High-quality biological samples are required for the favorable outcome of research studies, and valid data sets are crucial for successful biomarker identification. Prolonged storage of biospecimens may have an artificial effect on compound levels. In order to investigate the potential effects of long-term storage on the metabolome, human ethylenediaminetetraacetic acid (EDTA) plasma samples stored for up to 16 years were analyzed by gas and liquid chromatography-tandem mass spectrometry-based metabolomics. Only 2% of 231 tested plasma metabolites were altered in the first seven years of storage. However, upon longer storage periods of up to 16 years and more time differences of few years significantly affected up to 26% of the investigated metabolites when analyzed within subject age groups. Ontology classes that were most affected included complex lipids, fatty acids, energy metabolism molecules, and amino acids. In conclusion, the human plasma metabolome is adequately stable to long-term storage at −80 °C for up to seven years but significant changes occur upon longer storage. However, other biospecimens may display different sensitivities to long-term storage. Therefore, in retrospective studies on EDTA plasma samples, analysis is best performed within the first seven years of storage.

Metabolomics is a powerful technology with broad applications in life science that, like other -omics approaches, requires high-quality samples to achieve reliable results and ensure reproducibility. Therefore, along with quality assurance, methods to assess sample quality regarding pre-analytical confounders are urgently needed. In this study, we analyzed the response of the human serum metabolome to pre-analytical variations comprising prolonged blood incubation and extended serum storage at room temperature by using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) -based metabolomics. We found that the prolonged incubation of blood results in a statistically significant 20% increase and 4% decrease of 225 tested serum metabolites. Extended serum storage affected 21% of the analyzed metabolites (14% increased, 7% decreased). Amino acids and nucleobases showed the highest percentage of changed metabolites in both confounding conditions, whereas lipids were remarkably stable. Interestingly, the amounts of taurine and O-phosphoethanolamine, which have both been discussed as biomarkers for various diseases, were 1.8- and 2.9-fold increased after 6 h of blood incubation. Since we found that both are more stable in ethylenediaminetetraacetic acid (EDTA) blood, EDTA plasma should be the preferred metabolomics matrix.

Discrepancies in blood sample collection and processing could have a significant impact on levels of metabolites, peptides, and protein biomarkers of inflammation in the blood; thus, sample quality control is critical for successful biomarker identification and validation. In this study, we analyzed the effects of several preanalytical processing conditions, including different storage times and temperatures for blood or plasma samples and different centrifugation forces on the levels of metabolites, peptides, and inflammation biomarkers in human plasma samples using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Temperature was found to be the major factor for metabolite variation, and both time and temperature were identified as major factors for peptide variation. For inflammation biomarkers, temperature played different roles depending on the sample type (blood or plasma). Low temperature affected inflammation biomarkers in blood, while room temperature impacted inflammation biomarkers in plasma.

The kinase AKT2 (PKB) is an important mediator of insulin signaling, for which loss-of-function knockout (KO) mutants lead to early onset diabetes mellitus, and dominant active mutations lead to early development of obesity and endothelial cell (EC) dysfunction. To model EC dysfunction, we used edited human pluripotent stem cells (hPSCs) that carried either a homozygous deletion of AKT2 (AKT2 KO) or a dominant active mutation (AKT2 E17K), which, along with the parental wild type (WT), were differentiated into ECs. Profiling of EC lines indicated an increase in proinflammatory and a reduction in anti-inflammatory fatty acids, an increase in inflammatory chemokines in cell supernatants, increased expression of proinflammatory genes, and increased binding to the EC monolayer in a functional leukocyte adhesion assay for both AKT2 KO and AKT2 E17K. Collectively, these findings suggest that vascular endothelial inflammation that results from dysregulated insulin signaling (homeostasis) may contribute to coronary artery disease, and that either downregulation or upregulation of the insulin pathway may lead to inflammation of endothelial cells. This suggests that the standard of care for patients must be expanded from control of metabolic parameters to include control of inflammation, such that endothelial dysfunction and cardiovascular disorders can ultimately be prevented.

Short-term feeding studies have highlighted a phenomenon in Ca regulation that raises concerns around Ca absorption in dogs that may make an impact on commercial diets near to the maximum recommended level. A recent study to determine responses in dogs fed one of two diets differing in dietary Ca over 40 weeks found no evidence to suggest a concern across a range of biological parameters hypothesised to be affected by Ca. Unforeseen consequences of dietary Ca could have occurred and metabolic profiling was deemed a suitable data-driven approach to identify effects of dietary Ca. The objectives were to compare the fasted plasma metabolome (sampled at 8-week intervals over 40 weeks) of dogs fed one of two diets, near to the minimum and maximum recommended levels of dietary Ca. Comparisons with the control diet were also investigated across the postprandial time course (1–4 h) following acute (1 d) and long-term (24 weeks) feeding of the test diet. Comparing fasted plasma samples at each time point, no significant effect (adjusted P < 0·05) of diet on metabolites was observed. In the postprandial state, only phosphate was consistently different between diets and was explained by additional dietary P to maintain Ca:P. Metabolic profiling analysis supports the view that the dietary Ca upper limit is safe. Additionally, the canine plasma metabolome was characterised, providing insights into the stability of individual profiles across 40 weeks, the response to consumption of a nutritionally complete meal over a 4 h postprandial time course and different kinetic categories of postprandial absorption.

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