Pharmacometabolomic profiles in type 2 diabetic subjects treated with liraglutide or glimepiride.

BACKGROUND: Treatment with glucagon-like peptide-1 receptor agonists (GLP-1 RAs) leads to multiple metabolic changes, reduction in glucose levels and body weight are well established. In people with type 2 diabetes, GLP-1 RAs reduce the risk of cardiovascular (CV) disease and may also potentially represent a treatment for fatty liver disease. The mechanisms behind these effects are still not fully elucidated. The aim of the study was to investigate whether treatment with liraglutide is associated with favourable metabolic changes in cases of both CV disease and fatty liver disease. METHODS: In a prespecified post-hoc analysis of a double-blind, placebo-controlled trial in 62 individuals with type 2 diabetes (GLP-1 RA liraglutide or glimepiride, both in combination with metformin), we evaluated the changes in plasma molecular lipids and polar metabolites after 18 weeks of treatment. The lipids and polar metabolites were measured by using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS). RESULTS: In total, 340 lipids and other metabolites were identified, covering 14 lipid classes, bile acids, free fatty acids, amino acids and other polar metabolites. We observed more significant changes in the metabolome following liraglutide treatment compared to with glimepiride, particularly as regards decreased levels of cholesterol esters hexocyl-ceramides, lysophosphatidylcholines, sphingolipids and phosphatidylcholines with alkyl ether structure. In the liraglutide-treated group, lipids were reduced by approximately 15% from baseline, compared to a 10% decrease in the glimepiride group. At the pathway level, the liraglutide treatment was associated with lipid, bile acid as well as glucose metabolism, while glimepiride treatment was associated with tryptophan metabolism, carbohydrate metabolism, and glycerophospholipid metabolism. CONCLUSIONS: Compared with glimepiride, liraglutide treatment led to greater changes in the circulating metabolome, particularly regarding lipid metabolism involving sphingolipids, including ceramides. Our findings are hypothesis-generating and shed light on the underlying biological mechanisms of the CV benefits observed with GLP-1 RAs in outcome studies. Further studies investigating the role of GLP-1 RAs on ceramides and CV disease including fatty liver disease are warranted. TRIAL REGISTRATION: NCT01425580.

Perfluorooctanoic acid induces liver and serum dyslipidemia in humanized PPARα mice fed an American diet.

Per- and polyfluoroalkyl substances (PFAS) are pervasive in the environment resulting in nearly universal detection in people. Human serum PFAS concentrations are strongly associated with increased serum low-density lipoprotein cholesterol (LDL-C), and growing evidence suggests an association with serum triacylglycerides (TG). Here, we tested the hypothesis that perfluorooctanoic acid (PFOA) dysregulates liver and serum triacylglycerides in human peroxisome proliferator activated receptor α (hPPARα)-expressing mice fed an American diet. Mice were exposed to PFOA (3.5 mg/L) in drinking water for 6 weeks resulting in a serum concentration of 48 ± 9 µg/ml. In male and female hPPARα mice, PFOA increased total liver TG and TG substituted with saturated and monounsaturated fatty acids. Lack of expression of PPARα alone also increased total liver TG, and PFOA treatment had little effect on liver TG in PPARα null mice. In hPPARα mice, PFOA neither significantly increased nor decreased serum TG; however, there was a modest increase in TG associated with very low-density cholesterol particles in both sexes. Intriguingly, in female PPARα null mice, PFOA significantly increased serum TG, with a similar trend in males. PFOA also modified fatty acid and TG homeostasis-related gene expression in liver, in a hPPARα-dependent manner, but not in adipose. The results of our study and others reveal the importance of context (serum concentration and genotype) in determining the effect of PFOA on lipid homeostasis.

Characterising metabolically healthy obesity in weight-discordant monozygotic twins.

AIMS/HYPOTHESIS: Not all obese individuals display the metabolic disturbances commonly associated with excess fat accumulation. Mechanisms maintaining this 'metabolically healthy obesity' (MHO) are as yet unknown. We aimed to study different fat depots and transcriptional pathways in subcutaneous adipose tissue (SAT) as related to the MHO phenomenon. METHODS: Sixteen rare young adult obesity-discordant monozygotic (MZ) twin pairs (intra-pair difference (∆) in BMI ≥ 3 kg/m(2)), aged 22.8-35.8 years, were examined for detailed characteristics of metabolic health (subcutaneous, intra-abdominal and liver fat [magnetic resonance imaging/spectroscopy]), OGTT, lipids, adipokines and C-reactive protein (CRP). Affymetrix U133 Plus 2.0 chips were used to analyse transcriptomics pathways related to mitochondrial function and inflammation in SAT. RESULTS: Based on liver fat accumulation, two metabolically different subgroups emerged. In half (8/16) of the pairs (∆weight 17.1 ± 2.0 kg), the obese co-twin had significantly higher liver fat (∆718%), 78% increase in AUC insulin during OGTT and CRP, significantly more disturbance in the lipid profile and greater tendency for hypertension compared with the lean co-twin. In these obese co-twins, SAT expression of mitochondrial oxidative phosphorylation, branched-chain amino acid catabolism, fatty acid oxidation and adipocyte differentiation pathways were downregulated and chronic inflammation upregulated. In the other eight pairs (∆weight 17.4 ± 2.8 kg), the obese co-twin did not differ from the non-obese co-twin in liver fat (∆8%), insulin sensitivity, CRP, lipids, blood pressure or SAT transcriptomics. CONCLUSIONS/INTERPRETATION: Our results suggest that maintenance of high mitochondrial transcription and lack of inflammation in SAT are associated with low liver fat and MHO.

Controlled mechanical stimuli reveal novel associations between basil metabolism and sensory quality.

There is an increasing interest in the use of automation in plant production settings. Here, we employed a robotic platform to induce controlled mechanical stimuli (CMS) aiming to improve basil quality. Semi-targeted UHPLC-qToF-MS analysis of organic acids, amino acids, phenolic acids, and phenylpropanoids revealed changes in basil secondary metabolism under CMS, which appear to be associated with changes in taste, as revealed by different means of sensory evaluation (overall liking, check-all-that-apply, and just-about-right analysis). Further network analysis combining metabolomics and sensory data revealed novel links between plant metabolism and sensory quality. Amino acids and organic acids including maleic acid were negatively associated with basil quality, while increased levels of secondary metabolites, particularly linalool glucoside, were associated with improved basil taste. In summary, by combining metabolomics and sensory analysis we reveal the potential of automated CMS on crop production, while also providing new associations between plant metabolism and sensory quality.

From farm to fork and beyond! UV enhances Aryl hydrocarbon receptor-mediated activity of cruciferous vegetables in human intestinal cells upon colonic fermentation.

While the "farm to fork" strategy ticks many boxes in the sustainability agenda, it does not go far enough in addressing how we can improve crop nutraceutical quality. Here, we explored whether supplementary ultraviolet (UV) radiation exposure during growth of broccoli and Chinese cabbage can induce bioactive tryptophan- and glucosinolate-specific metabolite accumulation thereby enhancing Aryl hydrocarbon receptor (AhR) activation in human intestinal cells. By combining metabolomics analysis of both plant extracts and in vitro human colonic fermentation extracts with AhR reporter cell assay, we reveal that human colonic fermentation of UVB-exposed Chinese cabbage led to enhanced AhR activation in human intestinal cells by 23% compared to plants grown without supplementary UV. Thus, by exploring aspects beyond "from farm to fork", our study highlights a new strategy to enhance nutraceutical quality of Brassicaceae, while also providing new insights into the effects of cruciferous vegetables on human intestinal health.

Salinomycin inhibits prostate cancer growth and migration via induction of oxidative stress.

BACKGROUND: We have shown that a sodium ionophore monensin inhibits prostate cancer cell growth. A structurally related compound to monensin, salinomycin, was recently identified as a putative cancer stem cell inhibitor. METHODS: The growth inhibitory potential of salinomycin was studied in a panel of prostate cells. To get insights into the mechanism of action, a variety of assays such as gene expression and steroid profiling were performed in salinomycin-exposed prostate cancer cells. RESULTS: Salinomycin inhibited the growth of prostate cancer cells, but did not affect non-malignant prostate epithelial cells. Salinomycin impacted on prostate cancer stem cell functions as evidenced by reduced aldehyde dehydrogenase activity and the fraction of CD44(+) cells. Moreover, salinomycin reduced the expression of MYC, AR and ERG, induced oxidative stress as well as inhibited nuclear factor-κB activity and cell migration. Furthermore, profiling steroid metabolites revealed increased levels of oxidative stress-inducing steroids 7-ketocholesterol and aldosterone and decreased levels of antioxidative steroids progesterone and pregnenolone in salinomycin-exposed prostate cancer cells. CONCLUSION: Our results indicate that salinomycin inhibits prostate cancer cell growth and migration by reducing the expression of key prostate cancer oncogenes, inducing oxidative stress, decreasing the antioxidative capacity and cancer stem cell fraction.

Correlations between microbiota and metabolites after faecal microbiota transfer in irritable bowel syndrome.

Faecal microbiota transfer (FMT) consists of the infusion of donor faecal material into the intestine of a patient with the aim to restore a disturbed gut microbiota. In this study, it was investigated whether FMT has an effect on faecal microbial composition, its functional capacity, faecal metabolite profiles and their interactions in 16 irritable bowel syndrome (IBS) patients. Faecal samples from eight different time points before and until six months after allogenic FMT (faecal material from a healthy donor) as well as autologous FMT (own faecal material) were analysed by 16S RNA gene amplicon sequencing and gas chromatography coupled to mass spectrometry (GS-MS). The results showed that the allogenic FMT resulted in alterations in the microbial composition that were detectable up to six months, whereas after autologous FMT this was not the case. Similar results were found for the functional profiles, which were predicted from the phylogenetic sequencing data. While both allogenic FMT as well as autologous FMT did not have an effect on the faecal metabolites measured in this study, correlations between the microbial composition and the metabolites showed that the microbe-metabolite interactions seemed to be disrupted after allogenic FMT compared to autologous FMT. This shows that FMT can lead to altered interactions between the gut microbiota and its metabolites in IBS patients. Further research should investigate if and how this affects efficacy of FMT treatments.

Identification of a plasma signature of psychotic disorder in children and adolescents from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort.

The identification of an early biomarker of psychotic disorder is important as early treatment is associated with improved patient outcome. Metabolomic and lipidomic approaches in combination with multivariate statistical analysis were applied to identify plasma alterations in children (age 11) (38 cases vs 67 controls) and adolescents (age 18) (36 cases vs 117 controls) preceeding or coincident with the development of psychotic disorder (PD) at age 18 in the Avon Longitudinal Study of Parents and Children (ALSPAC). Overall, 179 lipids were identified at age 11, with 32 found to be significantly altered between the control and PD groups. Following correction for multiple comparisons, 8 of these lipids remained significant (lysophosphatidlycholines (LPCs) LPC(18:1), LPC(18:2), LPC(20:3); phosphatidlycholines (PCs) PC(32:2; PC(34:2), PC(36:4), PC(0-34-3) and sphingomyelin (SM) SM(d18:1/24:0)), all of which were elevated in the PD group. At age 18, 23 lipids were significantly different between the control and PD groups, although none remained significant following correction for multiple comparisons. In conclusion, the findings indicate that the lipidome is altered in the blood during childhood, long before the development of psychotic disorder. LPCs in particular are elevated in those who develop PD, indicating inflammatory abnormalities and altered phospholipid metabolism. These findings were not found at age 18, suggesting there may be ongoing alterations in the pathophysiological processes from prodrome to onset of PD.

Serum metabolite profile associates with the development of metabolic co-morbidities in first-episode psychosis.

Psychotic patients are at high risk for developing obesity, metabolic syndrome and type 2 diabetes. These metabolic co-morbidities are hypothesized to be related to both treatment side effects as well as to metabolic changes occurring during the psychosis. Earlier metabolomics studies have shown that blood metabolite levels are predictive of insulin resistance and type 2 diabetes in the general population as well as sensitive to the effects of antipsychotics. In this study, we aimed to identify the metabolite profiles predicting future weight gain and other metabolic abnormalities in psychotic patients. We applied comprehensive metabolomics to investigate serum metabolite profiles in a prospective study setting in 36 first-episode psychosis patients during the first year of the antipsychotic treatment and 19 controls. While corroborating several earlier findings when comparing cases and controls and the effects of the antipsychotic medication, we also found that prospective weight gain in psychotic patients was associated with increased levels of triacylglycerols with low carbon number and double-bond count at baseline, that is, lipids known to be associated with increased liver fat. Our study suggests that metabolite profiles may be used to identify the psychotic patients most vulnerable to develop metabolic co-morbidities, and may point to a pharmacological approach to counteract the antipsychotic-induced weight gain.

Determination of morphine analogues, caffeine and amphetamine in biological fluids by capillary electrophoresis with the marker technique.

A reliable method was sought for the fast screening and simultaneous determination of amphetamine, morphine, heroin (acetomorphine), codeine (methylmorphine) and caffeine in biological fluids and drug seizures. Capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC), with detection at 200 and 220 nm, were investigated for analytes in human serum and urine. When adequate separation was not achieved in preliminary studies with CZE, further development was focused on the MEKC method. Glycine buffer containing sodium lauryl sulfate (pH 10.5) was used for the MEKC separations. The analytes and carboxylic acids used as marker compounds could be screened by a short-capillary method in less than 2 min. In the simultaneous determination of the drugs in urine and serum a longer separation of 18 min was preferred so that all the compounds, the markers and the endogenous compounds absorbing at the detection wavelength could be adequately separated in a single run. The migration times of the compounds increased in the order caffeine, morphine, heroin, codeine and amphetamine. The repeatability of the separation was tested by using two carboxylic acids as marker compounds in the determination of the migration indices of the analytes. The relative standard deviations for the migration indices were less than 1%, which is accurate enough for the determination of the drugs in biological fluids.

Analysis of pesticides in red wines by on-line coupled reversed-phase liquid chromatography-gas chromatography with vaporiser/precolumn solvent split/gas discharge interface.

Pesticides in red wines were analysed by on-line coupled reversed-phase liquid chromatography-gas chromatography where a vaporiser/precolumn solvent split/gas discharge interface enabled direct transfer of aqueous eluent to the GC system. The LC part of the system provided sample clean-up and re-concentration, and the GC the final analytical step. The method developed allowed automated and quantitative analysis of the wine samples, where the only manual step was filtration. The limits of quantification were clearly below the maximum residue limits established for grapes, being lower than 10 micrograms l-1 for all pesticides studied.

Analysis of phenols in pyrolysis oils by gel permeation chromatography and multidimensional liquid chromatography.

A simple method with minimal manual sample preparation was developed for the analysis of phenols in pyrolysis oils. Sample pre-treatment was done by gel permeation chromatography (GPC), where the high-molecular-mass lignins were separated from the phenols. Multidimensional liquid chromatography (LC-LC) was used in the analysis of the phenolic fraction. The pre-column was used for sample clean-up and pre-fractionation before introduction of the phenolic fraction to the analytical column. The repeatability and linearity of the total GPC and LC-LC methods were excellent. The results were in accordance with the reference method in which the sample pre-treatment was done by precipitating the lignins with water, and the phenols were extracted with toluene and analysed by GC-MS.

Determination of pesticides in red wines with on-line coupled microporous membrane liquid-liquid extraction-gas chromatography.

Microporous membrane liquid-liquid extraction (MMLLE) was coupled on-line with gas chromatography for the determination of pesticides in wine. The MMLLE-GC provided to be efficient and selective and the method was linear, repeatable and sensitive. The limits of detection ranged from 0.05 to 2.3 microg/l and the limits of quantification were 0.2-7.5 microg/l for all the analytes using FID as detector. With MS detection LODs in the range 0.03-0.4 and LOQs of 0.3-3.5 microg/l were achieved. The method was applied to the determination of pesticides in several red wines of different origin.

Extremely high electric field strengths in non-aqueous capillary electrophoresis.

The influence of high electric field strength on the separation of basic analytes in non-aqueous alcohol background electrolyte (BGE) solutions was investigated. Increasing the separation voltage in capillary electrophoresis (CE) may be advantageous if the conductivity of the BGE solution is low enough to allow fast separations without excessive Joule heating or band broadening. The voltage range tested was 20-60 kV with methanol and ethanol, and 25-60 kV with propanol and butanol as solvent for BGE. The resulting electric field strengths ranged from 660 V cm(-1) to 2000 V cm(-1). Experiments were made with a special laboratory constructed CE instrument. The separation efficiency vs. voltage curve was found to vary with the alcohol BGE solution. The increase in voltage decreased the separation efficiency in the case of methanol BGE solution, but with the other BGEs a clear efficiency maximum was obtained above 30 kV. The highest separation efficiencies were achieved with propanol BGE solution, where the efficiency maximum was reached at 45 kV. However, reasonable efficiency was achieved even at 60 kV. The extent of Joule heating was determined by calculating the temperature inside the capillary and the observed plate heights were interpreted in terms of the Van Deemter equation. The decrease in the separation efficiency with higher voltage was attributed mainly to Joule heating in the case of methanol and ethanol BGE solution and to the analyte adsorption on the capillary wall with propanol and butanol BGE solutions.

Assessment of toxicity hazards of dredged lake sediment contaminated by creosote.

In order to predict the potential toxicity hazards of sediment remediation by dredging, an experimental laboratory simulation was made by investigating seven ratios of creosote-contaminated sediment (Lake Jämsänvesi, central Finland) and artificial lake water mixtures. Sediment was suspended in water at the ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 v/v. The elutriates were analysed for the acute toxicity by photoluminescence bacterial and waterflea (Daphnia magna Straus) tests. The concentrations of polycyclic aromatic hydrocarbons (PAHs) are determined by gas chromatography (GC/FID). The elutriate of ratio 1:2 was most toxic to bacteria (EC50 = 4.5%), whereas the ratio 1:4 was most toxic to waterfleas (EC50 = 21%). The elutriate of 1:1 contained the highest total PAH-concentration (1.67 mg/l) and total organic carbon (TOC) content (39.4 mg/l). When compared to the 1:1 ratio, taken as unity, the relative toxic emission yield (RTE) for bacteria was 307 for the ratio 1:128, so the high mixing ratio may cause a considerable ecotoxicological hazard. The highest amounts of PAHs were desorbed from sediment to water layer when the sediment was mixed with water at the ratios 1:1, 1:2 and 1:4 (v/v). It is assumed that dredging of creosote-contaminated sediment can potentially cause an ecotoxicological risk for a lake system at wide range of suspension ratios. We recommended that basic knowledge for these risks can be produced by simple laboratory simulation.

The toxicity and concentrations of PAHs in creosote-contaminated lake sediment.

Sediment samples, divided into three layers (0-10, 10-20 and 20-30 cm), were collected from 16 sites in Lake Jämsänvesi, Central Finland. The acute toxicity of pore waters and elutriates (sediment + water 1:4 v/v) were studied by bioluminescence inhibition test and by immobilisation of water fleas (Daphnia magna Straus). Concentrations of polycyclic aromatic hydrocarbons (PAHs) in sediments and elutriates were measured by gas chromatography using flame ionization detection (GC/FID). The highest total PAH concentration was 3.3 mg/g dry weight in the sediment and up to 1.7 mg/l in the elutriate of the uppermost (0-10 cm) layer, also being the most toxic to photoluminencent bacteria and water flea. When sediment and water mix, like during dredging operations, toxic compounds may spread from the sediment to the water column and can pose on environmental risk.

Liquid chromatographic sample cleanup coupled on-line with gas chromatography in the analysis of beta-blockers in human serum and urine.

An on-line coupled reversed-phase liquid chromatographic-gas chromatographic (LC-GC) method with minimal manual sample preparation is developed for the analysis of metoprolol, oxprenolol, propranolol, timolol, and codeine (as an internal standard) in human serum and urine. The method is based on a loop-type interface and concurrent eluent evaporation technique. On-line liquid-liquid extraction (LLE) is used to extract the analytes from aqueous eluent to organic solvent before injection onto the GC, and the two phases are separated with a sandwich-type phase separator. The LC is used for cleanup, and the GC is used for the final separation and detection of the analytes. Total analysis time is less than 45 min, which is much less than those of traditional analysis methods. Recoveries in LC cleanup and on-line LLE are excellent. A marked increase in the recoveries with on-line LLE is obtained by heating the aqueous eluent and the extraction coil. Linearity and repeatability of the method are good for both serum and urine, and the limits of quantitation for the analytes are 18-44 ng/mL.

Lipid-Filled semipermeable membrane devices and mussels as samplers of organochlorine compounds in lake water.

Semipermeable membrane sampling devices (SPMDs) and caged lake mussels (Anodonta piscinalis) were simultaneously deployed at four lake watercourse sites in Central Finland four weeks in August 1992. This study was part of the regular annual monitoring of the organochlorine compounds (OCC) in pulp-mill recipient watercourses of Finland with bivalves. Chlorohydrocarbons (CHCs), chlorophenol compounds (PCPs), chloroanisoles (PCAs) and chloroveratroles (PCVs) were analyzed from lipid extract of mussels and from the synthetic triolein lipid of the SPMDs. Hexane-diethyl ether (9:1, v/v) dialysis using polyethylene membrane was applied in dean up of the SPMD lipids and, for comparison, to six sets of the mussel fat. Dialysis recovered CHCs but not PCPs from the mussel fat. CHCs, PCPs, PCAs and PCVs were all recovered in dialysis of the SPMD lipid. Handling of SPMDs in the transport and deployment operations caused significant OCC contamination for the blank SPMDs. Similar trends were revealed in the OCC profiles for mussels ans SPMDs. An exception was the lack of PCPs appearing in SPMDs that did appear in mussels and in a complementary manner the appearance of the PCAs and PCVs in SPMDs.

Metabolome in progression to Alzheimer's disease.

Mild cognitive impairment (MCI) is considered as a transition phase between normal aging and Alzheimer's disease (AD). MCI confers an increased risk of developing AD, although the state is heterogeneous with several possible outcomes, including even improvement back to normal cognition. We sought to determine the serum metabolomic profiles associated with progression to and diagnosis of AD in a prospective study. At the baseline assessment, the subjects enrolled in the study were classified into three diagnostic groups: healthy controls (n=46), MCI (n=143) and AD (n=47). Among the MCI subjects, 52 progressed to AD in the follow-up. Comprehensive metabolomics approach was applied to analyze baseline serum samples and to associate the metabolite profiles with the diagnosis at baseline and in the follow-up. At baseline, AD patients were characterized by diminished ether phospholipids, phosphatidylcholines, sphingomyelins and sterols. A molecular signature comprising three metabolites was identified, which was predictive of progression to AD in the follow-up. The major contributor to the predictive model was 2,4-dihydroxybutanoic acid, which was upregulated in AD progressors (P=0.0048), indicating potential involvement of hypoxia in the early AD pathogenesis. This was supported by the pathway analysis of metabolomics data, which identified upregulation of pentose phosphate pathway in patients who later progressed to AD. Together, our findings primarily implicate hypoxia, oxidative stress, as well as membrane lipid remodeling in progression to AD. Establishment of pathogenic relevance of predictive biomarkers such as ours may not only facilitate early diagnosis, but may also help identify new therapeutic avenues.

Pressurised hot water extraction-microporous membrane liquid-liquid extraction coupled on-line with gas chromatography-mass spectrometry in the analysis of pesticides in grapes.

Pressurised hot water extraction-microporous membrane liquid-liquid extraction was coupled on-line with gas chromatography-mass spectrometry (PHWE-MMLLE-GC-MS) for the analysis of pesticides in grapes. MMLLE serves as a trapping step after PHWE. Water from PHWE is directed to the donor side of the membrane unit and the analytes are extracted to the acceptor solution on the other side. The role of MMLLE is to clean and concentrate the extract before on-line transfer to the GC via a sample loop and an on-column interface using partially concurrent solvent evaporation. The extraction conditions were investigated, and then the quantitative features such as linearity, limit of quantification (LOQ), extraction yield and enrichment factors. LOQs in the range 0.3-1.8 microg kg(-1) were achieved. Procymidone and tetradifon were found in the skins of the grapes. The results were in good agreement with those obtained by liquid-solid and ultrasonic extractions.