in retina, eye and brain, respectively. Racemic VGB (nmol/g; plasma, nmol/mL, range of means for dose) content was: retina, 25-36; eye (minus retina), 4.8-8.0; brain, 3.1-6.8 and plasma, 8.7-14.9. GABA tissue content (nmol/g) was 1246-3335, 18-64 and 2615-3200 as a function of VGB dose for retina, eye (minus retina) and brain, respectively. The retinal glial cell toxin 2-aminoadipic acid also increased with VGB dose (76-96 nmol/g). Partitioning of active (S)-(+) VGB to retina suggests the involvement of a stereospecific transporter, the identification of which could reveal new therapeutic paradigms that might mitigate VGB's well-known retinal toxicity and expand its clinical utility.

Hepatocyte transplantation (HTx) corrects selected neurometabolic abnormalities in murine intermediate maple syrup urine disease (iMSUD).

Skvorak et al. [1] demonstrated the therapeutic efficacy of HTx in a murine model of iMSUD, confirming significant metabolic improvement and survival. To determine the effect of HTx on extrahepatic organs, we examined the metabolic effects of HTx in brain from iMSUD animals. Amino acid analysis revealed that HTx corrected increased ornithine, partially corrected depleted glutamine, and revealed a trend toward alloisoleucine correction. For amino acid and monoamine neurotransmitters, decreased GABA was partially corrected with HTx, while the l-histidine dipeptide of GABA, homocarnosine, was decreased in iMSUD mice and hypercorrected following HTx. Elevated branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in MSUD can deplete brain tyrosine and tryptophan (the precursors of monoamine neurotransmitters, dopamine (DA) and serotonin (5-hydroxytryptamine; 5-HT)) through competition via the large neutral amino acid transporter. HTx corrected decreased DA levels and the DA metabolite, 3-methoxytyramine, and partially corrected the DA intermediate 3,4-dihydroxyphenylacetate (DOPAC) and 5-HT levels, despite normal tyrosine and tryptophan levels in iMSUD mouse brain. We further observed enhanced intracellular turnover of both DA and 5-HT in iMSUD mouse brain, both of which partially corrected with HTx. Our results suggest new pathomechanisms of neurotransmitter metabolism in this disorder and support the therapeutic relevance of HTx in iMSUD mice, while providing proof-of-principle that HTx has corrective potential in extrahepatic organs.

Metabolomic analyses of vigabatrin (VGB)-treated mice: GABA-transaminase inhibition significantly alters amino acid profiles in murine neural and non-neural tissues.

The anticonvulsant vigabatrin (VGB; SabrilR) irreversibly inhibits GABA transaminase to increase neural GABA, yet its mechanism of retinal toxicity remains unclear. VGB is suggested to alter several amino acids, including homocarnosine, ß-alanine, ornithine, glycine, taurine, and 2-aminoadipic acid (AADA), the latter a homologue of glutamic acid. Here, we evaluate the effect of VGB on amino acid concentrations in mice, employing a continuous VGB infusion (subcutaneously implanted osmotic minipumps), dose-escalation paradigm (35-140 mg/kg/d, 12 days), and amino acid quantitation in eye, visual and prefrontal cortex, total brain, liver and plasma. We hypothesized that continuous VGB dosing would reveal numerous hitherto undescribed amino acid disturbances. Consistent amino acid elevations across tissues included GABA, ß-alanine, carnosine, ornithine and AADA, as well as neuroactive aspartic and glutamic acids, serine and glycine. Maximal increase of AADA in eye occurred at 35 mg/kg/d (41 ±â€¯2 nmol/g (n = 21, vehicle) to 60 ±â€¯8.5 (n = 8)), and at 70 mg/kg/d for brain (97 ±â€¯6 (n = 21) to 145 ±â€¯6 (n = 6)), visual cortex (128 ±â€¯6 to 215 ±â€¯19) and prefrontal cortex (124 ±â€¯11 to 200 ±â€¯13; mean ±â€¯SEM; p < 0.05), the first demonstration of tissue AADA accumulation with VGB in mammal. VGB effects on basic amino acids, including guanidino-species, suggested the capacity of VGB to alter urea cycle function and nitrogen disposal. The known toxicity of AADA in retinal glial cells highlights new avenues for assessing VGB retinal toxicity and other off-target effects.

Preclinical tissue distribution and metabolic correlations of vigabatrin, an antiepileptic drug associated with potential use-limiting visual field defects.

Vigabatrin (VGB; (S)-(+)/(R)-(-) 4-aminohex-5-enoic acid), an antiepileptic irreversibly inactivating GABA transaminase (GABA-T), manifests use-limiting ocular toxicity. Hypothesizing that the active S enantiomer of VGB would preferentially accumulate in eye and visual cortex (VC) as one potential mechanism for ocular toxicity, we infused racemic VGB into mice via subcutaneous minipump at 35, 70, and 140 mg/kg/d (n = 6-8 animals/dose) for 12 days. VGB enantiomers, total GABA and ß-alanine (BALA), 4-guanidinobutyrate (4-GBA), and creatine were quantified by mass spectrometry in eye, brain, liver, prefrontal cortex (PFC), and VC. Plasma VGB concentrations increased linearly by dose (3 ± 0.76 (35 mg/kg/d); 15.1 ± 1.4 (70 mg/kg/d); 34.6 ± 3.2 µmol/L (140 mg/kg/d); mean ± SEM) with an S/R ratio of 0.74 ± 0.02 (n = 14). Steady state S/R ratios (35, 70 mg/kg/d doses) were highest in eye (5.5 ± 0.2; P < 0.0001), followed by VC (3.9 ± 0.4), PFC (3.6 ± 0.3), liver (2.9 ± 0.1), and brain (1.5 ± 0.1; n = 13-14 each). Total VGB content of eye exceeded that of brain, PFC and VC at all doses. High-dose VGB diminished endogenous metabolite production, especially in PFC and VC. GABA significantly increased in all tissues (all doses) except brain; BALA increases were confined to liver and VC; and 4-GBA was prominently increased in brain, PFC and VC (and eye at high dose). Linear correlations between enantiomers and GABA were observed in all tissues, but only in PFC/VC for BALA, 4-GBA, and creatine. Preferential accumulation of the VGB S isomer in eye and VC may provide new insight into VGB ocular toxicity.

Pre-analytical stability of novel cerebrospinal fluid biomarkers.

Stability of the cerebrospinal fluid (CSF) composition under different pre-analytical conditions is relevant for the diagnostic potential of biomarkers. Our aim was to examine the pre-analytical stability of promising CSF biomarkers that are currently evaluated for their discriminative use in various neurological diseases. Pooled CSF was aliquoted and experimentally exposed to delayed storage: 0, 1, 2, 4, 24, 72, or 168 h at 4 °C or room temperature (RT), or 1-4 months at -20 °C; or up to 7 freeze/thaw (f/t) cycles, before final storage at -80 °C. Eleven CSF biomarkers were screened using immunoassays, liquid chromatography, or enzymatic methods. Levels of neurogranin (truncP75), chitinase-3-like protein (YKL-40), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), acetylcholinesterase (AChE) enzymatic activity, theobromine, secreted protein acidic and rich in cysteine-like 1 (SPARCL-1) and homovanillic acid (HVA) levels were not affected by the applied storage conditions. 3-Methoxy-4-hydroxyphenylglycol (MHPG) levels linearly and strongly decreased after 4 h at RT (-10%) or 24 h at 4 °C (-27%), and with 6% after every f/t cycle. 5-Methyltetrahydrofolate (5-MTHF) (-29% after 1 week at RT) and 5-hydroxyindoleacetic acid levels (5-HIAA) (-16% after 1 week at RT) were reduced and 3,4-dihydroxyphenylacetic acid (DOPAC) levels (+22% after 1 week at RT) increased, but only after >24 h at RT. Ten out of eleven potential CSF novel biomarkers showed very limited change under common storage and f/t conditions, suggesting that these CSF biomarkers can be trustfully tested under the pre-analytical conditions present across different cohorts.

Sedoheptulokinase deficiency due to a 57-kb deletion in cystinosis patients causes urinary accumulation of sedoheptulose: elucidation of the CARKL gene.

The most common mutation in the nephropathic cystinosis (CTNS) gene is a homozygous 57-kb deletion that also includes an adjacent gene carbohydrate kinase-like (CARKL). The latter gene encodes a protein that is predicted to function as a carbohydrate kinase. Cystinosis patients with the common 57-kb deletion had strongly elevated urinary concentrations of sedoheptulose (28-451 mmol/mol creatinine; controls and other cystinosis patients <9) and erythritol (234-1110 mmol/mol creatinine; controls and other cystinosis patients <148). Enzyme studies performed on fibroblast homogenates derived from patients carrying the 57-kb deletion revealed 80% reduction in their sedoheptulose phosphorylating activity compared to cystinosis patients with other mutations and controls. This indicates that the CARKL-encoded protein, sedoheptulokinase (SHK), is responsible for the reaction: sedoheptulose + ATP --> sedoheptulose-7-phosphate + ADP and that deletion of CARKL causes urinary accumulation of sedoheptulose and erythritol.

Neurotransmitter alterations in embryonic succinate semialdehyde dehydrogenase (SSADH) deficiency suggest a heightened excitatory state during development.

BACKGROUND: SSADH (aldehyde dehydrogenase 5a1 (Aldh5a1); gamma-hydroxybutyric (GHB) aciduria) deficiency is a defect of GABA degradation in which the neuromodulators GABA and GHB accumulate. The human phenotype is that of nonprogressive encephalopathy with prominent bilateral discoloration of the globi pallidi and variable seizures, the latter displayed prominently in Aldh5a1-/- mice with lethal convulsions. Metabolic studies in murine neural tissue have revealed elevated GABA [and its derivatives succinate semialdehyde (SSA), homocarnosine (HC), 4,5-dihydroxyhexanoic acid (DHHA) and guanidinobutyrate (GB)] and GHB [and its analogue D-2-hydroxyglutarate (D-2-HG)] at birth. Because of early onset seizures and the neurostructural anomalies observed in patients, we examined metabolite features during Aldh5a1-/- embryo development. METHODS: Embryos were obtained from pregnant dams sacrificed at E (embryo day of life) 10-13, 14-15, 16-17, 18-19 and newborn mice. Intact embryos were extracted and metabolites quantified by isotope dilution mass spectrometry (n = 5-15 subjects, Aldh5a1+/+ and Aldh5a1-/-) for each gestational age group. Data was evaluated using the t test and one-way ANOVA with Tukey post hoc analysis. Significance was set at the 95th centile. RESULTS: GABA and DHHA were significantly elevated at all gestational ages in Aldh5a1-/- mice, while GB was increased only late in gestation; SSA was not elevated at any time point. GHB and D-2-HG increased in an approximately linear fashion with gestational age. Correlative studies in human amniotic fluid from SSADH-deficient pregnancies (n = 5) also revealed significantly increased GABA. CONCLUSION: Our findings indicate early GABAergic alterations in Aldh5a1-/- mice, possibly exacerbated by other metabolites, which likely induce a heightened excitatory state that may predispose neural networks to epilepsy in these animals.