A global metabolomics minefield: Confounding effects of preanalytical factors when studying rare disorders.

A common challenge when studying rare diseases or medical conditions is the limited number of patients, usually resulting in long inclusion periods as well as unequal sampling and storage conditions. The main purpose of this study was to demonstrate the challenges when comparing samples subject to different preanalytical conditions. We performed a global (commonly referred to as "untargeted") liquid chromatography-high resolution mass spectrometry metabolomics analysis of blood samples from cases of sudden infant death syndrome and controls stored as dried blood spots on a chemical-free filter card for 15 years at room temperature compared with the same blood samples stored as whole blood at -80°C before preparing new dried blood spots using a chemically treated filter card. Principal component analysis plots distinctly separated the samples based on the type of filter card and storage, but not sudden infant death syndrome versus controls. Note that, 1263 out of 5161 and 642 out of 1587 metabolite features detected in positive and negative ionization mode, respectively, were found to have significant 2-fold changes in amounts corresponding to different preanalytical conditions. The study demonstrates that the dried blood spot metabolome is largely affected by preanalytical factors. This emphasizes the importance of thoroughly addressing preanalytical factors during study design and interpretation, enabling identification of real, biological differences between sample groups whilst preventing other factors or random variation to be falsely interpreted as positive results.

Monitoring Farmed Fish Welfare by Measurement of Cortisol as a Stress Marker in Fish Feces by Liquid Chromatography Coupled with Tandem Mass Spectrometry.

The aquaculture industry has become a sustainable source of food for humans. Remaining challenges include disease issues and ethical concerns for the discomfort and stress of farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid−liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g, the limit of quantification was 0.34 ng/g, and the method was linear (R2 > 0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a coefficient of variation (CV) of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

Void Content Determination of Carbon Fiber Reinforced Polymers: A Comparison between Destructive and Non-Destructive Methods.

The properties of composite materials are highly dependent on the fiber and matrix fraction and on the porosity resulting from micro voids. This paper addresses void content characterization and the constituent content of composite materials by resorting to a comparison of destructive and non-destructive methods. The work presents the detailed procedures of two destructive methods, using acid digestion of epoxy resins matrices, and compares their processes. It also presents the results of a non-destructive method, by means of Micro Computed Tomography (MicroCT). The results of both destructive and non-destructive methods are compared, and a recommendation is made based on the application and the type of composite being analyzed. The MicroCT showed better and more consistent results in detecting voids in the material, while the acid digestion tests provided better results about the fiber and matrix percentage. Exported results from the MicroCT scanning with actual locations of voids were used in numerical analysis, to examine the feasibility of using them, whether by developing models that map damage in the proximity of the void, or by developing models that predict the properties of the entire material with respect to the content, shape, and distribution in the material.

Bridging the Polar and Hydrophobic Metabolome in Single-Run Untargeted Liquid Chromatography-Mass Spectrometry Dried Blood Spot Metabolomics for Clinical Purposes.

Dried blood spot (DBS) metabolite analysis is a central tool for the clinic, e.g., newborn screening. Instead of applying multiple analytical methods, a single liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolites spanning from highly polar glucose to hydrophobic long-chain acylcarnitines. For liquid chromatography, a diphenyl column and a multi-linear solvent gradient operated at elevated flow rates allowed for an even-spread resolution of diverse metabolites. Injecting moderate volumes of DBS organic extracts directly, in contrast to evaporation and reconstitution, provided substantial increases in analyte recovery. Q Exactive MS settings were also tailored for sensitivity increases, and the method allowed for analyte retention time and peak area repeatabilities of 0.1-0.4 and 2-10%, respectively, for a wide polarity range of metabolites (log P -4.4 to 8.8). The method's performance was suited for both untargeted analysis and targeted approaches evaluated in clinically relevant experiments.

Caffeine increases strength and power performance in resistance-trained females during early follicular phase.

The effects of 4 mg·kg-1 caffeine ingestion on strength and power were investigated for the first time, in resistance-trained females during the early follicular phase utilizing a randomized, double-blind, placebo-controlled, crossover design. Fifteen females (29.8 ± 4.0 years, 63.8 ± 5.5 kg [mean ± SD]) ingested caffeine or placebo 60 minutes before completing a test battery separated by 72 hours. One-repetition maximum (1RM), repetitions to failure (RTF) at 60% of 1RM, was assessed in the squat and bench press. Maximal voluntary contraction torque (MVC) and rate of force development (RFD) were measured during isometric knee extensions, while utilizing interpolated twitch technique to measure voluntary muscle activation. Maximal power and jump height were assessed during countermovement jumps (CMJ). Caffeine metabolites were measured in plasma. Adverse effects were registered after each trial. Caffeine significantly improved squat (4.5 ± 1.9%, effect size [ES]: 0.25) and bench press 1RM (3.3 ± 1.4%, ES: 0.20), and squat (15.9 ± 17.9%, ES: 0.31) and bench press RTF (9.8 ± 13.6%, ES: 0.31), compared to placebo. MVC torque (4.6 ± 7.3%, ES: 0.26), CMJ height (7.6 ± 4.0%, ES: 0.50), and power (3.8 ± 2.2%, ES: 0.24) were also significantly increased with caffeine. There were no differences in RFD or muscle activation. Plasma [caffeine] was significantly increased throughout the protocol, and mild side effects of caffeine were experienced by only 3 participants. This study demonstrated that 4 mg·kg-1 caffeine ingestion enhanced maximal strength, power, and muscular endurance in resistance-trained and caffeine-habituated females during the early follicular phase, with few adverse effects. Female strength and power athletes may consider using this dose pre-competition and -training as an effective ergogenic aid.