Short Chain Fatty Acids (SCFAs) are produced by the gut microbiota and are present in varying concentrations in the intestinal lumen, in feces but also in the circulatory system. By interacting with different cell types in the body, they have a great impact on host metabolism and their exact quantification is indispensable. Here, we present a derivatization-free method for the gas chromatography mass spectrometry (GC-MS) based quantification of SCFAs in plasma, feces, cecum, liver and adipose tissue. SCFAs were extracted using ethanol and concentrated by alkaline vacuum centrifugation. To allow volatility for separation by GC, samples were acidified with succinic acid. Analytes were detected in selected ion monitoring (SIM) mode and quantified using deuterated internal standards and external calibration curves. Method validation rendered excellent linearity (R² > 0.99 for most analytes), good recovery rates (95–117%), and good reproducibility (RSD: 1–4.5%). Matrix effects were ruled out in plasma, feces, cecum, liver and fat tissues where most abundant SCFAs were detected and accurately quantified. Finally, applicability of the method was assessed using samples derived from conventionally raised versus germ-free mice or mice treated with antibiotics. Altogether, a reliable, fast, derivatization-free GC-MS method for the quantification of SCFAs in different biological matrices was developed allowing for the study of the (patho)physiological role of SCFAs in metabolic health.

Ultrashort pulse infrared lasers can simultaneously sample and homogenize biological tissue using desorption by impulsive vibrational excitation (DIVE). With growing attention on alterations in lipid metabolism in malignant disease, mass spectrometry (MS)-based lipidomic analysis has become an emerging topic in cancer research. In this pilot study, we investigated the feasibility of tissue sampling with a nanosecond infrared laser (NIRL) for the subsequent lipidomic analysis of oropharyngeal tissues, and its potential to discriminate oropharyngeal squamous cell carcinoma (OPSCC) from non-tumorous oropharyngeal tissue. Eleven fresh frozen oropharyngeal tissue samples were ablated. The produced aerosols were collected by a glass fiber filter, and the lipidomes were analyzed with mass spectrometry. Data was evaluated by principal component analysis and Welch’s t-tests. Lipid profiles comprised 13 lipid classes and up to 755 lipid species. We found significant inter- and intrapatient alterations in lipid profiles for tumor and non-tumor samples (p-value < 0.05, two-fold difference). Thus, NIRL tissue sampling with consecutive MS lipidomic analysis is a feasible and promising approach for the differentiation of OPSCC and non-tumorous oropharyngeal tissue and may provide new insights into lipid composition alterations in OPSCC.

Background/Objectives: AKT signaling is a central regulator of cancer metabolism; however, the isoform-specific roles of AKT1, AKT2, and AKT3 in fatty acid metabolism remain incompletely understood in hepatocellular carcinoma (HCC). This study aimed to investigate the contributions of individual AKT isoforms to fatty acid regulation in an HCC cells model. Methods: Stable knockdown models of AKT1, AKT2, and AKT3 were established in Mahlavu cells, whereas stable knockdown models of AKT1 and AKT2 were established in Huh7 cells because endogenous AKT3 expression was very low. Targeted fatty acid profiling was performed using gas chromatography–mass spectrometry (GC–MS) to evaluate alterations in fatty acid composition following AKT isoform knockdown. Results: Knockdown of AKT1, AKT2, and AKT3 in Mahlavu cells, and AKT1 and AKT2 in Huh7 cells, was associated with reduced relative abundance of several common fatty acids, including myristic acid (C14:0), myristoleic acid (C14:1), 7-hexadecenoic acid (C16:1), arachidic acid (C20:0), eicosenoic acid (C20:1), and erucic acid (C22:1). In addition, isoform-specific differences in fatty acids composition were also observed in both HCC cell lines. Compared with scrambled control cells, knockdown of AKT1 or AKT3, but not AKT2, was associated with significant reductions in palmitic acid (C16:0) and stearic acid (C18:0) in Mahlavu cells. In contrast, Huh7 cells exhibited distinct changes in fatty acid composition, showing unchanged or increased relative abundance of palmitic acid (C16:0) and stearic acid (C18:0) following AKT1 or AKT2 knockdown. Moreover, in Huh7 cells very-long-chain fatty acids (VLCFAs), behenic acid (C22:0) and nervonic acid (C24:1) were significantly reduced following AKT2 knockdown, but not AKT1 knockdown. Conclusions: These findings suggest that AKT isoforms play a role regarding fatty acid composition in HCC cells, with both shared and isoform-associated patterns which should be investigated in further studies.

Accurate quantification of bile acids is vital as they are potential biomarkers for several diseases. To solubilize bile acids from tissues, homogenization is required, typically performed using mechanical methods such as the TissueLyser method. Drawbacks like available tissue amount and low abundance of bile acids interfere with the analysis. We aim to establish a nanosecond infrared laser (NIRL) as a possible tissue homogenization tool to circumvent problems associated with limited sample amounts for research and to target low abundance bile acids. We quantify bile acids from mouse and human liver tissues comparing both the classic TissueLyser and the NIRL methods for homogenization prior to LC-MS/MS analysis. The applicability of our approach is tested using mice lacking Cyp2c70, a well-established mouse model with an altered bile acid composition compared to wild type mice. We found the NIRL method to require an almost 14 times smaller starting tissue amount than the TissueLyser method. The NIRL is also comparable to the TissueLyser for high abundance bile acids. While the TissueLyser provides efficient mechanical homogenization, the NIRL potentially enables quantification of low abundance bile acids. The well-established biological differences in bile acid profiles from the Cyp2c70 knockout mouse model were also observed with NIRL homogenization. These results show that the NIRL is potentially useful as smaller tissue amounts are required for analysis and low abundance bile acids are quantifiable.