Volumetric absorptive microsampling as an effective microsampling technique for LC-MS/MS bioanalysis of biomarkers in drug discovery.

Aim: Develop and validate a volumetric absorptive microsampling (VAMS)-based LC-MS/MS method to support the bioanalysis of amino acid and carboxylic acid biomarkers in mouse whole blood. Method: Mouse whole blood was collected using a 10 µl VAMS device. The analytes in VAMS were extracted and analyzed using an LC-MS/MS method. Results: The VAMS-based LC-MS/MS assay exhibited a linearity range of 10.0-10,000 ng/ml with acceptable precision and accuracy and consistent recovery. The analyte stability in mouse whole blood VAMS was shown for 7 days at ambient conditions and at -80°C, as well as with three freeze/thaw cycles. Conclusion: A simple and robust VAMS-based LC-MS/MS method was developed and further validated for simultaneous bioanalysis of nine biomarkers in mouse whole blood.

Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring.

Accurate reconstruction of metabolic pathways is an important prerequisite for interpreting metabolomics changes and understanding the diverse biological processes in disease models. A tracer-based metabolomics strategy utilizes stable isotope-labeled precursors to resolve complex pathways by tracing the labeled atom(s) to downstream metabolites through enzymatic reactions. Isotope enrichment analysis is informative and achieved by counting total labeled atoms and acquiring the mass isotopologue distribution (MID) of the intact metabolite. However, quantitative analysis of labeled metabolite substructures/moieties (MS2 fragments) can offer more valuable insights into the reaction connections through measuring metabolite transformation. In order to acquire the isotopic labeling information at the intact metabolite and moiety level simultaneously, we developed a method that couples hydrophilic interaction liquid chromatography (HILIC) with Zeno trap-enabled high-resolution multiple reaction monitoring (MRMHR). The method enabled accurate and reproducible MID quantification for intact metabolites as well as their fragmented moieties, with notably high sensitivity in the MS2 fragmentation mode based on the measurement of 13C- or 15N-labeled cellular samples. The method was applied to human-induced pluripotent stem cell-derived neurons to trace the fate of 13C/15N atoms from D-13C6-glucose/L-15N2-glutamine added to the media. With the MID analysis of both intact metabolites and fragmented moieties, we validated the pathway reconstruction of de novo glutathione synthesis in mid-brain neurons. We discovered increased glutathione oxidization from both basal and newly synthesized glutathione pools under neuronal oxidative stress. Furthermore, the significantly decreased de novo glutathione synthesis was investigated and associated with altered activities of several key enzymes, as evidenced by suppressed glutamate supply via glucose metabolism and a diminished flux of glutathione synthetic reaction in the neuronal model of rotenone-induced neurodegeneration.

High-throughput proteomics of nanogram-scale samples with Zeno SWATH MS.

The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.

Relation of plasma ceramides to visceral adiposity, insulin resistance and the development of type 2 diabetes mellitus: the Dallas Heart Study.

AIMS/HYPOTHESIS: Ceramides are sphingolipids that contribute to insulin resistance in preclinical studies. We hypothesised that plasma ceramides would be associated with body fat distribution, insulin resistance and incident type 2 diabetes in a multi-ethnic cohort. METHODS: A total of 1557 participants in the Dallas Heart Study without type 2 diabetes underwent measurements of metabolic biomarkers, fat depots by MRI and plasma ceramides by liquid chromatography-mass spectrometry. Diabetes outcomes were assessed after 7 years. Associations of body fat and insulin resistance with ceramides at baseline and of ceramides with incident diabetes outcomes were analysed. RESULTS: The cohort had a mean age of 43 years, with 58% women, 45% black participants and a mean BMI of 28 kg/m2. Total cholesterol levels were associated with all ceramides, but higher triacylglycerols and lower HDL-cholesterol and adiponectin were associated only with saturated fatty acid chain ceramides (p < 0.0003). After adjusting for clinical characteristics and total body fat, visceral adipose tissue was positively associated with saturated fatty acid ceramides (per SD, ß = 0.16 to 0.18) and inversely associated with polyunsaturated fatty acid ceramides (ß = -0.14 to -0.16, p < 0.001 for all). Lower-body subcutaneous fat showed an opposite pattern to that for visceral fat. HOMA-IR was positively associated with saturated (ß = 0.08 to 0.09, p < 0.001) and inversely with polyunsaturated ceramides (ß = -0.06 to -0.07, p < 0.05). Ceramides were not associated with incident type 2 diabetes after adjustment for clinical factors. CONCLUSIONS/INTERPRETATION: Plasma ceramides demonstrate a biologically complex relationship with metabolic and imaging indicators of dysfunctional adiposity. The role of ceramides in a shared pathway of metabolic dysfunction linking visceral adiposity and insulin resistance requires further investigation.

Skin-specific regulation of SREBP processing and lipid biosynthesis by glycerol kinase 5.

The recessive N-ethyl-N-nitrosourea-induced phenotype toku is characterized by delayed hair growth, progressive hair loss, and excessive accumulation of dermal cholesterol, triglycerides, and ceramides. The toku phenotype was attributed to a null allele of Gk5, encoding glycerol kinase 5 (GK5), a skin-specific kinase expressed predominantly in sebaceous glands. GK5 formed a complex with the sterol regulatory element-binding proteins (SREBPs) through their C-terminal regulatory domains, inhibiting SREBP processing and activation. In Gk5toku/toku mice, transcriptionally active SREBPs accumulated in the skin, but not in the liver; they were localized to the nucleus and led to elevated lipid synthesis and subsequent hair growth defects. Similar defective hair growth was observed in kinase-inactive GK5 mutant mice. Hair growth defects of homozygous toku mice were partially rescued by treatment with the HMG-CoA reductase inhibitor simvastatin. GK5 exists as part of a skin-specific regulatory mechanism for cholesterol biosynthesis, independent of cholesterol regulation elsewhere in the body.

Cyp8b1 ablation prevents Western diet-induced weight gain and hepatic steatosis because of impaired fat absorption.

Bile acids (BAs) are cholesterol derivatives that regulate lipid metabolism, through their dual abilities to promote lipid absorption and activate BA receptors. However, different BA species have varying abilities to perform these functions. Eliminating 12α-hydroxy BAs in mice via Cyp8b1 knockout causes low body weight and improved glucose tolerance. The goal of this study was to determine mechanisms of low body weight in Cyp8b1-/- mice. We challenged Cyp8b1-/- mice with a Western-type diet and assessed body weight and composition. We measured energy expenditure, fecal calories, and lipid absorption and performed lipidomic studies on feces and intestine. We investigated the requirement for dietary fat in the phenotype using a fat-free diet. Cyp8b1-/- mice were resistant to Western diet-induced body weight gain, hepatic steatosis, and insulin resistance. These changes were associated with increased fecal calories, due to malabsorption of hydrolyzed dietary triglycerides. This was reversed by treating the mice with taurocholic acid, the major 12α-hydroxylated BA species. The improvements in body weight and steatosis were normalized by feeding mice a fat-free diet. The effects of BA composition on intestinal lipid handling are important for whole body energy homeostasis. Thus modulating BA composition is a potential tool for obesity or diabetes therapy.

Duodenal-jejunal bypass surgery induces hepatic lipidomic alterations associated with ameliorated hepatic steatosis in mice.

OBJECTIVE: Bariatric surgery induces weight loss and improvement of insulin resistance; one aspect of both is an amelioration of hepatic steatosis. This study was undertaken to assess the changes in the hepatic lipidome after duodenal-jejunal bypass (DJB) surgery. METHODS: A DJB surgical model was developed and characterized in diet-induced obese mice. In comparison with sham-operated mice, an unbiased lipidomic profiling of hepatic lipids was performed together with measurements of gene expression within key pathways of hepatic lipid metabolism. RESULTS: In the liver of DJB mice, a dramatic reduction (by 77%) in hepatic triacylglycerols was observed. Global lipidomic profiling identified marked decreases of triacylglycerols comprised of medium length fatty acids and with low double bond content. Specific diacylglycerol species were also among the most dramatic decreases in hepatic lipids, whereas lysophosphatidic acids and phosphatidic acids were increased. Expression of fatty acid transporter and lipogenic genes was down-regulated. CONCLUSIONS: From in-depth analysis of hepatic lipid composition, specific lipid intermediates were identified that are preferentially changed following DJB surgery. These changes were most likely due to DJB-induced weight loss, and only further studies will be able to distinguish weight loss-dependent from weight loss-independent changes.

The MBOAT7-TMC4 Variant rs641738 Increases Risk of Nonalcoholic Fatty Liver Disease in Individuals of European Descent.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver damage and is characterized by steatosis. Genetic factors increase risk for progressive NAFLD. A genome-wide association study showed that the rs641738 C>T variant in the locus that contains the membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7, also called LPIAT1) and transmembrane channel-like 4 gene (TMC4) increased the risk for cirrhosis in alcohol abusers. We investigated whether the MBOAT7-TMC4 is a susceptibility locus for the development and progression of NAFLD. METHODS: We genotyped rs641738 in DNA collected from 3854 participants from the Dallas Heart Study (a multi-ethnic population-based probability sample of Dallas County residents) and 1149 European individuals from the Liver Biopsy Cross-Sectional Cohort. Clinical and anthropometric data were collected, and biochemical and lipidomics were measured in plasma samples from participants. A total of 2736 participants from the Dallas Heart Study also underwent proton magnetic resonance spectroscopy to measure hepatic triglyceride content. In the Liver Biopsy Cross-Sectional Cohort, a total of 1149 individuals underwent liver biopsy to diagnose liver disease and disease severity. RESULTS: The genotype rs641738 at the MBOAT7-TMC4 locus associated with increased hepatic fat content in the 2 cohorts, and with more severe liver damage and increased risk of fibrosis compared with subjects without the variant. MBOAT7, but not TMC4, was found to be highly expressed in the liver. The MBOAT7 rs641738 T allele was associated with lower protein expression in the liver and changes in plasma phosphatidylinositol species consistent with decreased MBOAT7 function. CONCLUSIONS: We provide evidence for an association between the MBOAT7 rs641738 variant and the development and severity of NAFLD in individuals of European descent. This association seems to be mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling.

Multiplexed Quantification of Proglucagon-Derived Peptides by Immunoaffinity Enrichment and Tandem Mass Spectrometry after a Meal Tolerance Test.

BACKGROUND: Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity. METHODS: We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy. RESULTS: Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay. CONCLUSIONS: IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal.

Quantitative profiling of oxylipins in plasma and atherosclerotic plaques of hypercholesterolemic rabbits.

Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that affect a broad range of physiological processes, including cell proliferation, inflammation, inflammation resolution, and vascular function. Moreover, oxylipins are readily detectable in plasma, and certain subsets of oxylipins have been detected in human atherosclerotic lesions. Taken together, we set out to produce a detailed quantitative assessment of plasma and plaque oxylipins in a widely used model of atherosclerosis, to identify potential biomarkers of disease progression. We administered regular chow or regular chow supplemented with 0.5% cholesterol (HC) to male New Zealand white rabbits for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our targeted lipidomic analyses of oxylipins on plaques isolated from rabbits fed the HC diet detected 34 oxylipins, 28 of which were in compliance with our previously established quality control acceptance criteria. The arachidonic acid (AA) metabolite derived from the COX pathway, 6-keto-PGF1α was the most abundant plaque oxylipin, followed by the linoleic acid (LA) metabolites 9-HODE, 13-HODE and 9,12,13-TriHOME and the arachidonic acid (AA)-derivatives 11-HETE and 12-HETE. We additionally found that the most abundant oxylipins in plasma were three of the five most abundant oxylipins in plaque, namely 11-HETE, 13-HODE, and 9-HODE. The studies reported here make the first step towards a comprehensive characterization of oxylipins as potentially translatable biomarkers of atherosclerosis.

Evaluation of CETP activity in vivo under non-steady-state conditions: influence of anacetrapib on HDL-TG flux.

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.

Increased Bile Acid Synthesis and Impaired Bile Acid Transport in Human Obesity.

CONTEXT: Alterations in bile acid (BA) synthesis and transport have the potential to affect multiple metabolic pathways in the pathophysiology of obesity. OBJECTIVE: The objective of the study was to investigate the effects of obesity on serum fluctuations of BAs and markers of BA synthesis. DESIGN: We measured BA fluctuations in 11 nonobese and 32 obese subjects and BA transporter expression in liver specimens from 42 individuals and specimens of duodenum, jejunum, ileum, colon, and pancreas from nine individuals. MAIN OUTCOME MEASURES: We analyzed serum BAs and markers of BA synthesis after overnight fasting, during a hyperinsulinemic-euglycemic clamp, or a mixed-meal tolerance test and the association of BA transporter expression with body mass index. RESULTS: BA synthesis markers were 2-fold higher (P < .01) and preferentially 12α-hydroxylated (P < .05) in obese subjects, and both measures were correlated with clamp-derived insulin sensitivity (r = -0.62, P < .0001, and r = -0.39, P = .01, respectively). Insulin infusion acutely reduced serum BAs in nonobese subjects, but this effect was blunted in obese subjects (δBAs -44.2% vs -4.2%, P < .05). The rise in serum BAs postprandially was also relatively blunted in obese subjects (δBAs +402% vs +133%, P < .01). Liver expression of the Na+-taurocholate cotransporting polypeptide and the bile salt export pump were negatively correlated with body mass index (r = -0.37, P = .02, and r = -0.48, P = .001, respectively). CONCLUSIONS: Obesity is associated with increased BA synthesis, preferential 12α-hydroxylation, and impaired serum BA fluctuations. The findings reveal new pathophysiological aspects of BA action in obesity that may lend themselves to therapeutic targeting in metabolic disease.

Glucagon receptor antagonism induces increased cholesterol absorption.

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

Effect of Error Propagation in Stable Isotope Tracer Studies: An Approach for Estimating Impact on Apparent Biochemical Flux.

Stable isotope tracers are widely used to quantify metabolic rates, and yet a limited number of studies have considered the impact of analytical error on estimates of flux. For example, when estimating the contribution of de novo lipogenesis, one typically measures a minimum of four isotope ratios, i.e., the precursor and product labeling pre- and posttracer administration. This seemingly simple problem has 1 correct solution and 80 erroneous outcomes. In this report, we outline a methodology for evaluating the effect of error propagation on apparent physiological endpoints. We demonstrate examples of how to evaluate the influence of analytical error in case studies concerning lipid and protein synthesis; we have focused on (2)H2O as a tracer and contrast different mass spectrometry platforms including GC-quadrupole-MS, GC-pyrolysis-IRMS, LC-quadrupole-MS, and high-resolution FT-ICR-MS. The method outlined herein can be used to determine how to minimize variations in the apparent biology by altering the dose and/or the type of tracer. Likewise, one can facilitate biological studies by estimating the reduction in the noise of an outcome that is expected for a given increase in the number of replicate injections.

Discovery of Triazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys.

Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Inhibition of cholesteryl ester transfer protein increases cholesteryl ester content of large HDL independently of HDL-to-HDL homotypic transfer: in vitro vs in vivo comparison using anacetrapib and dalcetrapib.

The increase in high density lipoprotein (HDL)-cholesterol observed with cholesteryl ester transfer protein (CETP) inhibition is commonly attributed to blockade of cholesteryl ester (CE) transfer from HDL to low density lipoprotein particles. In vitro, it has been observed that CETP can mediate transfer of CE between HDL particles ("homotypic transfer"), and it is postulated that this contributes to HDL remodeling and generation of anti-atherogenic pre-beta HDL. Inhibition of CETP could limit this beneficial remodeling and reduce pre-beta HDL levels. We observed that anacetrapib does not reduce pre-beta HDL in vivo, but the role of HDL homotypic transfer was not examined. This study evaluated the effects of anacetrapib on homotypic transfer from HDL3 to HDL2 in vivo using deuterium-labeled HDL3, and compared this to in vitro settings, where homotypic transfer was previously described. In vitro, both anacetrapib and dalcetrapib inhibited transfer of CE from HDL3 to HDL2 particles. In CETP transgenic mice, anacetrapib did not inhibit the appearance of labeled CE derived from HDL3 in HDL2 particles, but rather promoted the appearance of labeled CE in HDL2. We concluded that inhibition of CETP by anacetrapib promoted HDL particle remodeling, and does not impair the flux of cholesterol ester into larger HDL particles when studied in vivo, which is not consistent with in vitro observations. We further conclude, therefore, that the in vitro conditions used to examine HDL-to-HDL homotypic transfer may not recapitulate the in vivo condition, where multiple mechanisms contribute to cholesteryl ester flux into and out of the HDL pool.

Discovery of Benzimidazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys.

We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Increased Bile Acid Synthesis and Deconjugation After Biliopancreatic Diversion.

Biliopancreatic diversion (BPD) improves insulin sensitivity and decreases serum cholesterol out of proportion with weight loss. Mechanisms of these effects are unknown. One set of proposed contributors to metabolic improvements after bariatric surgeries is bile acids (BAs). We investigated the early and late effects of BPD on plasma BA levels, composition, and markers of BA synthesis in 15 patients with type 2 diabetes (T2D). We compared these to the early and late effects of Roux-en-Y gastric bypass (RYGB) in 22 patients with T2D and 16 with normal glucose tolerance. Seven weeks after BPD, insulin sensitivity had doubled and serum cholesterol had halved. At this time, BA synthesis markers and total plasma BAs, particularly unconjugated BAs, had markedly risen; this effect could not be entirely explained by low FGF19. In contrast, after RYGB, insulin sensitivity improved gradually with weight loss and cholesterol levels declined marginally; BA synthesis markers were decreased at an early time point (2 weeks) after surgery and returned to the normal range 1 year later. These findings indicate that BA synthesis contributes to the decreased serum cholesterol after BPD. Moreover, they suggest a potential role for altered enterohepatic circulation of BAs in improving insulin sensitivity and cholesterol metabolism after BPD.

Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans.

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys the endogenous OATP1B substrates that are potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the 18 biomarkers tested, RIF (18 mg/kg, oral) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate whether cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we determined the inhibitory effect of RIF on in vitro transport and pharmacokinetics of rosuvastatin (RSV) and atorvastatin (ATV). RIF strongly inhibited the uptake of RSV and ATV by cOATP1B1 and cOATP1B3 in vitro. In agreement with clinical observations, RIF (18 mg/kg, oral) significantly decreased plasma clearance and increased the area under the plasma concentration curve (AUC) of intravenously administered RSV by 2.8- and 2.7-fold, and increased the AUC and maximum plasma concentration of orally administered RSV by 6- and 10.3-fold, respectively. In contrast to clinical findings, RIF did not significantly increase plasma exposure of either intravenous or orally administered ATV, indicating species differences in the rate-limiting elimination pathways.

Lipidome of atherosclerotic plaques from hypercholesterolemic rabbits.

The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome.