Variability of the Plasma Lipidome and Subclinical Coronary Atherosclerosis.

OBJECTIVE: While the risk of acute coronary events has been associated with biological variability of circulating cholesterol, the association with variability of other atherogenic lipids remains less understood. We evaluated the longitudinal variability of 284 lipids and investigated their association with asymptomatic coronary atherosclerosis. Approach and Results: Circulating lipids were extracted from fasting blood samples of 83 community-sampled symptom-free participants (age 41-75 years), collected longitudinally over 6 months. Three types of coronary plaque volume (calcified, lipid-rich, and fibrotic) were quantified using computed tomography coronary angiogram. We first deconvoluted between-subject (CVg) and within-subject (CVw) lipid variabilities. We then tested whether the mean lipid abundance was different across groups categorized by Framingham risk score and plaques phenotypes (lipid-rich, fibrotic, and calcified). Finally, we investigated whether visit-to-visit variability of each lipid was associated with plaque burden. Most lipids (72.5%) exhibited higher CVg than CVw. Among the lipids (n=145) with 1.2-fold higher CVg than CVw, 26 species including glycerides and ceramides were significantly associated with Framingham risk score and the 3 plaque phenotypes (false discovery rate <0.05). In an exploratory analysis of person-specific visit-to-visit variability without multiple testing correction, high variability of 3 lysophospholipids (lysophosphatidylethanolamines 16:0, 18:0, and lysophosphatidylcholine O-18:1) was associated with lipid-rich and fibrotic (noncalcified) plaque volume while high variability of diacylglycerol 18:1_20:0, triacylglycerols 52:2, 52:3, and 52:4, ceramide d18:0/20:0, dihexosylceramide d18:1/16:0, and sphingomyelin 36:3 was associated with calcified plaque volume. CONCLUSIONS: High person-specific longitudinal variation of specific nonsterol lipids is associated with the burden of subclinical coronary atherosclerosis. Larger studies are needed to confirm these exploratory findings.

Population-based plasma lipidomics reveals developmental changes in metabolism and signatures of obesity risk: a mother-offspring cohort study.

BACKGROUND: Lipids play a vital role in health and disease, but changes to their circulating levels and the link with obesity remain poorly characterized in expecting mothers and their offspring in early childhood. METHODS: LC-MS/MS-based quantitation of 480 lipid species was performed on 2491 plasma samples collected at 4 time points in the mother-offspring Asian cohort GUSTO (Growing Up in Singapore Towards healthy Outcomes). These 4 time points constituted samples collected from mothers at 26-28 weeks of gestation (n=752) and 4-5 years postpartum (n=650), and their offspring at birth (n=751) and 6 years of age (n=338). Linear regression models were used to identify the pregnancy and developmental age-specific variations in the plasma lipidomic profiles, and their association with obesity risk. An independent birth cohort (n=1935), the Barwon Infant Study (BIS), comprising mother-offspring dyads of Caucasian origin was used for validation. RESULTS: Levels of 36% of the profiled lipids were significantly higher (absolute fold change > 1.5 and Padj < 0.05) in antenatal maternal circulation as compared to the postnatal phase, with phosphatidylethanolamine levels changing the most. Compared to antenatal maternal lipids, cord blood showed lower concentrations of most lipid species (79%) except lysophospholipids and acylcarnitines. Changes in lipid concentrations from birth to 6 years of age were much higher in magnitude (log2FC=-2.10 to 6.25) than the changes observed between a 6-year-old child and an adult (postnatal mother) (log2FC=-0.68 to 1.18). Associations of cord blood lipidomic profiles with birth weight displayed distinct trends compared to the lipidomic profiles associated with child BMI at 6 years. Comparison of the results between the child and adult BMI identified similarities in association with consistent trends (R2=0.75). However, large number of lipids were associated with BMI in adults (67%) compared to the children (29%). Pre-pregnancy BMI was specifically associated with decrease in the levels of phospholipids, sphingomyelin, and several triacylglycerol species in pregnancy. CONCLUSIONS: In summary, our study provides a detailed landscape of the in utero lipid environment provided by the gestating mother to the growing fetus, and the magnitude of changes in plasma lipidomic profiles from birth to early childhood. We identified the effects of adiposity on the circulating lipid levels in pregnant and non-pregnant women as well as offspring at birth and at 6 years of age. Additionally, the pediatric vs maternal overlap of the circulating lipid phenotype of obesity risk provides intergenerational insights and early opportunities to track and intervene the onset of metabolic adversities. CLINICAL TRIAL REGISTRATION: This birth cohort is a prospective observational study, which was registered on 1 July 2010 under the identifier NCT01174875 .

LICAR: An Application for Isotopic Correction of Targeted Lipidomic Data Acquired with Class-Based Chromatographic Separations Using Multiple Reaction Monitoring.

Lipidomics is developing as an important area in biomedical and clinical research. Reliable quantification of lipid species is required for clinical translation of lipidomic studies. Hydrophilic interaction chromatography (HILIC), normal-phase liquid chromatography (NPLC), and supercritical fluid chromatography (SFC) are commonly used techniques in lipidomics and provide class-based separation of lipids. While co-elution of lipid species and their internal standards is an advantage for accurate quantification, it leads to isotopic overlap between species of the same lipid class. In shotgun lipidomics, isotopic correction is typically done based on elemental formulas of precursor ions. In multiple reaction monitoring (MRM) analyses, however, this approach should not be used, as the overall contribution of heavy isotopes to the MRM transitions' intensities depends on their location in the molecule with respect to the fragmentation pattern. We present an algorithm, provided in the R programming language, for isotopic correction in class-based separation using MRM, extracting relevant structural information from MRM transitions to apply adequate isotopic correction factors. Using standards, we show that our algorithm accurately estimates the isotopic contribution of isotopologues to MRM transitions' measured intensities. Using human plasma as an example, we demonstrate the necessity of adequate isotopic correction for accurate quantitation of lipids measured by MRM with class-based chromatographic separation. We show that over a third of the measured phosphatidylcholine species had their intensity corrected by more than 10%. This isotopic correction algorithm and R-implemented application enable a more accurate quantification of lipids in class-based separation-MRM, a prerequisite for successful translation of lipidomic applications.

Shared reference materials harmonize lipidomics across MS-based detection platforms and laboratories.

Quantitative MS of human plasma lipids is a promising technology for translation into clinical applications. Current MS-based lipidomic methods rely on either direct infusion (DI) or chromatographic lipid separation methods (including reversed phase and hydrophilic interaction LC). However, the use of lipid markers in laboratory medicine is limited by the lack of reference values, largely because of considerable differences in the concentrations measured by different laboratories worldwide. These inconsistencies can be explained by the use of different sample preparation protocols, method-specific calibration procedures, and other experimental and data-reporting parameters, even when using identical starting materials. Here, we systematically investigated the roles of some of these variables in multiple approaches to lipid analysis of plasma samples from healthy adults by considering: 1) different sample introduction methods (separation vs. DI methods); 2) different MS instruments; and 3) between-laboratory differences in comparable analytical platforms. Each of these experimental variables resulted in different quantitative results, even with the inclusion of isotope-labeled internal standards for individual lipid classes. We demonstrated that appropriate normalization to commonly available reference samples (i.e., "shared references") can largely correct for these systematic method-specific quantitative biases. Thus, to harmonize data in the field of lipidomics, in-house long-term references should be complemented by a commonly available shared reference sample, such as NIST SRM 1950, in the case of human plasma.

MRMkit: Automated Data Processing for Large-Scale Targeted Metabolomics Analysis.

MRMkit is an open-source software package designed for automated processing of large-scale targeted mass spectrometry-based metabolomics data. With improvements in the automation of sample preparation for LC-MS analysis, a challenging next step is to fully automate the workflow to process raw data and ensure the quality of measurements in large-scale analysis settings. MRMkit capitalizes on the richness of large-sample data in capturing peak shapes and interference patterns of transitions across many samples and delivers fully automated, reproducible peak integration results in a scalable and time-efficient manner. In addition to fast and accurate peak integration, the tool also provides reliable data normalization functions and quality metrics along with visualizations for fast data quality evaluation. In addition, MRMkit learns retention time offset patterns by user-specified compound classes and makes recommendations for peak picking in multimodal ion chromatograms. In summary, MRMkit offers highly consistent and scalable data processing capacity for targeted metabolomics, substantially curtailing the time required to produce the final quantification results after LC-MS analysis.

SmartPeak Automates Targeted and Quantitative Metabolomics Data Processing.

Technological advances in high-resolution mass spectrometry (MS) vastly increased the number of samples that can be processed in a life science experiment, as well as volume and complexity of the generated data. To address the bottleneck of high-throughput data processing, we present SmartPeak (https://github.com/AutoFlowResearch/SmartPeak), an application that encapsulates advanced algorithms to enable fast, accurate, and automated processing of capillary electrophoresis-, gas chromatography-, and liquid chromatography (LC)-MS(/MS) data and high-pressure LC data for targeted and semitargeted metabolomics, lipidomics, and fluxomics experiments. The application allows for an approximate 100-fold reduction in the data processing time compared to manual processing while enhancing quality and reproducibility of the results.

Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics.

We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.

Dual mass spectrometry as a tool to improve annotation and quantification in targeted plasma lipidomics.

INTRODUCTION: High quality data, based on reliable quantification and clear identification of the reported lipid species, are required for the clinical translation of human plasma lipidomic studies. OBJECTIVE: Lipid quantification can be efficiently performed on triple quadrupole (QqQ) mass spectrometers in targeted multiple reaction monitoring (MRM) mode. However, a series of issues can be encountered when aiming at unambiguous identification and accurate quantification, including (i) resolving peaks of polyunsaturated species, (ii) discriminating between plasmanyl-, plasmenyl- and odd chain species and (iii) resolving the isotopic overlap between co-eluting lipid species. METHODS: As a practical tool to improve the quality of targeted lipidomics studies, we applied a Dual MS platform by simultaneously coupling a reversed-phase liquid chromatography separation to a QqQ and a quadrupole-time of flight (Q-ToF) mass spectrometers. In one single experiment, this platform allows to correctly identify, by high-resolution MS and MS/MS, the peaks that are quantified by MRM. RESULTS: As proof of concept, we applied the platform on glycerophosphocholines (GPCs) and sphingomyelins (SMs), which are highly abundant in human plasma and play crucial roles in various physiological functions. Our results demonstrated that Dual MS could provide a higher level of confidence in the identification and quantification of GPCs and SMs in human plasma. The same approach can also be applied to improve the study of other lipid classes and expanded for the identification of novel lipid molecular species. CONCLUSIONS: This methodology might have a great potential to achieve a better specificity in the quantification of lipids by targeted lipidomics in high-throughput studies.

Serum lipidome analysis of healthy beagle dogs receiving different diets.

INTRODUCTION: Food and dietary ingredients have significant effects on metabolism and health. OBJECTIVE: To evaluate whether and how different diets affected the serum lipidomic profile of dogs. METHODS: Sixteen healthy beagles were fed a commercial dry diet for 3 months (control diet). After an overnight fasting period, a blood sample was taken for serum lipidomic profile analysis, and each dog was then randomly assigned to one of two groups. Group 1 was fed a commercial diet (Diet 1) and group 2 was fed a self-made, balanced diet supplemented with linseed oil and salmon oil (Diet 2) for 3 months. After an overnight fasting period, a blood sample was taken from each dog. Serum cholesterol and triacylglycerol analyses were performed and the serum lipidomic profiles were analyzed using targeted liquid chromatography-mass spectrometry. RESULTS: Dogs fed the supplemented self-made diet (Diet 2) had significantly higher omega-3 fatty acid-containing lipids species and significantly lower saturated and mono- and di-unsaturated lipid species. Concentrations of sphingosine 1-phosphate species S1P d16:1 and S1P d17:1 were significantly increased after feeding Diet 2. CONCLUSION: This study found that different diets had significant effects on the dog's serum lipidomic profile. Therefore, in studies that include lipidomic analyses, diet should be included as a confounding factor.

MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines.

Human blood is a self-regenerating lipid-rich biological fluid that is routinely collected in hospital settings. The inventory of lipid molecules found in blood plasma (plasma lipidome) offers insights into individual metabolism and physiology in health and disease. Disturbances in the plasma lipidome also occur in conditions that are not directly linked to lipid metabolism; therefore, plasma lipidomics based on MS is an emerging tool in an array of clinical diagnostics and disease management. However, challenges exist in the translation of such lipidomic data to clinical applications. These relate to the reproducibility, accuracy, and precision of lipid quantitation, study design, sample handling, and data sharing. This position paper emerged from a workshop that initiated a community-led process to elaborate and define a set of generally accepted guidelines for quantitative MS-based lipidomics of blood plasma or serum, with harmonization of data acquired on different instrumentation platforms across independent laboratories as an ultimate goal. We hope that other fields may benefit from and follow such a precedent.

ABCC1, an ATP binding cassette protein from grape berry, transports anthocyanidin 3-O-Glucosides.

Accumulation of anthocyanins in the exocarp of red grapevine (Vitis vinifera) cultivars is one of several events that characterize the onset of grape berry ripening (véraison). Despite our thorough understanding of anthocyanin biosynthesis and regulation, little is known about the molecular aspects of their transport. The participation of ATP binding cassette (ABC) proteins in vacuolar anthocyanin transport has long been a matter of debate. Here, we present biochemical evidence that an ABC protein, ABCC1, localizes to the tonoplast and is involved in the transport of glucosylated anthocyanidins. ABCC1 is expressed in the exocarp throughout berry development and ripening, with a significant increase at véraison (i.e., the onset of ripening). Transport experiments using microsomes isolated from ABCC1-expressing yeast cells showed that ABCC1 transports malvidin 3-O-glucoside. The transport strictly depends on the presence of GSH, which is cotransported with the anthocyanins and is sensitive to inhibitors of ABC proteins. By exposing anthocyanin-producing grapevine root cultures to buthionine sulphoximine, which reduced GSH levels, a decrease in anthocyanin concentration is observed. In conclusion, we provide evidence that ABCC1 acts as an anthocyanin transporter that depends on GSH without the formation of an anthocyanin-GSH conjugate.

Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous B1/B2 globin gene deletion) mice.

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.

Wounding of Arabidopsis halleri leaves enhances cadmium accumulation that acts as a defense against herbivory.

Approximately 0.2% of all angiosperms are classified as metal hyperaccumulators based on their extraordinarily high leaf metal contents, for example >1% zinc, >0.1% nickel or >0.01% cadmium (Cd) in dry biomass. So far, metal hyperaccumulation has been considered to be a taxon-wide, constitutively expressed trait, the extent of which depends solely on available metal concentrations in the soil. Here we show that in the facultative metallophyte Arabidopsis halleri, both insect herbivory and mechanical wounding of leaves trigger an increase specifically in leaf Cd accumulation. Moreover, the Cd concentrations accumulated in leaves can serve as an elemental defense against herbivory by larvae of the Brassicaceae specialist small white (Pieris rapae), thus allowing the plant to take advantage of this non-essential trace element and toxin. Metal homeostasis genes are overrepresented in the systemic transcriptional response of roots to the wounding of leaves in A. halleri, supporting that leaf Cd accumulation is preceded by systemic signaling events. A similar, but quantitatively less pronounced transcriptional response was observed in A. thaliana, suggesting that the systemically regulated modulation of metal homeostasis in response to leaf wounding also occurs in non-hyperaccumulator plants. This is the first report of an environmental stimulus influencing metal hyperaccumulation.

Vacuolar transport of abscisic acid glucosyl ester is mediated by ATP-binding cassette and proton-antiport mechanisms in Arabidopsis.

Abscisic acid (ABA) is a key plant hormone involved in diverse physiological and developmental processes, including abiotic stress responses and the regulation of stomatal aperture and seed germination. Abscisic acid glucosyl ester (ABA-GE) is a hydrolyzable ABA conjugate that accumulates in the vacuole and presumably also in the endoplasmic reticulum. Deconjugation of ABA-GE by the endoplasmic reticulum and vacuolar ß-glucosidases allows the rapid formation of free ABA in response to abiotic stress conditions such as dehydration and salt stress. ABA-GE further contributes to the maintenance of ABA homeostasis, as it is the major ABA catabolite exported from the cytosol. In this work, we identified that the import of ABA-GE into vacuoles isolated from Arabidopsis (Arabidopsis thaliana) mesophyll cells is mediated by two distinct membrane transport mechanisms: proton gradient-driven and ATP-binding cassette (ABC) transporters. Both systems have similar Km values of approximately 1 mm. According to our estimations, this low affinity appears nevertheless to be sufficient for the continuous vacuolar sequestration of ABA-GE produced in the cytosol. We further demonstrate that two tested multispecific vacuolar ABCC-type ABC transporters from Arabidopsis exhibit ABA-GE transport activity when expressed in yeast (Saccharomyces cerevisiae), which also supports the involvement of ABC transporters in ABA-GE uptake. Our findings suggest that the vacuolar ABA-GE uptake is not mediated by specific, but rather by several, possibly multispecific, transporters that are involved in the general vacuolar sequestration of conjugated metabolites.

Effects of wolfberry (Lycium barbarum) consumption on the human plasma lipidome and its association with cardiovascular disease risk factors: a randomized controlled trial of middle-aged and older adults.

Background: Long-term wolfberry intake as part of a healthy dietary pattern was recognized to have beneficial vascular outcomes. Characterization of the plasma lipidome may further provide comprehensive insights into pathways underlying these cardiovascular protective effects. Objective: We analyzed the plasma lipidome of subjects who adhered to a healthy dietary pattern either with or without wolfberry and investigated the associations between the plasma lipidomic profile and cardiovascular health-related indicators. Methods: In this 16-week, parallel design, randomized controlled trial, middle-aged and older adults (n = 41) were provided dietary counseling and assigned to either consume or not consume 15 g of wolfberry daily. At baseline and post-intervention, plasma lipidomics was assayed, and its relationships with classical CVD risk factors, vascular health, oxidant burden, carotenoids status, body composition, and anthropometry were examined. Results: From the plasma lipidome, 427 lipid species from 26 sub-classes were quantified. In the wolfberry and control groups, significant changes were prominent for 27 and 42 lipid species, respectively (P < 0.05 with > 0.2-fold change). Fold changes for seven lipid species were also markedly different between the two groups. Examining the relationships between the plasma lipidome and CVD-related risk factors, total cholesterol revealed a marked positive correlation with 13 ceramide species, while HDL-cholesterol which was notably increased with wolfberry consumption showed a positive correlation with 10 phosphatidylcholine species. Oxidant burden, as represented by plasma 8-isoprostanes, was also inversely associated with lipidomic triglycerides and ether-triglycerides (41 species) and directly associated with hexosylceramides (eight species) and sphingomyelins (six species). There were no differential associations with CVD risk detected between groups. Conclusion: Characteristic alterations to the plasma lipidome were observed with healthy dietary pattern adherence and wolfberry consumption. An examination of these fluctuations suggests potential biochemical mechanisms that may mediate the antioxidant and cardiovascular protective effects of healthy dietary pattern adherence and wolfberry intake. This study was registered at clinicaltrials.gov as NCT0353584.

Plasma and platelet lipidome changes in Fabry disease.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage disorder characterized by the progressive accumulation of globotriaosylceramide (Gb3) leading to systemic manifestations such as chronic kidney disease, cardiomyopathy, and stroke. There is still a need for novel markers for improved FD screening and prognosis. Moreover, the pathological mechanisms in FD, which also include systemic inflammation and fibrosis are not yet fully understood. METHODS: Plasma and platelets were obtained from 11 ERT (enzyme-replacement therapy)-treated symptomatic, 4 asymptomatic FD patients, and 13 healthy participants. A comprehensive targeted lipidomics analysis was conducted quantitating more than 550 lipid species. RESULTS: Sphingadiene (18:2;O2)-containing sphingolipid species, including Gb3 and galabiosylceramide (Ga2), were significantly increased in FD patients. Plasma levels of lyso-dihexosylceramides, sphingoid base 1-phosphates (S1P), and GM3 ganglioside were also altered in FD patients, as well as specific plasma ceramide ratios used in cardiovascular disease risk prediction. Gb3 did not increase in patients' platelets but displayed a high inter-individual variability in patients and healthy participants. Platelets accumulated, however, lyso-Gb3, acylcarnitines, C16:0-sphingolipids and S1P. CONCLUSIONS: This study identified new aspects of the systemic and cellular lipid metabolism in FD, a possible involvement of platelets in FD, and potential novel markers for FD screening and monitoring.

An integrated signature of extracellular matrix proteins and a diastolic function imaging parameter predicts post-MI long-term outcomes.

Background: Patients suffering from acute myocardial infarction (AMI) are at risk of secondary outcomes including major adverse cardiovascular events (MACE) and heart failure (HF). Comprehensive molecular phenotyping and cardiac imaging during the post-discharge time window may provide cues for risk stratification for the outcomes. Materials and methods: In a prospective AMI cohort in New Zealand (N = 464), we measured plasma proteins and lipids 30 days after hospital discharge and inferred a unified partial correlation network with echocardiographic variables and established clinical biomarkers (creatinine, c-reactive protein, cardiac troponin I and natriuretic peptides). Using a network-based data integration approach (iOmicsPASS+), we identified predictive signatures of long-term secondary outcomes based on plasma protein, lipid, imaging markers and clinical biomarkers and assessed the prognostic potential in an independent cohort from Singapore (N = 190). Results: The post-discharge levels of plasma proteins and lipids showed strong correlations within each molecular type, reflecting concerted homeostatic regulation after primary MI events. However, the two molecular types were largely independent with distinct correlation structures with established prognostic imaging parameters and clinical biomarkers. To deal with massively correlated predictive features, we used iOmicsPASS + to identify subnetwork signatures of 211 and 189 data features (nodes) predictive of MACE and HF events, respectively (160 overlapping). The predictive features were primarily imaging parameters, including left ventricular and atrial parameters, tissue Doppler parameters, and proteins involved in extracellular matrix (ECM) organization, cell differentiation, chemotaxis, and inflammation. The network signatures contained plasma protein pairs with area-under-the-curve (AUC) values up to 0.74 for HF prediction in the validation cohort, but the pair of NT-proBNP and fibulin-3 (EFEMP1) was the best predictor (AUC = 0.80). This suggests that there were a handful of plasma proteins with mechanistic and functional roles in predisposing patients to the secondary outcomes, although they may be weaker prognostic markers than natriuretic peptides individually. Among those, the diastolic function parameter (E/e' - an indicator of left ventricular filling pressure) and two ECM proteins, EFEMP1 and follistatin-like 3 (FSTL3) showed comparable performance to NT-proBNP and outperformed left ventricular measures as benchmark prognostic factors for post-MI HF. Conclusion: Post-discharge levels of E/e', EFEMP1 and FSTL3 are promising complementary markers of secondary adverse outcomes in AMI patients.

Malate transport by the vacuolar AtALMT6 channel in guard cells is subject to multiple regulation.

Gas exchange in plants is controlled by guard cells, specialized cells acting as turgor pressure-driven valves. Malate is one of the major anions accumulated inside the vacuole during stomatal opening counteracting the positive charge of potassium. AtALMT6, a member of the aluminum-activated malate transporter family, is expressed in guard cells of leaves and stems as well as in flower organs of Arabidopsis thaliana. An AtALMT6-GFP fusion protein was targeted to the vacuolar membrane both in transient and stable expression systems. Patch-clamp experiments on vacuoles isolated from AtALMT6-GFP over-expressing Arabidopsis plants revealed large inward-rectifying malate currents only in the presence of micromolar cytosolic calcium concentrations. Further analyses showed that vacuolar pH and cytosolic malate regulate the threshold of activation of AtALMT6-mediated currents. The interplay of these two factors determines the AtALMT6 function as a malate influx or efflux channel depending on the tonoplast potential. Guard cell vacuoles isolated from Atalmt6 knock-out plants displayed reduced malate currents compared with wild-type vacuoles. This reduction, however, was not accompanied by phenotypic differences in the stomatal movements in knock-out plants, probably because of functional redundancy of malate transporters in guard cell vacuoles.

Serum Lipidome Signatures of Dogs with Different Endocrinopathies Associated with Hyperlipidemia.

Hyperlipidemia (hypertriglyceridemia, hypercholesterolemia) is a common finding in human and veterinary patients with endocrinopathies (e.g., hypothyroidism and hypercortisolism (Cushing's syndrome; CS)). Despite emerging use of lipidomics technology in medicine, the lipid profiles of these endocrinopathies have not been evaluated and characterized in dogs. The aim of this study was to compare the serum lipidomes of dogs with naturally occurring CS or hypothyroidism with those of healthy dogs. Serum samples from 39 dogs with CS, 45 dogs with hypothyroidism, and 10 healthy beagle dogs were analyzed using a targeted lipidomics approach with liquid chromatography-mass spectrometry. There were significant differences between the lipidomes of dogs with CS, hypothyroidism, and the healthy dogs. The most significant changes were found in the lysophosphatidylcholines, lysophosphatidylethanolamines, lysophosphatidylinositols, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, ceramides, and sphingosine 1-phosphates. Lipid alterations were especially pronounced in dogs with hypothyroidism. Several changes suggested a more atherogenic lipid profile in dogs with HT than in dogs with CS. In this study, we found so far unknown effects of naturally occurring hypothyroidism and CS on lipid metabolism in dogs. Our findings provide starting points to further examine differences in occurrence of atherosclerotic lesion formation between the two diseases.

Evaluation of Normalization Approaches for Quantitative Analysis of Bile Acids in Human Feces.

Quantitative analysis of bile acids in human feces can potentially help to better understand the influence of the gut microbiome and diet on human health. Feces is a highly heterogeneous sample matrix, mainly consisting of water and indigestible solid material (as plant fibers) that show high inter-individual variability. To compare bile acid concentrations among different individuals, a reliable normalization approach is needed. Here, we compared the impact of three normalization approaches, namely sample wet weight, dry weight, and protein concentration, on the absolute concentrations of fecal bile acids. Bile acid concentrations were determined in 70 feces samples from healthy humans. Our data show that bile acid concentrations normalized by the three different approaches are substantially different for each individual sample. Fecal bile acid concentrations normalized by wet weight show the narrowest distribution. Therefore, our analysis will provide the basis for the selection of a suitable normalization approach for the quantitative analysis of bile acids in feces.