Cold-induced changes in plasma signaling lipids are associated with a healthier cardiometabolic profile independently of brown adipose tissue.

Cold exposure activates brown adipose tissue (BAT) and potentially improves cardiometabolic health through the secretion of signaling lipids by BAT. Here, we show that 2 h of cold exposure in young adults increases the levels of omega-6 and omega-3 oxylipins, the endocannabinoids (eCBs) anandamide and docosahexaenoylethanolamine, and lysophospholipids containing polyunsaturated fatty acids. Contrarily, it decreases the levels of the eCBs 1-LG and 2-LG and 1-OG and 2-OG, lysophosphatidic acids, and lysophosphatidylethanolamines. Participants overweight or obese show smaller increases in omega-6 and omega-3 oxylipins levels compared to normal weight. We observe that only a small proportion (∼4% on average) of the cold-induced changes in the plasma signaling lipids are slightly correlated with BAT volume. However, cold-induced changes in omega-6 and omega-3 oxylipins are negatively correlated with adiposity, glucose homeostasis, lipid profile, and liver parameters. Lastly, a 24-week exercise-based randomized controlled trial does not modify plasma signaling lipid response to cold exposure.

Omega-6 and omega-3 oxylipins as potential markers of cardiometabolic risk in young adults.

OBJECTIVE: Omega-6 and omega-3 oxylipins are known to play a role in inflammation and cardiometabolic diseases in preclinical models. The associations between plasma levels of omega-6 and omega-3 polyunsaturated fatty acid-derived oxylipins and body composition and cardiometabolic risk factors in young adults were assessed. METHODS: Body composition, brown adipose tissue, traditional serum cardiometabolic risk factors, inflammatory markers, and a panel of 83 oxylipins were analyzed in 133 young adults (age 22.1[SD 2.2] years, 67% women). RESULTS: Plasma levels of four omega-6 oxylipins (15-HeTrE, 5-HETE, 14,15-EpETrE, and the oxidative stress-derived 8,12-iso-iPF2α -VI) correlated positively with adiposity, prevalence of metabolic syndrome, fatty liver index, and homeostatic model assessment of insulin resistance index and lipid parameters. By contrast, the plasma levels of three omega-3 oxylipins (14,15-DiHETE, 17,18-DiHETE, and 19,20-DiHDPA) were negatively correlated with adiposity, prevalence of metabolic syndrome, fatty liver index, homeostatic model assessment of insulin resistance index, and lipid parameters. The panel of seven oxylipins predicted adiposity better than traditional inflammatory markers such as interferon gamma or tumor necrosis factor-alpha. Pathway analyses revealed that individuals with obesity had higher plasma levels of omega-6 and lower plasma levels of omega-3 oxylipins than normal-weight individuals. CONCLUSION: Plasma levels of seven omega-6 and omega-3 oxylipins may have utility as early markers of cardiometabolic risk in young adults.

Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening.

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Mechanistic and Quantitative Understanding of Pharmacokinetics in Zebrafish Larvae through Nanoscale Blood Sampling and Metabolite Modeling of Paracetamol.

Zebrafish larvae are increasingly used for pharmacological research, but internal drug exposure is often not measured. Understanding pharmacokinetics is necessary for reliable translation of pharmacological results to higher vertebrates, including humans. Quantification of drug clearance and distribution requires measurements of blood concentrations. Additionally, measuring drug metabolites is of importance to understand clearance in this model organism mechanistically. We therefore mechanistically studied and quantified pharmacokinetics in zebrafish larvae, and compared this to higher vertebrates, using paracetamol (acetaminophen) as a paradigm compound. A method was developed to sample blood from zebrafish larvae 5 days post fertilization. Blood concentrations of paracetamol and its major metabolites, paracetamol-glucuronide and paracetamol-sulfate, were measured. Blood concentration data were combined with measured amounts in larval homogenates and excreted amounts and simultaneously analyzed through nonlinear mixed-effects modeling, quantifying absolute clearance and distribution volume. Blood sampling from zebrafish larvae was most successful from the posterior cardinal vein, with a median volume (interquartile range) of 1.12 nl (0.676-1.66 nl) per blood sample. Samples were pooled (n = 15-35) to reach measurable levels. Paracetamol blood concentrations at steady state were only 10% of the external paracetamol concentration. Paracetamol-sulfate was the major metabolite, and its formation was quantified using a time-dependent metabolic formation rate. Absolute clearance and distribution volume correlated well with reported values in higher vertebrates, including humans. Based on blood concentrations and advanced data analysis, the mechanistic and quantitative understanding of paracetamol pharmacokinetics in zebrafish larvae has been established. This will improve the translational value of this vertebrate model organism in drug discovery and development. SIGNIFICANCE STATEMENT: In early phases of drug development, new compounds are increasingly screened in zebrafish larvae, but the internal drug exposure is often not taken into consideration. We developed innovative experimental and computational methods, including a blood-sampling technique, to measure the paradigm drug paracetamol (acetaminophen) and its major metabolites and quantify pharmacokinetics (absorption, distribution, elimination) in zebrafish larvae of 5 days post fertilization with a total volume of only 300 nl. These parameter values were scaled to higher vertebrates, including humans.

Role of amino acids in rheumatoid arthritis studied by metabolomics.

BACKGROUND: Rheumatoid arthritis (RA) is a complex, chronic autoimmune disease characterized by various inflammatory symptoms, including joint swelling, joint pain, and both structural and functional joint damage. The most commonly used animal model for studying RA is mice with collagen-induced arthritis (CIA); the wide use of this model is due primarily to many similarities with RA in human patients. Metabolomics is used increasingly in biological studies for diagnosing disease and for predicting and evaluating drug interventions, as a large number of disease-associated metabolites can be analyzed and interpreted from a biological perspective. AIM: To profile free amino acids and their biogenic metabolites in CIA mice plasma. METHOD: Ultra-high-performance liquid chromatography/tandem mass spectrometry coupled with multiple reaction monitoring (MRM) was used for metabolomics study. RESULTS: Profile of 45 amine metabolites, including free amino acids and their biogenic metabolites in plasma was obtained from CIA mice. We found that the plasma levels of 20 amine metabolites were significantly decreased in the CIA group. CONCLUSION: The results suggest that a disordered amine response is linked to RA-associated muscle wasting and energy expenditure.

Fusing metabolomics data sets with heterogeneous measurement errors.

Combining different metabolomics platforms can contribute significantly to the discovery of complementary processes expressed under different conditions. However, analysing the fused data might be hampered by the difference in their quality. In metabolomics data, one often observes that measurement errors increase with increasing measurement level and that different platforms have different measurement error variance. In this paper we compare three different approaches to correct for the measurement error heterogeneity, by transformation of the raw data, by weighted filtering before modelling and by a modelling approach using a weighted sum of residuals. For an illustration of these different approaches we analyse data from healthy obese and diabetic obese individuals, obtained from two metabolomics platforms. Concluding, the filtering and modelling approaches that both estimate a model of the measurement error did not outperform the data transformation approaches for this application. This is probably due to the limited difference in measurement error and the fact that estimation of measurement error models is unstable due to the small number of repeats available. A transformation of the data improves the classification of the two groups.

A metabolomics study of Qiliqiangxin in a rat model of heart failure: a reverse pharmacology approach.

The Chinese medicine Qiliqiangxin (QL) has been shown to have a protective role in heart failure. Here, we explore the underlying working mechanism of the key therapeutic component in QL using a rat model of heart failure. Heart failure after myocardial infarction was induced surgically and confirmed using echocardiography; a separate group of rats underwent sham surgery. The rats with heart failure were randomly assigned to receive QL, the angiotensin-converting enzyme inhibitor benazepril, or placebo groups. Blood samples were collected from the rats at four time points for up to 8 weeks and used for biochemical analysis and mass spectrometry‒based metabolomics profiling. In total, we measured nine well-known biochemical parameters of heart failure and 147 metabolites. In the rats with heart failure, QL significantly improved these biochemical parameters and metabolomics profiles, significantly increasing the cardioprotective parameter angiopoietin-like 4 and significantly lowering inflammation-related oxylipins and lysophosphatidic acids compared to benazepril. Mechanistically, QL may improve outcome in heart failure by controlling inflammatory process and cardiac hypertrophy. Clinical studies should be designed in order to investigate these putative mechanisms in patients.

Global testing of shifts in metabolic phenotype.

INTRODUCTION: Current metabolomics approaches to unravel impact of diet- or lifestyle induced phenotype variation and shifts predominantly deploy univariate or multivariate approaches, with a posteriori interpretation at pathway level. This however often provides only a fragmented view on the involved metabolic pathways. OBJECTIVES: To demonstrate the feasibility of using Goeman's global test (GGT) for assessment of variation and shifts in metabolic phenotype at the level of a priori defined pathways. METHODS: Two intervention studies with identified phenotype variations and shifts were examined. In a weight loss (WL) intervention study obese subjects received a mixed meal challenge before and after WL. In a polyphenol (PP) intervention study obese subjects received a high fat mixed meal challenge (61E% fat) before and after a PP intervention. Plasma samples were obtained at fasting and during the postprandial response. Besides WL- and PP-induced phenotype shifts, also correlation of plasma metabolome with phenotype descriptors was assessed at pathway level. The plasma metabolome covered organic acids, amino acids, biogenic amines, acylcarnitines and oxylipins. RESULTS: For the population of the WL study, GGT revealed that HOMA correlated with the fasting levels of the TCA cycle, BCAA catabolism, the lactate, arginine-proline and phenylalanine-tyrosine pathways. For the population of the PP study, HOMA correlated with fasting metabolite levels of TCA cycle, fatty acid oxidation and phenylalanine-tyrosine pathways. These correlations were more pronounced for metabolic pathways in the fasting state, than during the postprandial response. The effect of the WL and PP intervention on a priori defined metabolic pathways, and correlation of pathways with insulin sensitivity as described by HOMA was in line with previous studies. CONCLUSION: GGT confirmed earlier biological findings in a hypothesis led approach. A main advantage of GGT is that it provides a direct view on involvement of a priori defined pathways in phenotype shifts.

Characterization of the therapeutic properties of Chinese herbal materials by measuring delayed luminescence and dendritic cell-based immunomodulatory response.

Based on the traditional Chinese medicine theory, the Chinese pharmacopeia assigns a therapeutic description of "taste" to all herbs; thus, an herb's "taste" is valued in traditional Chinese medicine as a major ethnopharmacological category and reflects the herb's therapeutic properties. These properties guide the practitioner with respect to preparing a specific herbal formula in order to provide each patient with a personalized intervention. The key challenge in evidence-based medicine is to characterize herbal therapeutic properties from a multi-target, multi-dimensional systems pharmacology perspective. Here, we used delayed luminescence (DL, the slowly decaying emission of photons following excitation with light) as a rapid, direct, highly sensitive indicator to characterize the properties of herbal medicines. The DL parameters were able to reliably identify a specific category of herbal materials with the so-called "sweet" taste. To support the DL results and provide biological relevance to the DL results, we used a murine bone marrow-derived dendritic cell-based assay to examine the immunomodulatory effects of herbal extracts from various "taste" categories. Our results indicate that DL may serve as a robust and sensitive tool for evaluating the therapeutic properties of herbs based on the traditional Chinese medicine classification of "taste". Thus, DL provides a promising technological platform for investigating the properties of Chinese herbal medicines both qualitatively and quantitatively.

Measuring ultra-weak photon emission as a non-invasive diagnostic tool for detecting early-stage type 2 diabetes: A step toward personalized medicine.

The global prevalence of type 2 diabetes is estimated to reach 4.4% by 2030, placing a significant burden on our healthcare system. Therefore, the ability to identify patients in early stages of the disease is essential for both prevention and effective management, and diagnostic methods based on traditional Chinese medicine (TCM) may be suitable for identifying patients with early-stage type 2 diabetes. Here, a panel of three physicians trained in TCM classified 44 pre-diabetic subjects into three syndrome subtypes using TCM-based diagnostics. In addition, ultra-weak photon emission (UPE) was measured at four anatomical sites in each subject. Ten properties encompassing 40 parameters were then extracted from the UPE time series. Statistical analyses, including multinomial logistic regression, were performed using the results of each parameter measured at the four sites. Sixteen UPE parameters were then selected and used to discriminate between the three subtypes of pre-diabetic subjects. Next, Spearman's correlation coefficient was used to quantify the correlation between the 16 UPE parameters and the TCM-based diagnoses. The resulting correlation networks accurately reflected the differences between the three syndrome subtypes. These results suggest that UPE is a suitable tool for detecting subtypes in early-stage type 2 diabetes. In addition, our results provide evidence that TCM may represent an important step toward personalized medicine.

Pharmacokinetic Modeling of Paracetamol Uptake and Clearance in Zebrafish Larvae: Expanding the Allometric Scale in Vertebrates with Five Orders of Magnitude.

Zebrafish larvae (Danio rerio) are increasingly used to translate findings regarding drug efficacy and safety from in vitro-based assays to vertebrate species, including humans. However, the limited understanding of drug exposure in this species hampers its implementation in translational research. Using paracetamol as a paradigm compound, we present a novel method to characterize pharmacokinetic processes in zebrafish larvae, by combining sensitive bioanalytical methods and nonlinear mixed effects modeling. The developed method allowed quantification of paracetamol and its two major metabolites, paracetamol-sulfate and paracetamol-glucuronide in pooled samples of five lysed zebrafish larvae of 3 days post-fertilization. Paracetamol drug uptake was quantified to be 0.289 pmole/min and paracetamol clearance was quantified to be 1.7% of the total value of the larvae. With an average volume determined to be 0.290 µL, this yields an absolute clearance of 2.96 × 107 L/h, which scales reasonably well with clearance rates in higher vertebrates. The developed methodology will improve the success rate of drug screens in zebrafish larvae and the translation potential of findings, by allowing the establishment of accurate exposure profiles and thereby also the establishment of concentration-effect relationships.

Effects of growth altitude on chemical constituents and delayed luminescence properties in medicinal rhubarb.

To improve the quality control of herbal drugs, there has been a major shift from evaluating individual chemicals to evaluating multiple-constituent chemicals, given the multi-pharmacology nature of herbal drugs. Therefore, rapid, systematic assays are needed in order to assess the quality of medicinal herbs using a comprehensive, integrated approach. Light-induced delayed luminescence (DL) is used to measure decaying long-term ultra-weak photon emissions following excitation with light. DL is considered to be a sensitive indicator for characterizing the properties of biological systems and herbal medicines with various therapeutic properties. The aim of this study was to examine the feasibility of using DL as a novel quality-assessment tool using rhubarb material as a model system, and to establish the correlation between DL parameters and the chemical constituents of rhubarb. Raw roots and rhizomes were collected from rhubarb (Rheum palmatum L.) at various elevations in western China. HPLC analysis was used to identify fourteen bioactive constituents. Five DL parameters were calculated from the DL decay curves of the rhubarb samples. Statistical tools, including principal component analysis, were used to classify the rhubarb samples using data obtained using two different assays. Finally, Spearman's correlation coefficient was calculated to quantify the correlation between the bioactive compounds and corresponding DL parameters. We found that both the chemical analysis and DL measurements reflect variations in the quality of rhubarb due to environment factor. The DL parameters were correlated significantly with the bioactive chemical constituents. Our results indicate that DL is a promising tool for evaluating multiple constituents and for assessing the therapeutic properties of herbal medicines. Thus, DL may be used as part of a comprehensive system for assessing the quality and/or therapeutic properties of herbal medicines.

A Chinese literature overview on ultra-weak photon emission as promising technology for studying system-based diagnostics.

To present the possibilities pertaining to linking ultra-weak photon emission (UPE) with Chinese medicine-based diagnostics principles, we conducted a review of Chinese literature regarding UPE with respect to a systems view of diagnostics. Data were summarized from human clinical studies and animal models published from 1979 through 1998. The research fields can be categorized as follows: (1) human physiological states measured using UPE; (2) characteristics of human UPE in relation to various pathological states; and (3) the relationship between diagnosis (e.g., Chinese syndromes) and the dynamics of UPE in animal models. We conclude that UPE has clear potential in terms of understanding the systems view on health and disease as described using Chinese medicine-based diagnostics, particularly from a biochemistry-based regulatory perspective. Linking UPE with metabolomics can further bridge biochemistry-based Western diagnostics with the phenomenology-based Chinese diagnostics, thus opening new avenues for studying systems diagnostics in the early stage of disease, for prevention-based strategies, as well as for systems-based intervention in chronic disease.

Delayed luminescence: an experimental protocol for Chinese herbal medicines.

In Chinese medicine, raw herbal materials are used in processed and unprocessed forms aiming to meet the different requirements of clinical practice. To assure the chemical quality and therapeutic properties of the herbs, fast and integrated systematic assays are required. So far, such assays have not been established. Delayed luminescence (DL) refers to a decaying long-term ultraweak photon emission after exposure to light. Its decay kinetics under certain conditions may be a sensitive indicator reflecting the internal structural and chemical/physiological state of a biological system. DL measurements have been used in many applications for quality control. However, relatively little research has been reported on dried plant material such as Chinese herbs. The objective of the present study is to establish a protocol for direct and rapid DL measurements of dried Chinese herbal materials, including the determination of the dependence on: (a) the optimal excitation time utilizing a white light source; (b) the optimal size of the grinded herbal particle; and (c) the humidity conditions before and during measurement. Results indicate that stable and reproducible curves of DL photon emission depend mainly on the water content of herbal materials. To investigate the application of the established DL measurement protocol, non-processed and processed Aconitum (Aconitum carmichaelii Debx.), wild and cultivated rhubarb (Rheum palmatum L.) and ginseng (Panax ginseng C.A.Mey) of different ages were measured using DL. The results suggest that DL technology is a potential tool for assessment of dried Chinese herb qualities. The results warrant a further exploration of this technique in relation to therapeutic properties of the herbs. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Testing tuberculosis drug efficacy in a zebrafish high-throughput translational medicine screen.

The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

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.

Solvent exchange module for LC-NMR hyphenation using machine vision-controlled droplet evaporation.

We report the use of pendant droplet evaporation for exchange of eluents for (1)H nuclear magnetic resonance ((1)H NMR) purposes. Analytes are fed and retained in 500 nL droplets, which are concentrated by evaporation and subsequently redissolved in deuterated solvent. Droplet size is monitored by machine vision (MV), and heating rates are adjusted concordingly to maintain a stable droplet volume. Evaporation control is independent of solvent properties, and the setup handles feed rates up to 7 µL min(-1). The interface is capable of exchanging up to 90% of solvent for deuterated solvent, with a good recovery and repeatability for tomato extracts (Solanum lycopersicum). The system was capable of handling both polar and nonpolar analytes in one run. Volatiles such as formate, acetate, and lactate and the thermosensitive compound epigallocatechin gallate were recovered without significant losses. Ethanol and propionate were recovered with significant losses due to the formation of a minimum boiling azeotrope. The current setup is ideally suited for on- and off-line hyphenation of liquid chromatography and NMR, as it is comprehensive, fully automated, and easy to operate.

How do metabolites differ from their parent molecules and how are they excreted?

Understanding which physicochemical properties, or property distributions, are favorable for successful design and development of drugs, nutritional supplements, cosmetics, and agrochemicals is of great importance. In this study we have analyzed molecules from three distinct chemical spaces (i) approved drugs, (ii) human metabolites, and (iii) traditional Chinese medicine (TCM) to investigate four aspects determining the disposition of small organic molecules. First, we examined the physicochemical properties of these three classes of molecules and identified characteristic features resulting from their distinctive biological functions. For example, human metabolites and TCM molecules can be larger and more hydrophobic than drugs, which makes them less likely to cross membranes. We then quantified the shifts in physicochemical property space induced by metabolism from a holistic perspective by analyzing a data set of several thousand experimentally observed metabolic trees. Results show how the metabolic system aims to retain nutrients/micronutrients while facilitating a rapid elimination of xenobiotics. In the third part we compared these global shifts with the contributions made by individual metabolic reactions. For better resolution, all reactions were classified into phase I and phase II biotransformations. Interestingly, not all metabolic reactions lead to more hydrophilic molecules. We were able to identify biotransformations leading to an increase of logP by more than one log unit, which could be used for the design of drugs with enhanced efficacy. The study closes with the analysis of the physicochemical properties of metabolites found in the bile, faeces, and urine. Metabolites in the bile can be large and are often negatively charged. Molecules with molecular weight >500 Da are rarely found in the urine, and most of these large molecules are charged phase II conjugates.