The emergence of proton nuclear magnetic resonance metabolomics in the cardiovascular arena as viewed from a clinical perspective

The ability to phenotype metabolic profiles in serum has increased substantially in recent years with the advent of metabolomics. Metabolomics is the study of the metabolome, defined as those molecules with an atomic mass less than 1.5 kDa. There are two main metabolomics methods: mass spectrometry (MS) and proton nuclear magnetic resonance (1H NMR) spectroscopy, each with its respective benefits and limitations. MS has greater sensitivity and so can detect many more metabolites. However, its cost (especially when heavy labelled internal standards are required for absolute quantitation) and quality control is sub-optimal for large cohorts. 1H NMR is less sensitive but sample preparation is generally faster and analysis times shorter, resulting in markedly lower analysis costs. 1H NMR is robust, reproducible and can provide absolute quantitation of many metabolites. Of particular relevance to cardio-metabolic disease is the ability of 1H NMR to provide detailed quantitative data on amino acids, fatty acids and other metabolites as well as lipoprotein subparticle concentrations and size. Early epidemiological studies suggest promise, however, this is an emerging field and more data is required before we can determine the clinical utility of these measures to improve disease prediction and treatment. This review describes the theoretical basis of 1H NMR; compares MS and 1H NMR and provides a tabular overview of recent 1H NMR-based research findings in the atherosclerosis field, describing the design and scope of studies conducted to date. 1H NMR metabolomics-CVD related research is emerging, however further large, robustly conducted prospective, genetic and intervention studies are needed to advance research on CVD risk prediction and to identify causal pathways amenable to intervention

Development of a new SonovueTM contrast-enhanced ultrasound approach reveals temporal and age-related features of muscle microvascular responses to feeding

Compromised limb blood flow in aging may contribute to the development of sarcopenia, frailty, and the metabolic syndrome. We developed a novel contrast-enhanced ultrasound technique using Sonovue™ to characterize muscle microvasculature responses to an oral feeding stimulus (15 g essential amino acids) in young (~20 years) and older (~70 years) men. Intensity-time replenishment curves were made via an ultrasound probe “fixed” over the quadriceps, with intermittent high mechanical index destruction of microbubbles within muscle vasculature. This permitted real-time measures of microvascular blood volume (MBV), microvascular flow velocity (MFV) and their product, microvascular blood flow (MBF). Leg blood flow (LBF) was measured by Doppler and insulin by enzyme-linked immunosorbent assay. Steady-state contrast concentrations needed for comparison between different physiological states were achieved <150 sec from commencing Sonovue™ infusion, and MFV and MBV measurements were completed <120 sec thereafter. Interindividual coefficients of variation in MBV and MFV were 35–40%, (N = 36). Younger men (N = 6) exhibited biphasic vascular responses to feeding with early increases in MBV (+36%, P < 0.008 45 min post feed) reflecting capillary recruitment, and late increases in MFV (+77%, P < 0.008) and MBF (+130%, P < 0.007 195 min post feed) reflecting more proximal vessel dilatation. Early MBV responses were synchronized with peak insulin but not increased LBF, while later changes in MFV and MBF occurred with insulin at post absorptive values but alongside increased LBF. All circulatory responses were absent in old men (N = 7). Thus, impaired postprandial circulation could impact age-related declines in muscle glucose disposal, protein anabolism, and muscle mass

An overview of technical considerations for Western blotting applications to physiological research

The applications of Western/immuno-blotting (WB) techniques have reached multiple layers of the scientific community and are now considered routine procedures in the field of physiology. This is none more so than in relation to skeletal muscle physiology (i.e. resolving the mechanisms underpinning adaptations to exercise). Indeed, the inclusion of WB data is now considered an essential aspect of many such physiological publications to provide mechanistic insight into regulatory processes. Despite this popularity, and due to the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and subsequent analysis and interpretation of the data can be variable, perhaps resulting in spurious conclusions. This may be due to poor laboratory technique and/or lack of comprehension of the critical steps involved in WB and what quality control procedures should be in place to ensure robust data generation. The present review aims to provide a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting and detection to analysis of the data collected. We aim to provide the reader with improved expertise to critically conduct, evaluate and troubleshoot the WB process, to produce reproducible and reliable blots