Katrin Strassburg

Gender-dependent associations of metabolite profiles and body fat distribution in a healthy population with central obesity: towards metabolomics diagnostics

Obesity is a risk factor for cardiovascular diseases and type 2 diabetes especially when the fat is accumulated to central depots. Novel biomarkers are crucial to develop diagnostics for obesity and related metabolic disorders. We evaluated the associations between metabolite profiles (136 lipid components, 12 lipoprotein subclasses, 17 low-molecular-weight metabolites, 12 clinical markers) and 28 phenotype parameters (including different body fat distribution parameters such as android (A), gynoid (G), abdominal visceral (VAT), subcutaneous (SAT) fat) in 215 plasma/serum samples from healthy overweight men (n=32) and women (n=83) with central obesity. (Partial) correlation analysis and partial least squares (PLS) regression analysis showed that only specific metabolites were associated to A:G ratio, VAT, and SAT, respectively. These association patterns were gender dependent. For example, insulin, cholesterol, VLDL, and certain triacylglycerols (TG 54:1-3) correlated to VAT in women, while in men VAT was associated with TG 50:1-5, TG 55:1, phosphatidylcholine (PC 32:0), and VLDL ((X)L). Moreover, multiple regression analysis revealed that waist circumference and total fat were sufficient to predict VAT and SAT in women. In contrast, only VAT but not SAT could be predicted in men and only when plasma metabolites were included, with PC 32:0 being most strongly associated with VAT. These findings collectively highlight the potential of metabolomics in obesity and that gender differences need to be taken into account for novel biomarker and diagnostic discovery for obesity and metabolic disorders.

Between Metabolite Relationships: an essential aspect of metabolic change

Not only the levels of individual metabolites, but also the relations between the levels of different metabolites may indicate (experimentally induced) changes in a biological system. Component analysis methods in current ‘standard’ use for metabolomics, such as Principal Component Analysis (PCA), do not focus on changes in these relations. We therefore propose the concept of ‘Between Metabolite Relationships’ (BMRs): common changes in the covariance (or correlation) between all metabolites in an organism. Such structural changes may indicate metabolic change brought about by experimental manipulation but which are lost with standard data analysis methods. These BMRs can be analysed by the INdividual Differences SCALing (INDSCAL) method. First the BMR quantification is described and subsequently the INDSCAL method. Finally, two studies illustrate the power and the applicability of BMRs in metabolomics. The first study is about the induced plant response of cabbage to herbivory, of which BMRs are a considerable part. In the second study—a human nutritional intervention study of green tea extract—standard data analysis tools did not reveal any metabolic change, although the BMRs were considerably affected. The presented results show that BMRs can be easily implemented in a wide variety of metabolomic studies. They provide a new source of information to describe biological systems in a way that fits flawlessly into the next generation of systems biology questions, dealing with personalized responses.

Comprehensive metabolomics to evaluate the impact of industrial processing on the phytochemical composition of vegetable purees

The effects of conventional industrial processing steps on global phytochemical composition of broccoli, tomato and carrot purees were investigated by using a range of complementary targeted and untargeted metabolomics approaches including LC-PDA for vitamins, (1)H NMR for polar metabolites, accurate mass LC-QTOF MS for semi-polar metabolites, LC-MRM for oxylipins, and headspace GC-MS for volatile compounds. An initial exploratory experiment indicated that the order of blending and thermal treatments had the highest impact on the phytochemicals in the purees. This blending-heating order effect was investigated in more depth by performing alternate blending-heating sequences in triplicate on the same batches of broccoli, tomato and carrot. For each vegetable and particularly in broccoli, a large proportion of the metabolites detected in the purees was significantly influenced by the blending-heating order, amongst which were potential health-related phytochemicals and flavour compounds like vitamins C and E, carotenoids, flavonoids, glucosinolates and oxylipins. Our metabolomics data indicates that during processing the activity of a series of endogenous plant enzymes, such as lipoxygenases, peroxidases and glycosidases, including myrosinase in broccoli, is key to the final metabolite composition and related quality of the purees.

Postprandial fatty acid specific changes in circulating oxylipins in lean and obese men after high-fat challenge tests

SCOPE Circulating oxylipins may affect peripheral tissues and are assumed to play an important role in endothelial function. They are esterified in triglyceride-rich lipoproteins that are increased after a high-fat (HF) meal, depending on BMI and fatty acid (FA) type. Yet, it is unclear which oxylipins appear in circulation after HF meals differing in FA composition. METHODS AND RESULTS In a double-blind randomized crossover challenge study, we characterized the postprandial oxylipin response after different HF challenges in lean and obese men receiving HF milkshakes, either high in saturated FAs (SFA), monounsaturated FAs (MUFA), or omega 3 (n-3) polyunsaturated FAs (PUFA). Plasma oxylipin profiles were significantly altered at 2 and 4 h after shake consumption when compared to baseline. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) derived oxylipins increased after n-3 PUFA shake consumption. MUFA shake consumption increased levels of cytochrome P450 mediated oxylipins. SFA shake consumption led to strong increases in linoleic acid (LA) derived HODEs. No differences were observed between lean and obese individuals at baseline and after any shake consumption. CONCLUSION We are the first demonstrating acute effects on circulating oxylipins after HF meal challenges. These changes were strongly influenced by different dietary FAs and may affect endothelial function.

Metabolic Phenotyping Reveals a Lipid Mediator Response to Ionizing Radiation

Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here, we screened mouse serum for metabolic alterations following an acute exposure to γ radiation using a multiplatform mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and low-abundance, oxygenated, polyunsaturated fatty acids (PUFAs) in the two groups of animals. Exposure to γ radiation induced a significant increase in the serum levels of ether phosphatidylcholines (PCs) while decreasing the levels of diacyl PCs carrying PUFAs. In exposed mice, levels of pro-inflammatory, oxygenated metabolites of arachidonic acid increased, whereas levels of anti-inflammatory metabolites of omega-3 PUFAs decreased. Our results indicate a specific serum lipidomic biosignature that could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine.

Plasma oxylipin profiling identifies polyunsaturated vicinal diols as responsive to arachidonic acid and docosahexaenoic acid intake in growing piglets

The dose-responsiveness of plasma oxylipins to incremental dietary intake of arachidonic acid (20:4n-6; ARA) and docosahexaenoic acid (22:6n-3; DHA) was determined in piglets. Piglets randomly received one of six formulas (n = 8 per group) from days 3 to 27 postnatally. Diets contained incremental ARA or incremental DHA levels as follows (% fatty acid, ARA/DHA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3–D3) 0.69/1.0; (A4) 1.1/1.0; (D1) 0.66/0.33; and (D2) 0.67/0.62, resulting in incremental intake (g/kg BW/day) of ARA: 0.07 ± 0.01, 0.43 ± 0.03, 0.55 ± 0.03, and 0.82 ± 0.05 at constant DHA intake (0.82 ± 0.05), or incremental intake of DHA: 0.27 ± 0.02, 0.49 ± 0.03, and 0.81 ± 0.05 at constant ARA intake (0.54 ± 0.04). Plasma oxylipin concentrations and free plasma PUFA levels were determined at day 28 using LC-MS/MS. Incremental dietary ARA intake dose-dependently increased plasma ARA levels. In parallel, ARA intake dose-dependently increased ARA-derived diols 5,6- and 14,15-dihydroxyeicosatrienoic acid (DiHETrE) and linoleic acid-derived 12,13-dihydroxyoctadecenoic acid (DiHOME), downstream metabolites of cytochrome P450 expoxygenase (CYP). The ARA epoxide products from CYP are important in vascular homeostatic maintenance. Incremental DHA intake increased plasma DHA and most markedly raised the eicosapentaenoic acid (EPA) metabolite 17,18-dihydroxyeicosatetraenoic acid (DiHETE) and the DHA metabolite 19,20-dihydroxydocosapentaenoic acid (DiHDPE). In conclusion, increasing ARA and DHA intake dose-dependently influenced endogenous n-6 and n-3 oxylipin plasma concentrations in growing piglets, although the biological relevance of these findings remains to be determined.

A Protective Lipidomic Biosignature Associated with a Balanced Omega-6/Omega-3 Ratio in fat-1 Transgenic Mice

A balanced omega-6/omega-3 polyunsaturated fatty acid (PUFA) ratio has been linked to health benefits and the prevention of many chronic diseases. Current dietary intervention studies with different sources of omega-3 fatty acids (omega-3) lack appropriate control diets and carry many other confounding factors derived from genetic and environmental variability. In our study, we used the fat-1 transgenic mouse model as a proxy for long-term omega-3 supplementation to determine, in a well-controlled manner, the molecular phenotype associated with a balanced omega-6/omega-3 ratio. The fat-1 mouse can convert omega-6 to omega-3 PUFAs, which protect against a wide variety of diseases including chronic inflammatory diseases and cancer. Both wild-type (WT) and fat-1 mice were subjected to an identical diet containing 10% corn oil, which has a high omega-6 content similar to that of the Western diet, for a six-month duration. We used a multi-platform lipidomic approach to compare the plasma lipidome between fat-1 and WT mice. In fat-1 mice, an unbiased profiling showed a significant increase in the levels of unesterified eicosapentaenoic acid (EPA), EPA-containing cholesteryl ester, and omega-3 lysophosphospholipids. The increase in omega-3 lipids is accompanied by a significant reduction in omega-6 unesterified docosapentaenoic acid (omega-6 DPA) and DPA-containing cholesteryl ester as well as omega-6 phospholipids and triacylglycerides. Targeted lipidomics profiling highlighted a remarkable increase in EPA-derived diols and epoxides formed via the cytochrome P450 (CYP450) pathway in the plasma of fat-1 mice compared with WT mice. Integration of the results of untargeted and targeted analyses has identified a lipidomic biosignature that may underlie the healthful phenotype associated with a balanced omega-6/omega-3 ratio, and can potentially be used as a circulating biomarker for monitoring the health status and the efficacy of omega-3 intervention in humans.

Global profiling of the muscle metabolome: method optimization, validation and application to determine exercise-induced metabolic effects

Skeletal muscle represents a crucial metabolic organ in the body characterized by a tremendous metabolic plasticity and the ability to influence important metabolic events elsewhere in the body. In order to understand the metabolic implications of skeletal muscle, it is imperative to characterize the metabolites within the tissue itself. In this work we aimed at developing a suitable analytical pipeline to analyze the metabolome of muscle tissue. Methanol/chloroform/water at neutral pH was selected as the method of choice for metabolite extraction prior to analysis by chromatographic-mass spectrometry systems in five different platforms covering a relevant part of the muscle metabolome: organic acids, amines, nucleotides, coenzymes, acylcarnitines and oxylipins. This analytical pipeline was extensively validated and proved to be robust, precise, accurate and biologically sound. The capability of our analytical method to capture metabolic alterations upon challenges was finally tested using a small proof-of-concept study involving an exercise intervention. Mild but consistent metabolic patterns were observed, allowing the discrimination between non-exercised and exercised muscles. Despite the low numbers of subjects enrolled in this study (5), these results are indicative that our method is suitable to determine intervention effects in skeletal muscle tissue whenever applied to adequately powered and well characterized studies.

Collagen Induced Arthritis in DBA/1J Mice Associates with Oxylipin Changes in Plasma

Oxylipins play important roles in various biological processes and are considered as mediators of inflammation for a wide range of diseases such as rheumatoid arthritis (RA). The purpose of this research was to study differences in oxylipin levels between a widely used collagen induced arthritis (CIA) mice model and healthy control (Ctrl) mice. DBA/1J male mice (age: 6-7 weeks) were selected and randomly divided into two groups, namely, a CIA and a Ctrl group. The CIA mice were injected intraperitoneally (i.p.) with the joint cartilage component collagen type II (CII) and an adjuvant injection of lipopolysaccharide (LPS). Oxylipin metabolites were extracted from plasma for each individual sample using solid phase extraction (SPE) and were detected with high performance liquid chromatography/tandem mass spectrometry (HPLC-ESI-MS/MS), using dynamic multiple reaction monitoring (dMRM). Both univariate and multivariate statistical analyses were applied. The results in univariate Students t-test revealed 10 significantly up- or downregulated oxylipins in CIA mice, which were supplemented by another 6 additional oxylipins, contributing to group clustering upon multivariate analysis. The dysregulation of these oxylipins revealed the presence of ROS-generated oxylipins and an increase of inflammation in CIA mice. The results also suggested that the collagen induced arthritis might associate with dysregulation of apoptosis, possibly inhibited by activated NF-κB because of insufficient PPAR-γ ligands.

Weight loss predictability by plasma metabolic signatures in adults with obesity and morbid obesity of the DiOGenes study

Aim is to predict successful weight loss by metabolic signatures at baseline and to identify which differences in metabolic status may underlie variations in weight loss success.