Human metabolomics: strategies to understand biology

Metabolomics provides a direct functional read-out of the physiological status of an organism and is in principle ideally suited to describe someone's health status. Whereas only a limited number of small metabolites are used in the clinics, in inborn errors of metabolism an extensive repertoire of metabolites are used as biomarkers. We discuss that the proper clinical phenotyping is crucial to find biomarkers and obtain biological insights for multifactorial diseases. This requires to study the phenotype dynamics including the concepts of homeostasis and allostasis, that is, the ability to adapt and cope with a challenge. We also elaborate that biology-driven metabolomics platforms (i.e. development of metabolomics technology driven by the need of studying and answering important biomedical questions) addressing clinically relevant pathways and at the same time providing absolute concentrations are key to allow discovery and validation of biomarkers across studies and labs. Following individuals over years will require high throughput metabolomics approaches, which are emerging for nuclear magnetic resonance spectroscopy and direct-infusion mass spectrometry, but should also include the biochemical networks needed for personalized health monitoring

Metabolomic analysis of dietary-restriction-induced attenuation of sarcopenia in prematurely aging DNA repair-deficient mice

Background: Sarcopenia is characterized by loss of skeletal muscle mass and function, and is a major risk factor for disability and independence in the elderly. Effective medication is not available. Dietary restriction (DR) has been found to attenuate aging and aging-related diseases, including sarcopenia, but the mechanism of both DR and sarcopenia are incompletely understood. Methods: In this study, mice body weight, fore and all limb grip strength, and motor learning and coordination performance were first analysed to evaluate the DR effects on muscle functioning. Liquid chromatography–mass spectrometry (LC–MS) was utilized for the metabolomics study of the DR effects on sarcopenia in progeroid DNA repair-deficient Ercc1∆/− and Xpg−/− mice, to identify potential biomarkers for attenuation of sarcopenia. Results: Muscle mass was significantly (P &lt; 0.05) decreased (13–20%) by DR; however, the muscle quality was improved with retained fore limbs and all limbs grip strength in Ercc1∆/− and Xpg−/− mice. The LC–MS results revealed that metabolites and pathways related to oxidative-stress, that is, GSSG/GSH (P &lt; 0.01); inflammation, that is, 9-HODE, 11-HETE (P &lt; 0.05), PGE2, PGD2, and TXB2 (P &lt; 0.01); and muscle growth (PGF2α) (P &lt; 0.01) and regeneration stimulation (PGE2) (P &lt; 0.05) are significantly downregulated by DR. On the other hand, anti-inflammatory indicator and several related metabolites, that is, β-hydroxybutyrate (P &lt; 0.01), 14,15-DiHETE (P &lt; 0.0001), 8,9-EET, 12,13-DiHODE, and PGF1 (P &lt; 0.05); consumption of sources of energy (i.e., muscle and liver glycogen); and energy production pathways, that is, glycolysis (glucose, glucose-6-P, fructose-6-P) (P &lt; 0.01), tricarboxylic acid cycle (succinyl-CoA, malate) (P &lt; 0.001), and gluconeogenesis-related metabolite, alanine (P &lt; 0.01), are significantly upregulated by DR. The notably (P &lt; 0.01) down-modulated muscle growth (PGF2α) and regeneration (PGE2) stimulation metabolite and the increased consumption of glycogen in muscle and liver may be related to the significantly (P &lt; 0.01) lower body weight and muscle mass by DR. The downregulated oxidative stress, pro-inflammatory mediators, and upregulated anti-inflammatory metabolites resulted in a lower energy expenditure, which contributed to enhanced muscle quality together with upregulated energy production pathways by DR. The improved muscle quality may explain why grip strength is maintained and motor coordination and learning performance are improved by DR in Ercc1∆/− and Xpg−/− mice. Conclusions: This study provides fundamental supporting information on biomarkers and pathways related to the attenuation of sarcopenia, which might facilitate its diagnosis, prevention, and clinical therapy.</p

Metabolomic analysis of dietary-restriction-induced attenuation of sarcopenia in prematurely aging DNA repair-deficient mice

Background: Sarcopenia is characterized by loss of skeletal muscle mass and function, and is a major risk factor for disability and independence in the elderly. Effective medication is not available. Dietary restriction (DR) has been found to attenuate aging and aging-related diseases, including sarcopenia, but the mechanism of both DR and sarcopenia are incompletely understood. Methods: In this study, mice body weight, fore and all limb grip strength, and motor learning and coordination performance were first analysed to evaluate the DR effects on muscle functioning. Liquid chromatography–mass spectrometry (LC–MS) was utilized for the metabolomics study of the DR effects on sarcopenia in progeroid DNA repair-deficient Ercc1∆/− and Xpg−/− mice, to identify potential biomarkers for attenuation of sarcopenia. Results: Muscle mass was significantly (P &lt; 0.05) decreased (13–20%) by DR; however, the muscle quality was improved with retained fore limbs and all limbs grip strength in Ercc1∆/− and Xpg−/− mice. The LC–MS results revealed that metabolites and pathways related to oxidative-stress, that is, GSSG/GSH (P &lt; 0.01); inflammation, that is, 9-HODE, 11-HETE (P &lt; 0.05), PGE2, PGD2, and TXB2 (P &lt; 0.01); and muscle growth (PGF2α) (P &lt; 0.01) and regeneration stimulation (PGE2) (P &lt; 0.05) are significantly downregulated by DR. On the other hand, anti-inflammatory indicator and several related metabolites, that is, β-hydroxybutyrate (P &lt; 0.01), 14,15-DiHETE (P &lt; 0.0001), 8,9-EET, 12,13-DiHODE, and PGF1 (P &lt; 0.05); consumption of sources of energy (i.e., muscle and liver glycogen); and energy production pathways, that is, glycolysis (glucose, glucose-6-P, fructose-6-P) (P &lt; 0.01), tricarboxylic acid cycle (succinyl-CoA, malate) (P &lt; 0.001), and gluconeogenesis-related metabolite, alanine (P &lt; 0.01), are significantly upregulated by DR. The notably (P &lt; 0.01) down-modulated muscle growth (PGF2α) and regeneration (PGE2) stimulation metabolite and the increased consumption of glycogen in muscle and liver may be related to the significantly (P &lt; 0.01) lower body weight and muscle mass by DR. The downregulated oxidative stress, pro-inflammatory mediators, and upregulated anti-inflammatory metabolites resulted in a lower energy expenditure, which contributed to enhanced muscle quality together with upregulated energy production pathways by DR. The improved muscle quality may explain why grip strength is maintained and motor coordination and learning performance are improved by DR in Ercc1∆/− and Xpg−/− mice. Conclusions: This study provides fundamental supporting information on biomarkers and pathways related to the attenuation of sarcopenia, which might facilitate its diagnosis, prevention, and clinical therapy.</p

A sample preparation method for the simultaneous profiling of signaling lipids and polar metabolites in small quantities of muscle tissues from a mouse model for sarcopenia

The metabolic profiling of a wide range of chemical classes relevant to understanding sarcopenia under conditions in which sample availability is limited, e.g., from mouse models, small muscles, or muscle biopsies, is desired. Several existing metabolomics platforms that include diverse classes of signaling lipids, energy metabolites, and amino acids and amines would be informative for suspected biochemical pathways involved in sarcopenia. The sample limitation requires an optimized sample preparation method with minimal losses during isolation and handling and maximal accuracy and reproducibility. Here, two developed sample preparation methods, BuOH-MTBE-Water (BMW) and BuOH-MTBE-More-Water (BMMW), were evaluated and compared with previously reported methods, Bligh-Dyer (BD) and BuOH-MTBE-Citrate (BMC), for their suitability for these classes. The most optimal extraction was found to be the BMMW method, with the highest extraction recovery of 63% for the signaling lipids and 81% for polar metabolites, and an acceptable matrix effect (close to 1.0) for all metabolites of interest. The BMMW method was applied on muscle tissues as small as 5 mg (dry weight) from the well-characterized, prematurely aging, DNA repair-deficient Ercc1 Delta/-mouse mutant exhibiting multiple-morbidities, including sarcopenia. We successfully detected 109 lipids and 62 polar targeted metabolites. We further investigated whether fast muscle tissue isolation is necessary for mouse sarcopenia studies. A muscle isolation procedure involving 15 min at room temperature revealed a subset of metabolites to be unstable; hence, fast sample isolation is critical, especially for more oxidative muscles. Therefore, BMMW and fast muscle tissue isolation are recommended for future sarcopenia studies. This research provides a sensitive sample preparation method for the simultaneous extraction of non-polar and polar metabolites from limited amounts of muscle tissue, supplies a stable mouse muscle tissue collection method, and methodologically supports future metabolomic mechanistic studies of sarcopenia.Analytical BioScience

Capillary electrophoresis in metabolomics

WOS: 000237665400014This article describes the potential of capillary electrophoresis (CE) in metabolomics, which concerns the comprehensive analysis of endogenous low-molecular weight compounds in a biological system. Consequently, metabolomic methods are often developed to separate and to detect as many endogenous metabolites as possible in a single analysis. CE is a very powerful technique for this type of profiling. We demonstrate the applicability of CE in different fields of metabolomics research. We show illustrative clinical, bacterial and plant examples and stress the information that CE systems obtain. We distinguish between targeted and non-targeted metabolomic studies in the bacterial and plant sections. We discuss general aspects of sample preparation of CE for biological samples, and pay special attention to the potential of CE-mass spectrometry in metabolomics. We also discuss future developments with regard to sample preparation and the use of hyphenated systems. (c) 2006 Elsevier Ltd. All rights reserved

Sheathless Capillary Electrophoresis–Mass Spectrometry for Metabolic Profiling of Biological Samples

PharmacologyAnalytical BioScience