Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography – tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.
Urea cycle disorders (UCD) are inherited defects in clearance of waste nitrogen with high morbidity and mortality. Novel and more effective therapies for UCD are needed. Studies in mice with constitutive activation of autophagy unravelled Beclin‐1 as druggable candidate for therapy of hyperammonemia. Next, we investigated efficacy of cell‐penetrating autophagy‐inducing Tat‐Beclin‐1 (TB‐1) peptide for therapy of the two most common UCD, namely ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) deficiencies. TB‐1 reduced urinary orotic acid and improved survival under protein‐rich diet in spf‐ash mice, a model of OTC deficiency (proximal UCD). In AslNeo/Neo mice, a model of ASL deficiency (distal UCD), TB‐1 increased ureagenesis, reduced argininosuccinate, and improved survival. Moreover, it alleviated hepatocellular injury and decreased both cytoplasmic and nuclear glycogen accumulation in AslNeo/Neo mice. In conclusion, Beclin‐1‐dependent activation of autophagy improved biochemical and clinical phenotypes of proximal and distal defects of the urea cycle.
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Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of β-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1−/− mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1−/− liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1−/− mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1−/− mice in better understanding disease mechanism in fatty acid α-oxidation disorders.
Argininosuccinate lyase (ASL) is a key enzyme integral to the hepatic urea cycle which is required for ammonia detoxification, and the citrulline-nitric oxide (NO) cycle for NO production. ASL deficient patients present with argininosuccinic aciduria (ASA), an inherited metabolic disease with hyperammonaemia and a chronic systemic phenotype with neurocognitive impairment and chronic liver disease. ASL deficiency as an inherited model of systemic NO deficiency, shows enhanced nitrosative and oxidative stress. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics andin vivopositron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-L-glutamate ([18F]FSPG). Upregulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways.hASLmRNA encapsulated in lipid nanoparticles corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis and glutathione metabolism and ameliorating chronic liver disease. We further present [18F]FSPG PET as a novel non-invasive diagnostic tool to assess liver disease and therapeutic efficacy in ASA. These findings support clinical translation of mRNA therapy for ASA.
The present study examined whether a single or multiple episode(s) of status epilepticus induced with kainic acid (KA) during the first 3 weeks of postnatal (P) development would aberrantly stimulate proliferation zones that alters migration to potentially injured areas and whether they would be blocked by selective Group I mGluR antagonists. mGluR1α (LY367385) and mGluR5 (MPEP) antagonists were administered 2h following KA-induced status epilepticus and animals were examined after 7days. Proliferating cells of the subventricular zone (SVZ), third ventricle, hippocampus, amygdala cortical complex were analyzed with the proliferative marker, Ki67; and two complementary retrograde dye tracers. Proliferation increased in extrahippocampal limbic structures when KA was administered on P13 or P20 which correlated with number of injured cells at the older age. LY367385 post-treatment caused striking decreases in proliferation in all limbic structures in the presence and absence of injury, whereas a reduction with MPEP was observed only within the amygdala cortical complex (Amg/ERcx) in the presence of multiple seizures (3×KA). After 3×KA and LY367385 post-treatments, diminished co-staining of dye tracers with Ki67 was observed within the Amg/ERcx despite high levels of progenitors marked by the retrograde tracers in this region. This indicates that not only was local proliferation within the SVZ and distant structures inhibited, but also that migration itself was reduced indirectly since there were less cells to migrate from the SVZ. Co-labeling with biomarkers provided evidence for neuronal differentiation suggesting potential aberrant integration may occur in distant locations, and that targeting of mGluR1α receptors may be a potential therapeutic strategy for future development.
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