Peripheral blood mononuclear cell mitochondrial dysfunction in acute alcohol-associated hepatitis.

BACKGROUND: Patients with acute alcohol-associated hepatitis (AH) have immune dysfunction. Mitochondrial function is critical for immune cell responses and regulates senescence. Clinical translational studies using complementary bioinformatics-experimental validation of mitochondrial responses were performed in peripheral blood mononuclear cells (PBMC) from patients with AH, healthy controls (HC), and heavy drinkers without evidence of liver disease (HD). METHODS: Feature extraction for differentially expressed genes (DEG) in mitochondrial components and telomere regulatory pathways from single-cell RNAseq (scRNAseq) and integrated 'pseudobulk' transcriptomics from PBMC from AH and HC (n = 4 each) were performed. After optimising isolation and processing protocols for functional studies in PBMC, mitochondrial oxidative responses to substrates, uncoupler, and inhibitors were quantified in independent discovery (AH n = 12; HD n = 6; HC n = 12) and validation cohorts (AH n = 10; HC n = 7). Intermediary metabolites (gas-chromatography/mass-spectrometry) and telomere length (real-time PCR) were quantified in subsets of subjects (PBMC/plasma AH n = 69/59; HD n = 8/8; HC n = 14/27 for metabolites; HC n = 13; HD n = 8; AH n = 72 for telomere length). RESULTS: Mitochondrial, intermediary metabolite, and senescence-regulatory genes were differentially expressed in PBMC from AH and HC in a cell type-specific manner at baseline and with lipopolysaccharide (LPS). Fresh PBMC isolated using the cell preparation tube generated optimum mitochondrial responses. Intact cell and maximal respiration were lower (p ≤ .05) in AH than HC/HD in the discovery and validation cohorts. In permeabilised PBMC, maximum respiration, complex I and II function were lower in AH than HC. Most tricarboxylic acid (TCA) cycle intermediates in plasma were higher while those in PBMC were lower in patients with AH than those from HC. Lower telomere length, a measure of cellular senescence, was associated with higher mortality in AH. CONCLUSION: Patients with AH have lower mitochondrial oxidative function, higher plasma TCA cycle intermediates, with telomere shortening in nonsurvivors.

Circadian clock controls rhythms in ketogenesis by interfering with PPARα transcriptional network.

Ketone bodies are energy-rich metabolites and signaling molecules whose production is mainly regulated by diet. Caloric restriction (CR) is a dietary intervention that improves metabolism and extends longevity across the taxa. We found that CR induced high-amplitude daily rhythms in blood ketone bodies (beta-hydroxybutyrate [ßOHB]) that correlated with liver ßOHB level. Time-restricted feeding, another periodic fasting-based diet, also led to rhythmic ßOHB but with reduced amplitude. CR induced strong circadian rhythms in the expression of fatty acid oxidation and ketogenesis genes in the liver. The transcriptional factor peroxisome-proliferator-activated-receptor α (PPARα) and its transcriptional target hepatokine fibroblast growth factor 21 (FGF21) are primary regulators of ketogenesis. Fgf21 expression and the PPARα transcriptional network became highly rhythmic in the CR liver, which implicated the involvement of the circadian clock. Mechanistically, the circadian clock proteins CLOCK, BMAL1, and cryptochromes (CRYs) interfered with PPARα transcriptional activity. Daily rhythms in the blood ßOHB level and in the expression of PPARα target genes were significantly impaired in circadian clock-deficient Cry1,2-/- mice. These data suggest that blood ßOHB level is tightly controlled and that the circadian clock is a regulator of diet-induced ketogenesis.

A liquid chromatography tandem mass spectrometry method for a semiquantitative screening of cellular acyl-CoA.

This study describes LC-ESI-MS/MS method that covers the analysis of various cellular acyl-CoA in a single injection. The method is based on a quick extraction step eliminating LLE/SPE clean up. Method performance characteristics were determined after spiking acyl-CoA standards in different concentrations into a surrogate matrix. The extensive matrix effect for most acyl-CoA except for palmitoyl-CoA was compensated by using isotopically labeled internal standard and matrix-matched calibration. As a result of the high matrix effect, the accuracy for palmitoyl-CoA at the low concentration deviated from the target range of ±20%. The developed method was applied to identify twenty-one cellular acyl-CoA in SK-HEP-1 cells and screening for alterations in acyl-CoA levels post Mito Q antioxidant intervention.

Metabolic Outcomes of Anaplerotic Dodecanedioic Acid Supplementation in Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Deficient Fibroblasts.

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD, OMIM 609575) is associated with energy deficiency and mitochondrial dysfunction and may lead to rhabdomyolysis and cardiomyopathy. Under physiological conditions, there is a fine balance between the utilization of different carbon nutrients to maintain the Krebs cycle. The maintenance of steady pools of Krebs cycle intermediates is critical formitochondrial energy homeostasis especially in high-energy demanding organs such as muscle and heart. Even-chain dicarboxylic acids are established as alternative energy carbon sources that replenish the Krebs cycle by bypassing a defective ß-oxidation pathway. Despite this, even-chain dicarboxylic acids are eliminated in the urine of VLCAD-affected individuals. In this study, we explore dodecanedioic acid (C12; DODA) supplementation and investigate its metabolic effect on Krebs cycle intermediates, glucose uptake, and acylcarnitine profiles in VLCAD-deficient fibroblasts. Our findings indicate that DODA supplementation replenishes the Krebs cycle by increasing the succinate pool, attenuates glycolytic flux, and reduces levels of toxic very long-chain acylcarnitines.

CR reprograms acetyl-CoA metabolism and induces long-chain acyl-CoA dehydrogenase and CrAT expression.

Calorie restriction (CR), an age delaying diet, affects fat oxidation through poorly understood mechanisms. We investigated the effect of CR on fat metabolism gene expression and intermediate metabolites of fatty acid oxidation in the liver. We found that CR changed the liver acylcarnitine profile: acetylcarnitine, short-chain acylcarnitines, and long-chain 3-hydroxy-acylcarnitines increased, and several long-chain acylcarnitines decreased. Acetyl-CoA and short-chain acyl-CoAs were also increased in CR. CR did not affect the expression of CPT1 and upregulated the expression of long-chain and very-long-chain Acyl-CoA dehydrogenases (LCAD and VLCAD, respectively). The expression of downstream enzymes such as mitochondrial trifunctional protein and enzymes in medium- and short-chain acyl-CoAs oxidation was not affected in CR. CR shifted the balance of fatty acid oxidation enzymes and fatty acid metabolites in the liver. Acetyl-CoA generated through beta-oxidation can be used for ketogenesis or energy production. In agreement, blood ketone bodies increased under CR in a time of the day-dependent manner. Carnitine acetyltransferase (CrAT) is a bidirectional enzyme that interconverts short-chain acyl-CoAs and their corresponding acylcarnitines. CrAT expression was induced in CR liver supporting the increased acetylcarnitine and short-chain acylcarnitine production. Acetylcarnitine can freely travel between cellular sub-compartments. Supporting this CR increased protein acetylation in the mitochondria, cytoplasm, and nucleus. We hypothesize that changes in acyl-CoA and acylcarnitine levels help to control energy metabolism and contribute to metabolic flexibility under CR.

A protocol for metabolic characterization of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CM).

Recent advances in human induced pluripotent stem cell-derived cardiomyocytes (iPSCM) field offer a novel platform for modeling cardiac metabolism, heart diseases drug candidates screening and cardiac toxicity assessments. These workflows require a fully functional characterization of iPSCMs. Here we report a step by step protocol for iPSCM metabolic characterization. The described assays cover analysis of small metabolites involved in a vital metabolic pathways.

Plasma Krebs Cycle Intermediates in Nonalcoholic Fatty Liver Disease.

Nonalcoholic liver disease (NAFLD) is manifested with a wide spectrum of clinical symptoms and is closely associated with the metabolic syndrome, inflammation, and mitochondrial dysfunction. Although the mechanism of mitochondrial dysfunction in NAFLD is still not fully elucidated, multiple studies have demonstrated evidence of molecular, biochemical, and biophysical mitochondrial abnormalities in NAFLD. Given the association between NAFLD and mitochondrial dysfunction, the aim of this study is to analyze circulating levels of Krebs cycle intermediates in a cohort of NAFLD-affected individuals and matching healthy controls and to correlate our findings with the liver function metrics. Standard serum biochemistry and Krebs cycle intermediates were analyzed in NAFLD (n = 22) and matched control (n = 67) cohorts. Circulating levels of isocitrate and citrate were significantly (p < 0.05) elevated in the NAFLD cohort of patients. The area under the curve (AUROC) for these two metabolites exhibited a moderate clinical utility. Correlations between plasma Krebs cycle intermediates and standard clinical plasma metrics were explored by Pearson's correlation coefficient. The data obtained for plasma Krebs cycle intermediates suggest pathophysiological insights that link mitochondrial dysfunction with NAFLD. Our findings reveal that plasma isocitrate and citrate can discriminate between normal and NAFLD cohorts and can be utilized as noninvasive markers of mitochondrial dysfunction in NAFLD. Future studies with large populations at different NAFLD stages are warranted.

Barth Syndrome: Exploring Cardiac Metabolism with Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Barth syndrome (BTHS) is an X-linked recessive multisystem disorder caused by mutations in the TAZ gene (TAZ, G 4.5, OMIM 300394) that encodes for the acyltransferase tafazzin. This protein is highly expressed in the heart and plays a significant role in cardiolipin biosynthesis. Heart disease is the major clinical manifestation of BTHS with a high incidence in early life. Although the genetic basis of BTHS and tetralinoleoyl cardiolipin deficiency in BTHS-affected individuals are well-established, downstream metabolic changes in cardiac metabolism are still uncovered. Our study aimed to characterize TAZ-induced metabolic perturbations in the heart. Control (PGP1-TAZWT) and TAZ mutant (PGP1-TAZ517delG) iPS-CM were incubated with 13C6-glucose and 13C5-glutamine and incorporation of 13C into downstream Krebs cycle intermediates was traced. Our data reveal that TAZ517delG induces accumulation of cellular long chain acylcarnitines and overexpression of fatty acid binding protein (FABP4). We also demonstrate that TAZ517delG induces metabolic alterations in pathways related to energy production as reflected by high glucose uptake, an increase in glycolytic lactate production and a decrease in palmitate uptake. Moreover, despite mitochondrial dysfunction, in the absence of glucose and fatty acids, TAZ517delG-iPS-CM can use glutamine as a carbon source to replenish the Krebs cycle.

Oxidative stress mediates ethanol-induced skeletal muscle mitochondrial dysfunction and dysregulated protein synthesis and autophagy.

Protein synthesis and autophagy are regulated by cellular ATP content. We tested the hypothesis that mitochondrial dysfunction, including generation of reactive oxygen species (ROS), contributes to impaired protein synthesis and increased proteolysis resulting in tissue atrophy in a comprehensive array of models. In myotubes treated with ethanol, using unbiased approaches, we identified defects in mitochondrial electron transport chain components, endogenous antioxidants, and enzymes regulating the tricarboxylic acid (TCA) cycle. Using high sensitivity respirometry, we observed impaired cellular respiration, decreased function of complexes I, II, and IV, and a reduction in oxidative phosphorylation in ethanol-treated myotubes and muscle from ethanol-fed mice. These perturbations resulted in lower skeletal muscle ATP content and redox ratio (NAD+/NADH). Ethanol also caused a leak of electrons, primarily from complex III, with generation of mitochondrial ROS and reverse electron transport. Oxidant stress with lipid peroxidation (thiobarbituric acid reactive substances) and protein oxidation (carbonylated proteins) were increased in myotubes and skeletal muscle from mice and humans with alcoholic liver disease. Ethanol also impaired succinate oxidation in the TCA cycle with decreased metabolic intermediates. MitoTEMPO, a mitochondrial specific antioxidant, reversed ethanol-induced mitochondrial perturbations (including reduced oxygen consumption, generation of ROS and oxidative stress), increased TCA cycle intermediates, and reversed impaired protein synthesis and the sarcopenic phenotype. We show that ethanol causes skeletal muscle mitochondrial dysfunction, decreased protein synthesis, and increased autophagy, and that these perturbations are reversed by targeting mitochondrial ROS.

The future perspective: metabolomics in laboratory medicine for inborn errors of metabolism.

Metabolomics can be described as a simultaneous and comprehensive analysis of small molecules in a biological sample. Recent technological and bioinformatics advances have facilitated large-scale metabolomic studies in many areas, including inborn errors of metabolism (IEMs). Despite significant improvements in the diagnosis and treatment of some IEMs, it is still challenging to understand how genetic variation affects disease progression and susceptibility. In addition, a search for new more personalized therapies and a growing demand for tools to monitor the long-term metabolic effects of existing therapies set the stage for metabolomics integration in preclinical and clinical studies. While targeted metabolomics approach is a common practice in biochemical genetics laboratories for biochemical diagnosis and monitoring of IEMs, applications of untargeted metabolomics in the clinical laboratories are still in infancy, facing some challenges. It is however, expected in the future to dramatically change the scope and utility of the clinical laboratory playing a significant role in patient management. This review provides an overview of targeted and global, large-scale metabolomic studies applied to investigate various IEMs. We discuss an existing and prospective clinical applications of metabolomics in IEMs for better diagnosis and deep understanding of complex metabolic perturbations associated with the etiology of inherited metabolic disorders.

Hyperammonaemia-induced skeletal muscle mitochondrial dysfunction results in cataplerosis and oxidative stress.

KEY POINTS: Hyperammonaemia occurs in hepatic, cardiac and pulmonary diseases with increased muscle concentration of ammonia. We found that ammonia results in reduced skeletal muscle mitochondrial respiration, electron transport chain complex I dysfunction, as well as lower NAD+ /NADH ratio and ATP content. During hyperammonaemia, leak of electrons from complex III results in oxidative modification of proteins and lipids. Tricarboxylic acid cycle intermediates are decreased during hyperammonaemia, and providing a cell-permeable ester of αKG reversed the lower TCA cycle intermediate concentrations and increased ATP content. Our observations have high clinical relevance given the potential for novel approaches to reverse skeletal muscle ammonia toxicity by targeting the TCA cycle intermediates and mitochondrial ROS. ABSTRACT: Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonaemia occurs in advanced hepatic, cardiac and pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non-hepatic ammonia disposal. Non-hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α-ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonaemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonaemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonaemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD+ /NADH ratio was observed during hyperammonaemia with reduced TCA cycle intermediates compared to controls. Generation of reactive oxygen species resulted in increased content of skeletal muscle carbonylated proteins and thiobarbituric acid reactive substances during hyperammonaemia. A cell-permeable ester of α-ketoglutarate reversed the low TCA cycle intermediates and ATP content in myotubes during hyperammonaemia. However, the mitochondrial antioxidant MitoTEMPO did not reverse the lower ATP content during hyperammonaemia. We provide for the first time evidence that skeletal muscle hyperammonaemia results in mitochondrial dysfunction and oxidative stress. Use of anaplerotic substrates to reverse ammonia-induced mitochondrial dysfunction is a novel therapeutic approach.

Metabolomics Reveals New Mechanisms for Pathogenesis in Barth Syndrome and Introduces Novel Roles for Cardiolipin in Cellular Function.

Barth Syndrome is the only known Mendelian disorder of cardiolipin remodeling, with characteristic clinical features of cardiomyopathy, skeletal myopathy, and neutropenia. While the primary biochemical defects of reduced mature cardiolipin and increased monolysocardiolipin are well-described, much of the downstream biochemical dysregulation has not been uncovered, and biomarkers are limited. In order to further expand upon the knowledge of the biochemical abnormalities in Barth Syndrome, we analyzed metabolite profiles in plasma from a cohort of individuals with Barth Syndrome compared to age-matched controls via 1H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry. A clear distinction between metabolite profiles of individuals with Barth Syndrome and controls was observed, and was defined by an array of metabolite classes including amino acids and lipids. Pathway analysis of these discriminating metabolites revealed involvement of mitochondrial and extra-mitochondrial biochemical pathways including: insulin regulation of fatty acid metabolism, lipid metabolism, biogenic amine metabolism, amino acid metabolism, endothelial nitric oxide synthase signaling, and tRNA biosynthesis. Taken together, this data indicates broad metabolic dysregulation in Barth Syndrome with wide cellular effects.

Glutathione species and metabolomic prints in subjects with liver disease as biological markers for the detection of hepatocellular carcinoma.

BACKGROUND: The incidence of liver disease is increasing in USA. Animal models had shown glutathione species in plasma reflects liver glutathione state and it could be a surrogate for the detection of hepatocellular carcinoma (HCC). METHODS: The present study aimed to translate methods to the human and to explore the role of glutathione/metabolic prints in the progression of liver dysfunction and in the detection of HCC. Treated plasma from healthy subjects (n = 20), patients with liver disease (ESLD, n = 99) and patients after transplantation (LTx, n = 7) were analyzed by GC- or LC/MS. Glutathione labeling profile was measured by isotopomer analyzes of 2H2O enriched plasma. Principal Component Analyzes (PCA) were used to determined metabolic prints. RESULTS: There was a significant difference in glutathione/metabolic profiles from patients with ESLD vs healthy subjects and patients after LTx. Similar significant differences were noted on patients with ESLD when stratified by the MELD score. PCA analyses showed myristic acid, citric acid, succinic acid, l-methionine, d-threitol, fumaric acid, pipecolic acid, isoleucine, hydroxy-butyrate and glycolic, steraric and hexanoic acids were discriminative metabolites for ESLD-HCC+ vs ESLD-HCC- subject status. CONCLUSIONS: Glutathione species and metabolic prints defined liver disease severity and may serve as surrogate for the detection of HCC in patients with established cirrhosis.

Metabolomics of ApcMin/+ mice genetically susceptible to intestinal cancer.

BACKGROUND: To determine how diets high in saturated fat could increase polyp formation in the mouse model of intestinal neoplasia, ApcMin/+, we conducted large-scale metabolome analysis and association study of colon and small intestine polyp formation from plasma and liver samples of ApcMin/+ vs. wild-type littermates, kept on low vs. high-fat diet. Label-free mass spectrometry was used to quantify untargeted plasma and acyl-CoA liver compounds, respectively. Differences in contrasts of interest were analyzed statistically by unsupervised and supervised modeling approaches, namely Principal Component Analysis and Linear Model of analysis of variance. Correlation between plasma metabolite concentrations and polyp numbers was analyzed with a zero-inflated Generalized Linear Model. RESULTS: Plasma metabolome in parallel to promotion of tumor development comprises a clearly distinct profile in ApcMin/+ mice vs. wild type littermates, which is further altered by high-fat diet. Further, functional metabolomics pathway and network analyses in ApcMin/+ mice on high-fat diet revealed associations between polyp formation and plasma metabolic compounds including those involved in amino-acids metabolism as well as nicotinamide and hippuric acid metabolic pathways. Finally, we also show changes in liver acyl-CoA profiles, which may result from a combination of ApcMin/+-mediated tumor progression and high fat diet. The biological significance of these findings is discussed in the context of intestinal cancer progression. CONCLUSIONS: These studies show that high-throughput metabolomics combined with appropriate statistical modeling and large scale functional approaches can be used to monitor and infer changes and interactions in the metabolome and genome of the host under controlled experimental conditions. Further these studies demonstrate the impact of diet on metabolic pathways and its relation to intestinal cancer progression. Based on our results, metabolic signatures and metabolic pathways of polyposis and intestinal carcinoma have been identified, which may serve as useful targets for the development of therapeutic interventions.

Clinical laboratory studies in Barth Syndrome.

Barth Syndrome is a rare X-linked disorder characterized principally by dilated cardiomyopathy, skeletal myopathy and neutropenia and caused by defects in tafazzin, an enzyme responsible for modifying the acyl chain moieties of cardiolipin. While several comprehensive clinical studies of Barth Syndrome have been published detailing cardiac and hematologic features, descriptions of its biochemical characteristics are limited. To gain a better understanding of the clinical biochemistry of this rare disease, we measured hematologic and biochemical values in a cohort of Barth Syndrome patients. We characterized multiple biochemical parameters, including plasma amino acids, plasma 3-methylglutaconic acid, cholesterol, cholesterol synthetic intermediates, and red blood cell membrane fatty acid profiles in 28 individuals with Barth Syndrome from ages 10 months to 30 years. We describe a unique biochemical profile for these patients, including decreased plasma arginine levels. We further studied the plasma amino acid profiles, cholesterol, cholesterol synthetic intermediates, and plasma 3-methylglutaconic acid levels in 8 female carriers and showed that they do not share any of the distinct, Barth Syndrome-specific biochemical laboratory abnormalities. Our studies augment and expand the biochemical profiles of individuals with Barth Syndrome, describe a unique biochemical profile for these patients, and provide insight into the possible underlying biochemical pathology in this disorder.

Time course of hepatic gluconeogenesis during hindlimb suspension unloading.

The goal of this work was to determine the time-dependent changes in fractional hepatic gluconeogenesis (GNG) during conditions of hindlimb suspension unloading (HSU), a 'ground-based' method for inducing muscular atrophy to simulate space flight. We hypothesized that GNG would increase in HSU conditions as a result of metabolic shifts in the liver and skeletal muscle. A significant and progressive atrophy was observed in the soleus (30, 47 and 55%) and gastrocnemius muscles (0, 15 and 17%) after 3, 7 and 14 days of HSU, respectively. Fractional hepatic GNG was determined following the incorporation of deuterium from deuterated water ((2)H(2)O) into C-H bonds of newly synthesized glucose after an 8 h fast. Enrichment of plasma glucose with (2)H was measured using the classic method of Landau et al. (the 'hexamethylenetetramine (HMT) method'), based on specific (2)H labelling of glucose carbons, and the novel method of Chacko et al. ('average method'), based on the assumption of equal (2)H enrichment on all glucose carbons (except C2). After 3 days of HSU, fractional GNG was significantly elevated in the HSU group, as determined by either method (∼13%, P < 0.05). After 7 and 14 days of HSU, gluconeogenesis was not significantly different. Both analytical methods yielded similar time-dependent trends in gluconeogenic rates, but GNG values determined using the average method were consistently lower (∼30%) than those found by the HMT method. To compare and validate the average method against the HMT method further, we starved animals for 13 h to allow for hepatic GNG to contribute 100% to endogenous glucose production. The HMT method yielded 100% GNG, while the average method yielded GNG of ∼70%. As both methods used the same values of precursor enrichment, we postulated that the underestimation of gluconeogenic rate was as a result of differences in the measurements of product enrichment ((2)H labelling of plasma glucose). This could be explained by the following factors: (i) loss of deuterium via exchange between acetate and glucose; (ii) interference caused by fragment m/z 169, representing multiple isobaric species; and (iii) interference from other sugars at m/z 169. In conclusion, HSU caused a time-dependent increase in hepatic gluconeogenesis, irrespective of the analytical methods used.

Combined extraction of acyl carnitines and 26:0 lysophosphatidylcholine from dried blood spots: prospective newborn screening for X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a severe genetic disorder that affects the nervous system, and the adrenal cortex. Newborn screening for X-ALD has been proposed to allow improved diagnosis along with prospective monitoring and treatment for this severe disorder. Newborn dried whole blood spot (DBS) 26:0 lysophosphatidyl choline was validated as a diagnostic marker for X-ALD and other peroxisomal disorders of peroxisomal ß-oxidation. In this study, we developed a new one step extraction procedure that simultaneously extracts acyl carnitines and the lysophosphatidyl cholines from DBS. Further analysis of these metabolites has been performed by two different high throughput LC-MS/MS methods. The 26:0 lysophosphatidyl choline levels in this study were consistent with previously published values and discriminate between healthy and abnormal profiles. There is a very minor modification to the original acyl carnitine extraction procedure and our data indicates that there is no significant effect on acyl carnitine levels in DBS. Our new method potentially can be complementary to the current newborn screening panel. It successfully combines the existing method for acyl carnitine analysis and 26:0 lysophosphatidyl choline that can be applied for prospective X-ALD newborn screening.

Profiling sterols in cerebrotendinous xanthomatosis: utility of Girard derivatization and high resolution exact mass LC-ESI-MS(n) analysis.

In this study we profile free 3-oxo sterols present in plasma from patients affected with the neurodegenerative disorder of sterol and bile acid metabolism cerebrotendinous xanthomatosis (CTX), utilizing a combination of charge-tagging and LC-ESI-MS(n) performed with an LTQ-Orbitrap Discovery instrument. In addition, we profile sterols in plasma from 24-month-old cyp27A1 gene knockout mice lacking the enzyme defective in CTX. Charge-tagging was accomplished by reaction with cationic Girard's P (GP) reagent 1-(carboxymethyl) pyridinium chloride hydrazide, an approach uniquely suited to studying the 3-oxo sterols that accumulate in CTX, as Girard's reagent reacts with the sterol oxo moiety to form charged hydrazone derivatives. The ability to selectively generate GP-tagged 3-oxo-4-ene and 3-oxo-5(H) saturated plasma sterols enabled ESI-MS(n) analysis of these sterols in the presence of a large excess (3 orders of magnitude) of cholesterol. Often cholesterol detected in biological samples makes it challenging to quantify minor sterols, with cholesterol frequently removed prior to analysis. We derivatized plasma (10 µl) without SPE removal of cholesterol to ensure detection of all sterols present in plasma. We were able to measure 4-cholesten-3-one in plasma from untreated CTX patients (1207±302 ng/ml, mean±SD, n=4), as well as other intermediates in a proposed pathway to 5α-cholestanol. In addition, a number of bile acid precursors were identified in plasma using this technique. GP-tagged sterols were identified utilizing high resolution exact mass spectra (±5 ppm), as well as MS(2) ([M](+)→) spectra that possessed characteristic neutral loss of 79Da (pyridine) fragment ions, and MS(3) ([M](+)→[M-79](+)→) spectra that provided additional structurally informative fragment ions.

Formation and stability of oxocarbenium ions from glycosides.

Structural, protecting group and leaving group effects in the formation of oxocarbenium intermediates were studied in the gas phase. It is found that significant stabilization of oxocarbenium cations is achieved by protecting groups that interact with the cationic center via neighboring group participation despite the electron-withdrawing character of these moieties. On the other hand, ethereal protecting groups do not facilitate the formation of oxocarbenium intermediates. The experimental findings are supported by DFT calculations that show the following order of stabilization by the group adjacent to the cationic center: RCO > SiR(3) > R, where R is an alkyl group. This indicates that the SN1-like mechanism that is commonly proposed for this reaction is not always valid. Moderate leaving group effect is also detected in a series of thioaryl glucopyranosides.