An Unbiased Lipid Phenotyping Approach To Study the Genetic Determinants of Lipids and Their Association with Coronary Heart Disease Risk Factors.

Direct infusion high-resolution mass spectrometry (DIHRMS) is a novel, high-throughput approach to rapidly and accurately profile hundreds of lipids in human serum without prior chromatography, facilitating in-depth lipid phenotyping for large epidemiological studies to reveal the detailed associations of individual lipids with coronary heart disease (CHD) risk factors. Intact lipid profiling by DIHRMS was performed on 5662 serum samples from healthy participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS). We developed a novel semi-targeted peak-picking algorithm to detect mass-to-charge ratios in positive and negative ionization modes. We analyzed lipid partial correlations, assessed the association of lipid principal components with established CHD risk factors and genetic variants, and examined differences between lipids for a common genetic polymorphism. The DIHRMS method provided information on 360 lipids (including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids), with a median coefficient of variation of 11.6% (range: 5.4-51.9). The lipids were highly correlated and exhibited a range of associations with clinical chemistry biomarkers and lifestyle factors. This platform can provide many novel insights into the effects of physiology and lifestyle on lipid metabolism, genetic determinants of lipids, and the relationship between individual lipids and CHD risk factors.

Interlaboratory Reproducibility of a Targeted Metabolomics Platform for Analysis of Human Serum and Plasma.

A critical question facing the field of metabolomics is whether data obtained from different centers can be effectively compared and combined. An important aspect of this is the interlaboratory precision (reproducibility) of the analytical protocols used. We analyzed human samples in six laboratories using different instrumentation but a common protocol (the AbsoluteIDQ p180 kit) for the measurement of 189 metabolites via liquid chromatography (LC) or flow injection analysis (FIA) coupled to tandem mass spectrometry (MS/MS). In spiked quality control (QC) samples 82% of metabolite measurements had an interlaboratory precision of <20%, while 83% of averaged individual laboratory measurements were accurate to within 20%. For 20 typical biological samples (serum and plasma from healthy individuals) the median interlaboratory coefficient of variation (CV) was 7.6%, with 85% of metabolites exhibiting a median interlaboratory CV of <20%. Precision was largely independent of the type of sample (serum or plasma) or the anticoagulant used but was reduced in a sample from a patient with dyslipidaemia. The median interlaboratory accuracy and precision of the assay for standard reference plasma (NIST SRM 1950) were 107% and 6.7%, respectively. Likely sources of irreproducibility were the near limit of detection (LOD) typical abundance of some metabolites and the degree of manual review and optimization of peak integration in the LC-MS/MS data after acquisition. Normalization to a reference material was crucial for the semi-quantitative FIA measurements. This is the first interlaboratory assessment of a widely used, targeted metabolomics assay illustrating the reproducibility of the protocol and how data generated on different instruments could be directly integrated in large-scale epidemiological studies.

Genetic Predisposition to an Impaired Metabolism of the Branched-Chain Amino Acids and Risk of Type 2 Diabetes: A Mendelian Randomisation Analysis.

BACKGROUND: Higher circulating levels of the branched-chain amino acids (BCAAs; i.e., isoleucine, leucine, and valine) are strongly associated with higher type 2 diabetes risk, but it is not known whether this association is causal. We undertook large-scale human genetic analyses to address this question. METHODS AND FINDINGS: Genome-wide studies of BCAA levels in 16,596 individuals revealed five genomic regions associated at genome-wide levels of significance (p < 5 × 10-8). The strongest signal was 21 kb upstream of the PPM1K gene (beta in standard deviations [SDs] of leucine per allele = 0.08, p = 3.9 × 10-25), encoding an activator of the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) responsible for the rate-limiting step in BCAA catabolism. In another analysis, in up to 47,877 cases of type 2 diabetes and 267,694 controls, a genetically predicted difference of 1 SD in amino acid level was associated with an odds ratio for type 2 diabetes of 1.44 (95% CI 1.26-1.65, p = 9.5 × 10-8) for isoleucine, 1.85 (95% CI 1.41-2.42, p = 7.3 × 10-6) for leucine, and 1.54 (95% CI 1.28-1.84, p = 4.2 × 10-6) for valine. Estimates were highly consistent with those from prospective observational studies of the association between BCAA levels and incident type 2 diabetes in a meta-analysis of 1,992 cases and 4,319 non-cases. Metabolome-wide association analyses of BCAA-raising alleles revealed high specificity to the BCAA pathway and an accumulation of metabolites upstream of branched-chain alpha-ketoacid oxidation, consistent with reduced BCKD activity. Limitations of this study are that, while the association of genetic variants appeared highly specific, the possibility of pleiotropic associations cannot be entirely excluded. Similar to other complex phenotypes, genetic scores used in the study captured a limited proportion of the heritability in BCAA levels. Therefore, it is possible that only some of the mechanisms that increase BCAA levels or affect BCAA metabolism are implicated in type 2 diabetes. CONCLUSIONS: Evidence from this large-scale human genetic and metabolomic study is consistent with a causal role of BCAA metabolism in the aetiology of type 2 diabetes.

Tile-based Fisher ratio analysis of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS) data using a null distribution approach.

Comprehensive two-dimensional (2D) gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOFMS) is a versatile instrumental platform capable of collecting highly informative, yet highly complex, chemical data for a variety of samples. Fisher-ratio (F-ratio) analysis applied to the supervised comparison of sample classes algorithmically reduces complex GC × GC-TOFMS data sets to find class distinguishing chemical features. F-ratio analysis, using a tile-based algorithm, significantly reduces the adverse effects of chromatographic misalignment and spurious covariance of the detected signal, enhancing the discovery of true positives while simultaneously reducing the likelihood of detecting false positives. Herein, we report a study using tile-based F-ratio analysis whereby four non-native analytes were spiked into diesel fuel at several concentrations ranging from 0 to 100 ppm. Spike level comparisons were performed in two regimes: comparing the spiked samples to the nonspiked fuel matrix and to each other at relative concentration factors of two. Redundant hits were algorithmically removed by refocusing the tiled results onto the original high resolution pixel level data. To objectively limit the tile-based F-ratio results to only features which are statistically likely to be true positives, we developed a combinatorial technique using null class comparisons, called null distribution analysis, by which we determined a statistically defensible F-ratio cutoff for the analysis of the hit list. After applying null distribution analysis, spiked analytes were reliably discovered at ∼1 to ∼10 ppm (∼5 to ∼50 pg using a 200:1 split), depending upon the degree of mass spectral selectivity and 2D chromatographic resolution, with minimal occurrence of false positives. To place the relevance of this work among other methods in this field, results are compared to those for pixel and peak table-based approaches.

Serial plasma metabolites following hypoxic-ischemic encephalopathy in a nonhuman primate model.

Biomarkers that indicate the severity of hypoxic-ischemic brain injury and response to treatment and that predict neurodevelopmental outcomes are urgently needed to improve the care of affected neonates. We hypothesize that sequentially obtained plasma metabolomes will provide indicators of brain injury and repair, allowing for the prediction of neurodevelopmental outcomes. A total of 33 Macaca nemestrina underwent 0, 15 or 18 min of in utero umbilical cord occlusion (UCO) to induce hypoxic-ischemic encephalopathy and were then delivered by hysterotomy, resuscitated and stabilized. Serial blood samples were obtained at baseline (cord blood) and at 0.1, 24, 48, and 72 h of age. Treatment groups included nonasphyxiated controls (n = 7), untreated UCO (n = 11), UCO + hypothermia (HT; n = 6), and UCO + HT + erythropoietin (n = 9). Metabolites were extracted and analyzed using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry and quantified by PARAFAC (parallel factor analysis). Using nontargeted discovery-based methods, we identified 63 metabolites as potential biomarkers. The changes in metabolite concentrations were characterized and compared between treatment groups. Further comparison determined that 8 metabolites (arachidonic acid, butanoic acid, citric acid, fumaric acid, lactate, malate, propanoic acid, and succinic acid) correlated with early and/or long-term neurodevelopmental outcomes. The combined outcomes of death or cerebral palsy correlated with citric acid, fumaric acid, lactate, and propanoic acid. This change in circulating metabolome after UCO may reflect cellular metabolism and biochemical changes in response to the severity of brain injury and have potential to predict neurodevelopmental outcomes.

Cardiac-specific deletion of acetyl CoA carboxylase 2 prevents metabolic remodeling during pressure-overload hypertrophy.

RATIONALE: Decreased fatty acid oxidation (FAO) with increased reliance on glucose are hallmarks of metabolic remodeling that occurs in pathological cardiac hypertrophy and is associated with decreased myocardial energetics and impaired cardiac function. To date, it has not been tested whether prevention of the metabolic switch that occurs during the development of cardiac hypertrophy has unequivocal benefits on cardiac function and energetics. OBJECTIVE: Because malonyl CoA production via acetyl CoA carboxylase 2 (ACC2) inhibits the entry of long chain fatty acids into the mitochondria, we hypothesized that mice with a cardiac-specific deletion of ACC2 (ACC2H-/-) would maintain cardiac FAO and improve function and energetics during the development of pressure-overload hypertrophy. METHODS AND RESULTS: ACC2 deletion led to a significant reduction in cardiac malonyl CoA levels. In isolated perfused heart experiments, left ventricular function and oxygen consumption were similar in ACC2H-/- mice despite an ≈60% increase in FAO compared with controls (CON). After 8 weeks of pressure overload via transverse aortic constriction (TAC), ACC2H-/- mice exhibited a substrate utilization profile similar to sham animals, whereas CON-TAC hearts had decreased FAO with increased glycolysis and anaplerosis. Myocardial energetics, assessed by 31P nuclear magnetic resonance spectroscopy, and cardiac function were maintained in ACC2H-/- after 8 weeks of TAC. Furthermore, ACC2H-/--TAC demonstrated an attenuation of cardiac hypertrophy with a significant reduction in fibrosis relative to CON-TAC. CONCLUSIONS: These data suggest that reversion to the fetal metabolic profile in chronic pathological hypertrophy is associated with impaired myocardial function and energetics and maintenance of the inherent cardiac metabolic profile and mitochondrial oxidative capacity is a viable therapeutic strategy.

Amelioration of age-related cognitive decline and anxiety in mice by Centella asiatica extract varies by sex, dose and mode of administration.

We have previously reported that a water extract (CAW) of the Ayurvedic plant Centella asiatica administered in drinking water can improve cognitive deficits in mouse models of aging and neurodegenerative diseases. Here we compared the effects of CAW administered in drinking water or the diet on cognition, measures of anxiety and depression-like behavior in healthy aged mice. Three- and eighteen-month-old male and female C57BL6 mice were administered rodent AIN-93M diet containing CAW (0, 0.2, 0.5 or 1% w/w) to provide 0, 200 mg/kg/d, 500 mg/kg/d or 1000 mg/kg/d for a total of 5 weeks. An additional group of eighteen-month-old mice were treated with CAW (10 mg/mL) in their drinking water for a total of five weeks to deliver the same exposure of CAW as the highest dietary dose (1000 mg/kg/d). CAW doses delivered were calculated based on food and water consumption measured in previous experiments. In the fourth and fifth weeks, mice underwent behavioral testing of cognition, anxiety and depression (n=12 of each sex per treatment group in each test). Aged mice of both sexes showed cognitive deficits relative to young mice while only female aged mice showed increased anxiety compared to the young female mice and no differences in depression were observed between the different ages. CAW (1000 mg/kg/d) in the drinking water improved deficits in aged mice in learning, executive function and recognition memory in both sexes and attenuated the increased measures of anxiety observed in the aged female mice. However, CAW in the diet only improved executive function in aged mice at the highest dose (1000 mg/kg/d) in both sexes and did so less robustly than when given in the water. There were no effects of CAW on depression-like behavior in aged animals regardless of whether it was administered in the diet or the water. These results suggest that CAW can ameliorate age-related changes in measures of anxiety and cognition and that the mode of administration is important for the effects of CAW on resilience to these age-related changes.

Amelioration of age-related cognitive decline and anxiety in mice by Centella asiatica extract varies by sex, dose and mode of administration.

Background: A water extract (CAW) of the Ayurvedic plant Centella asiatica administered in drinking water has been shown to improve cognitive deficits in mouse models of aging and neurodegenerative diseases. Here the effects of CAW administered in drinking water or the diet on cognition, measures of anxiety and depression-like behavior in healthy aged mice are compared. Methods: Three- and eighteen-month-old male and female C57BL6 mice were administered rodent AIN-93M diet containing CAW (0, 0.2, 0.5 or 1% w/w) to provide 0, 200 mg/kg/d, 500 mg/kg/d or 1,000 mg/kg/d CAW for a total of 5 weeks. An additional group of eighteen-month-old mice were treated with CAW (10 mg/mL) in their drinking water CAW for a total of 5 weeks to deliver the same exposure of CAW as the highest dietary dose (1,000 mg/kg/d). CAW doses delivered were calculated based on food and water consumption measured in previous experiments. In the fourth and fifth weeks, mice underwent behavioral testing of cognition, anxiety and depression (n = 12 of each sex per treatment group in each test). Results: Aged mice of both sexes showed cognitive deficits relative to young mice while only female aged mice showed increased anxiety compared to the young female mice and no differences in depression were observed between the different ages. CAW (1,000 mg/kg/d) in the drinking water improved deficits in aged mice in learning, executive function and recognition memory in both sexes and attenuated the increased measures of anxiety observed in the aged female mice. However, CAW in the diet only improved executive function in aged mice at the highest dose (1,000 mg/kg/d) in both sexes and did so less robustly than when given in the water. There were no effects of CAW on depression-like behavior in aged animals regardless of whether it was administered in the diet or the water. Conclusions: These results suggest that CAW can ameliorate age-related changes in measures of anxiety and cognition and that the mode of administration is important for the effects of CAW on resilience to these age-related changes.

Quantification of Caffeoylquinic Acids and Triterpenes as Targeted Bioactive Compounds of Centella asiatica in Extracts and Formulations by Liquid Chromatography Mass Spectrometry.

Centella asiatica (CA) is a culinary vegetable and well-known functional food that is widely used as a medicinal herb and dietary supplement. CA is rich in pentacyclic triterpenes (TTs), including asiaticoside (AS), madecassoside (MS) and the related aglycones asiatic acid (AA), madecassic acid (MA). Traditionally, TTs have been associated with the bioactivity and health promoting effect of CA. Recently, mono-caffeoylquinic acids (MonoCQAs) and di-caffeoylquinic acids (DiCQAs) have been found to contribute to the bioactivity of CA as well. This work reports an analytical strategy based on liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM-MS) for the simultaneous rapid and accurate quantification of 12 bioactive compounds in CA, namely AS, MS, AA, MA, 5-CQA, 4-CQA, 3-CQA, 1,3-DiCQA, 3,4-DiCQA, 1,5-DiCQA, 3,5-DiCQA, 4,5-DiCQA. Method selectivity, accuracy, precision, repeatability, robustness, linearity range, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The validated LC-MRM-MS method has been successfully applied to quantify the 12 bioactive compounds in CA aqueous extracts and two related formulations: a standardized CA product (CAP) used in a phase I clinical trial and formulated CA rodent diets used in preclinical studies. The validated method allows us to support the standardization of CA products used for clinical trials and conduct routine LC-MRM-MS analyses of formulated preclinical diets to confirm correct levels of CA phytochemical markers.

Regulation of expression quantitative trait loci by SVA retrotransposons within the major histocompatibility complex.

Genomic and transcriptomic studies of expression quantitative trait loci (eQTL) revealed that SINE-VNTR-Alu (SVA) retrotransposon insertion polymorphisms (RIPs) within human genomes markedly affect the co-expression of many coding and noncoding genes by coordinated regulatory processes. This study examined the polymorphic SVA modulation of gene co-expression within the major histocompatibility complex (MHC) genomic region where more than 160 coding genes are involved in innate and adaptive immunity. We characterized the modulation of SVA RIPs utilizing the genomic and transcriptomic sequencing data obtained from whole blood of 1266 individuals in the Parkinson's Progression Markers Initiative (PPMI) cohort that included an analysis of human leukocyte antigen (HLA)-A regulation in a subpopulation of the cohort. The regulatory properties of eight SVAs located within the class I and class II MHC regions were associated with differential co-expression of 71 different genes within and 75 genes outside the MHC region. Some of the same genes were affected by two or more different SVA. Five SVA are annotated in the human genomic reference sequence GRCh38.p14/hg38, whereas the other three were novel insertions within individuals. We also examined and found distinct structural effects (long and short variants and the CT internal variants) for one of the SVA (R_SVA_24) insertions on the differential expression of the HLA-A gene within a subpopulation (550 individuals) of the PPMI cohort. This is the first time that many HLA and non-HLA genes (multilocus expression units) and splicing mechanisms have been shown to be regulated by eight structurally polymorphic SVA within the MHC genomic region by applying precise statistical analysis of RNA data derived from the blood samples of a human cohort population. This study shows that SVA within the MHC region are important regulators or rheostats of gene co-expression that might have potential roles in diversity, health, and disease.

Chlorogenic Acids, Acting via Calcineurin, Are the Main Compounds in Centella asiatica Extracts That Mediate Resilience to Chronic Stress in Drosophila melanogaster.

Common symptoms of depressive disorders include anhedonia, sleep problems, and reduced physical activity. Drugs used to treat depression mostly aim to increase serotonin signaling but these can have unwanted side effects. Depression has also been treated by traditional medicine using plants like Centella asiatica (CA) and this has been found to be well tolerated. However, very few controlled studies have addressed CA's protective role in depression, nor have the active compounds or mechanisms that mediate this function been identified. To address this issue, we used Drosophila melanogaster to investigate whether CA can improve depression-associated symptoms like anhedonia and decreased climbing activity. We found that a water extract of CA provides resilience to stress induced phenotypes and that this effect is primarily due to mono-caffeoylquinic acids found in CA. Furthermore, we describe that the protective function of CA is due to a synergy between chlorogenic acid and one of its isomers also present in CA. However, increasing the concentration of chlorogenic acid can overcome the requirement for the second isomer. Lastly, we found that chlorogenic acid acts via calcineurin, a multifunctional phosphatase that can regulate synaptic transmission and plasticity and is also involved in neuronal maintenance.

Withania somnifera Extracts Promote Resilience against Age-Related and Stress-Induced Behavioral Phenotypes in Drosophila melanogaster; a Possible Role of Other Compounds besides Withanolides.

Withania somnifera (WS) extracts have been used in traditional medicine for millennia to promote healthy aging and wellbeing. WS is now also widely used in Western countries as a nutritional supplement to extend healthspan and increase resilience against age-related changes, including sleep deficits and depression. Although human trials have supported beneficial effects of WS, the study designs have varied widely. Plant material is intrinsically complex, and extracts vary widely with the origin of the plant material and the extraction method. Commercial supplements can contain various other ingredients, and the characteristics of the study population can also be varied. To perform maximally controlled experiments, we used plant extracts analyzed for their composition and stability. We then tested these extracts in an inbred Drosophila line to minimize effects of the genetic background in a controlled environment. We found that a water extract of WS (WSAq) was most potent in improving physical fitness, while an ethanol extract (WSE) improved sleep in aged flies. Both extracts provided resilience against stress-induced behavioral changes. WSE contained higher levels of withanolides, which have been proposed to be active ingredients, than WSAq. Therefore, withanolides may mediate the sleep improvement, whereas so-far-unknown ingredients enriched in WSAq likely mediate the effects on fitness and stress-related behavior.

Analytical Determination of the Severity of Potato Taste Defect in Roasted East African Arabica Coffee.

The quality of East African coffee beans has been significantly reduced by a flavor defect known as potato taste defect (PTD) due to the presence of 2-isopropyl-3-methoxypyrazine (IPMP) and 2-isobutyl-3-methoxypyrazine (IBMP). Therefore, the aims of this study were to determine the correlation between these methoxypyrazines and the severity of odor attributed to PTD and discover additional analytes that may be correlated with PTD using Fisher ratio analysis, a supervised discovery-based data analysis method. Specialty ground roasted coffees from East Africa were classified as clean (i.e., no off-odor), mild, medium, or strong PTD. For the samples examined, IPMP was found to discriminate between non-defective and defective samples, while IBMP did not do so. Samples affected by PTD exhibited a wide range of IPMP concentration (1.6-529.9 ng/g). Except for one sample, the IPMP concentration in defective samples was greater than the average IPMP concentration in the non-defective samples (2.0 ng/g). Also, an analysis of variance found that IPMP concentrations were significantly different based on the severity of odor attributed to PTD (p < 0.05). Fisher ratio analysis discovered 21 additional analytes whose concentrations were statistically different based on the severity of PTD odor (p < 0.05). Generally, analytes that were positively correlated with odor severity generally had unpleasant sensory descriptions, while analytes typically associated with desirable aromas were found to be negatively correlated with odor severity. These findings not only show that IPMP concentration can differentiate the severity of PTD but also that changes in the volatile analyte profile of coffee beans induced by PTD can contribute to odor severity.

Simultaneous quantification of apolipoprotein A-I and apolipoprotein B by liquid-chromatography-multiple- reaction-monitoring mass spectrometry.

BACKGROUND: If liquid-chromatography-multiple-reaction-monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS: We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS: We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay - 36.6; S(x|y) = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; S(x|y) = 7.9 for apoB). CONCLUSIONS: Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability.

Statistical inference of mass channel purity from Fisher ratio analysis using comprehensive two-dimensional gas chromatography with time of flight mass spectrometry data.

Tile-based Fisher ratio (F-ratio) analysis has recently been developed and validated for discovery-based studies of highly complex data collected using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS). In previous studies, interpretation and utilization of F-ratio hit lists has relied upon manual decomposition and quantification performed by chemometric methods such as parallel factor analysis (PARAFAC), or via manual translation of the F-ratio hit list information to peak table quantitative information provided by the instrument software (ChromaTOF). Both of these quantification approaches are bottlenecks in the overall workflow. In order to address this issue, a more automatable approach to provide accurate relative quantification for F-ratio analyses was investigated, based upon the mass spectral selectivity provided via the F-ratio spectral output. Diesel fuel spiked with 15 analytes at four concentration levels (80, 40, 20, and 10 ppm) produced three sets of two class comparisons that were submitted to tile-based F-ratio analysis to obtain three hit lists, with an F-ratio spectrum for each hit. A novel algorithm which calculates the signal ratio (S-ratio) between two classes (eg., 80 ppm versus 40 ppm) was applied to all mass channels (m/z) in the F-ratio spectrum for each hit. A lack of fit (LOF) metric was utilized as a measure of peak purity and combined with F-ratio and p-values to study the relationship of each of these metrics with m/z purity. Application of a LOF threshold coupled with a p-value threshold yielded a subset of the most pure m/z for each of the 15 spiked analytes, evident by the low deviations (< 5%) in S-ratio relative to the true concentration ratio. A key outcome of this study was to demonstrate the isolation of pure m/z without the need for higher level signal decomposition algorithms.

Isopropanol protein precipitation for the analysis of plasma free metanephrines by liquid chromatography-tandem mass spectrometry.

BACKGROUND: High-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS)1 analysis of plasma free metanephrines is the most diagnostically sensitive and specific screening test for the diagnosis of pheochromocytoma. We sought to develop an in-house method for this expensive test METHODS: We used off-line isopropanol protein precipitation of plasma to remove interfering substances before LC-MS/MS analysis. We compared the extraction efficiency and limits of quantification of protein precipitation to those of previously reported solid-phase techniques. RESULTS: The new method had limits of quantification of 0.09 nmol/L and 0.17 nmol/L for metanephrine and normetanephrine, respectively. Method comparison with a previously described solid-phase extraction method revealed Deming regression slopes of 0.904 and 0.994, intercepts of 0.007 and 0.023, and SEs of the residuals (S(y/x)) of 0.071 and 0.284 for metanephrine and normetanephrine, respectively. Extraction efficiency of isopropanol protein precipitation was 66% for metanephrine and 35% for normetanephrine, results that were superior to the efficiencies of 4% and 1% for our adapted solid-phase extraction method. No ion suppression was observed at the retention times for metanephrine and normetanephrine. CONCLUSIONS: Isopropanol protein precipitation is a novel and effective off-line sample preparation method for metanephrines that offers a less expensive alternative to on-line solid-phase extraction for low-volume testing and requires a sample volume of only 200 microL. The mass spectrometric analysis time is equivalent to that of solid-phase techniques.

Hepatic steatosis risk is partly driven by increased de novo lipogenesis following carbohydrate consumption.

BACKGROUND: Diet is a major contributor to metabolic disease risk, but there is controversy as to whether increased incidences of diseases such as non-alcoholic fatty liver disease arise from consumption of saturated fats or free sugars. Here, we investigate whether a sub-set of triacylglycerols (TAGs) were associated with hepatic steatosis and whether they arise from de novo lipogenesis (DNL) from the consumption of carbohydrates. RESULTS: We conduct direct infusion mass spectrometry of lipids in plasma to study the association between specific TAGs and hepatic steatosis assessed by ultrasound and fatty liver index in volunteers from the UK-based Fenland Study and evaluate clustering of TAGs in the National Survey of Health and Development UK cohort. We find that TAGs containing saturated and monounsaturated fatty acids with 16-18 carbons are specifically associated with hepatic steatosis. These TAGs are additionally associated with higher consumption of carbohydrate and saturated fat, hepatic steatosis, and variations in the gene for protein phosphatase 1, regulatory subunit 3b (PPP1R3B), which in part regulates glycogen synthesis. DNL is measured in hyperphagic ob/ob mice, mice on a western diet (high in fat and free sugar) and in healthy humans using stable isotope techniques following high carbohydrate meals, demonstrating the rate of DNL correlates with increased synthesis of this cluster of TAGs. Furthermore, these TAGs are increased in plasma from patients with biopsy-confirmed steatosis. CONCLUSION: A subset of TAGs is associated with hepatic steatosis, even when correcting for common confounding factors. We suggest that hepatic steatosis risk in western populations is in part driven by increased DNL following carbohydrate rich meals in addition to the consumption of saturated fat.

Mechanistic insights revealed by lipid profiling in monogenic insulin resistance syndromes.

BACKGROUND: Evidence from several recent metabolomic studies suggests that increased concentrations of triacylglycerols with shorter (14-16 carbon atoms), saturated fatty acids are associated with insulin resistance and the risk of type 2 diabetes. Although causality cannot be inferred from association studies, patients in whom the primary cause of insulin resistance can be genetically defined offer unique opportunities to address this challenge. METHODS: We compared metabolite profiles in patients with congenital lipodystrophy or loss-of-function insulin resistance (INSR gene) mutations with healthy controls. RESULTS: The absence of significant differences in triacylglycerol species in the INSR group suggest that changes previously observed in epidemiological studies are not purely a consequence of insulin resistance. The presence of triacylglycerols with lower carbon numbers and high saturation in patients with lipodystrophy suggests that these metabolite changes may be associated with primary adipose tissue dysfunction. The observed pattern of triacylglycerol species is indicative of increased de novo lipogenesis in the liver. To test this we investigated the distribution of these triacylglycerols in lipoprotein fractions using size exclusion chromatography prior to mass spectrometry. This associated these triacylglycerols with very low-density lipoprotein particles, and hence release of triacylglycerols into the blood from the liver. To test further the hepatic origin of these triacylglycerols we induced de novo lipogenesis in the mouse, comparing ob/ob and wild-type mice on a chow or high fat diet, confirming that de novo lipogenesis induced an increase in relatively shorter, more saturated fatty acids. CONCLUSIONS: Overall, these studies highlight hepatic de novo lipogenesis in the pathogenesis of metabolic dyslipidaemia in states where energy intake exceeds the capacity of adipose tissue.

Methods of discovery-based and targeted metabolite analysis by comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry detection.

The investigation of naturally volatile and derivatized metabolites in biological tissues by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOFMS) can provide highly complex and information-rich data for comprehensive metabolomics analysis. The addition of the second separation dimension with GC × GC provides additional chemical selectivity, and the fast scanning time of TOFMS offers benefits in chemical selectivity and overall peak capacity compared to traditional one-dimensional (1D) GC. Furthermore, methods of derivatization to facilitate volatility and thermal stability, the most prominent being the silylation of organic compounds, have extended the use of GC as an important metabolomics tool. The highly information-rich data from GC × GC-TOFMS benefits from sophisticated comprehensive targeted and nontargeted algorithmic software methods. Herein, we detail a robust derivatization and instrumental method for metabolomics analysis and provide a brief overview of possible methods for data analysis.

Correlation of rocket propulsion fuel properties with chemical composition using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry followed by partial least squares regression analysis.

There is an increased need to more fully assess and control the composition of kerosene-based rocket propulsion fuels such as RP-1. In particular, it is critical to make better quantitative connections among the following three attributes: fuel performance (thermal stability, sooting propensity, engine specific impulse, etc.), fuel properties (such as flash point, density, kinematic viscosity, net heat of combustion, and hydrogen content), and the chemical composition of a given fuel, i.e., amounts of specific chemical compounds and compound classes present in a fuel as a result of feedstock blending and/or processing. Recent efforts in predicting fuel chemical and physical behavior through modeling put greater emphasis on attaining detailed and accurate fuel properties and fuel composition information. Often, one-dimensional gas chromatography (GC) combined with mass spectrometry (MS) is employed to provide chemical composition information. Building on approaches that used GC-MS, but to glean substantially more chemical information from these complex fuels, we recently studied the use of comprehensive two dimensional (2D) gas chromatography combined with time-of-flight mass spectrometry (GC×GC-TOFMS) using a "reversed column" format: RTX-wax column for the first dimension, and a RTX-1 column for the second dimension. In this report, by applying chemometric data analysis, specifically partial least-squares (PLS) regression analysis, we are able to readily model (and correlate) the chemical compositional information provided by use of GC×GC-TOFMS to RP-1 fuel property information such as density, kinematic viscosity, net heat of combustion, and so on. Furthermore, we readily identified compounds that contribute significantly to measured differences in fuel properties based on results from the PLS models. We anticipate this new chemical analysis strategy will have broad implications for the development of high fidelity composition-property models, leading to an improved approach to fuel formulation and specification for advanced engine cycles.