Quantification Of Diacylglycerols Research Articles

Quantification of diacylglycerol and triacylglycerol species in human fecal samples by flow injection Fourier transform mass spectrometry

The intestinal microbiome plays an important role in human health and disease and fecal materials reflect the microbial activity. Thus, analysis of fecal metabolites provides insight in metabolic interactions between gut microbiota and host organism. In this work, we applied flow injection analysis coupled to Fourier transform mass spectrometry (FIA-FTMS) to identify and quantify lipid species in human fecal samples. Fecal homogenates were subjected to lipid extraction and analyzed by FIA-FTMS. The analysis of different subjects revealed a vast heterogeneity of lipid species abundance. The majority of samples displayed prominent signals of triacylglycerol (TG) and diacylglycerol (DG) species that could be verified by MS2 spectra. Therefore, we focused on the quantification of TG and DG. Method validation included limit of quantification, linearity, evaluation of matrix effects, recovery, and reproducibility. The validation experiments demonstrated the suitability of the method, with exception for approximately 10% of samples, where we observed coefficients of variation higher than 15%. Impaired reproducibility was related to sample inhomogeneity and could not be improved by additional sample preparation steps. Additionally, these experiments demonstrated that compared with aqueous samples, samples containing isopropanol showed higher amounts of DG, presumably due to lysis of bacteria and increased TG lipolysis. These effects were sample-specific and substantiate the high heterogeneity of fecal materials as well as the need for further evaluation of pre-analytic conditions. In summary, FIA-FTMS offers a fast and accurate tool to quantify DG and TG species and is suitable to provide insight into the fecal lipidome and its role in health and disease.

Quantification of Diacylglycerol Molecular Species in Biological Samples by Electrospray Ionization Mass Spectrometry after One-Step Derivatization

Diacylglycerols (DAGs) are important lipid intermediates in cellular trafficking and signaling. Their concentrations are altered in diabetes, cancer, and other disease states. Quantification of DAGs in biological samples may provide critical information to uncover molecular mechanisms leading to various cellular functional disorders. Recent advances in lipidomics using mass spectrometry have greatly accelerated global lipid analysis and quantification. Quantification of DAGs by electrospray mass spectrometry (ESI/MS), however, is challenged by the absence of a permanent charge on the molecule, its low proton affinity and acidity, and its low abundance under normal biological conditions. We describe here the introduction of a quaternary ammonium cation to DAG molecules, using N-chlorobetainyl chloride, to afford a derivatized DAG that gives 2 orders of magnitude higher signal intensities than their underivatized sodium adducts. A linear calibration curve in which peak intensity ratios are plotted versus molar ratios can be achieved by using ESI/MS with dilauroyl glycerol as the internal standard. Employing this new approach to this analyte, we found a 9-fold increase of total DAGs in the livers of obese db/db mice as compared to their heterozygous lean controls. This proven strategy can be used to detect and quantify DAG molecular species from biological samples using ESI/MS after one-step derivatization.

Light-mediated activation of diacylglycerol kinase in rat and bovine rod outer segments.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate is regulated by light in retinal rod outer segment (ROS) membranes. We recently reported that the activities of phosphatidylinositol synthetase and phosphatidylinositol 3-kinase are also higher in bleached (light-exposed) ROS (B-ROS). In this study, we investigated the effect of bleaching on diacylglycerol (DAG) kinase (DAG-kinase) activity in bovine and rat ROS membranes prepared from dark-adapted (D-ROS) or bleached (B-ROS) retinas. In bovine ROS, DAG-kinase activity toward endogenous DAG substrate was higher in B-ROS than in D-ROS. Quantification of DAG in both sets of membranes showed that the levels were the same, eliminating the possibility that the greater DAG-kinase activity was due to higher levels of endogenous substrate in B-ROS. DAG-kinase activity was also higher in B-ROS against an exogenous, water-soluable substrate (1, 2-didecanoyl-rac-glycerol), which competed with endogenous DAG substrate and saturated at approximately 2 mM. Immunoblot analysis with an anti-DAG-kinase gamma polyclonal antibody demonstrated that the gamma isoform was present in isolated bovine ROS. Immunocytochemistry of frozen bovine retinal sections confirmed the presence of DAG-kinase gamma immunoreactivity in ROS, as well as other retinal cells. Quantification of the immunoreactive products on western blots showed that more DAG-kinase gamma was present in B-ROS than in D-ROS. In an in vivo experiment, ROS prepared from rats exposed to 30 min of room light had greater DAG-kinase activity than ROS prepared from dark-adapted animals. Taken together, these data suggest that light exposure leads to the translocation of DAG-kinase from the cytosol to ROS membranes and that the greater DAG-kinase activity in B-ROS is due to the presence of more protein associated with ROS membranes.

Identification and quantification of diacylglycerols in HDL and accessibility to lipase

We have investigated the presence of diacylglycerols in lipoproteins and especially in HDL. Lipoprotein diacylglycerols are very difficult to isolate and to quantify using classical enzymatic techniques, as they are measured in the presence of triacylglycerols and monoacylglycerols. Using a rapid and very sensitive method of gas-liquid chromatography, developed for neutral lipid analysis on an Ultra 1 Hewlett-Packard fused silica capillary column, diacylglycerols (DG) were identified in HDL and classified into five groups: DG 14-16, DG 16-16, DG 16-18, DG 18-18, and DG 18-20. However, their quantitation was difficult due to only partial resolution of molecular species. HDL lipids were submitted to preparative gas-liquid chromatography and diacylglycerols were then silylated using trimethylsilyl reagents. The trimethylsilyl ethers were analyzed by gas-liquid chromatography on a Restek 50 capillary column and were resolved on the basis of carbon number, degree of unsaturation, and double bond positions. The amount of HDL diacylglycerols was twice that of triacylglycerols. The major molecular species of diacylglycerols consisted of 16:0-18:2n-6, 18:0-18:2n-6, and 16:0-18:1n-9 as the major molecular species (33.4, 22.2, and 16.1 mol % of total diacylglycerols, respectively). Using guinea pig cationic pancreatic lipase in order to test the accessibility of diacylglycerols at the surface of HDL, we measured 59% of diacylglycerol hydrolysis, whereas no triacylglycerol hydrolysis was obtained. In addition, most of diacylglycerols having long chain fatty acids, such as 18-20, were completely hydrolyzed, whereas 18-18 and 16-18 were only partially hydrolyzed (64 and 46% respectively). This reflects a different partition of diacylglycerol molecular species between the particle's surface and the lipid core in HDL. This is the first analysis of diacylglycerol molecular species and their distribution in native lipoprotein particles.

Quantification of Diacylglycerols by Capillary Gas Chromatography-Negative Ion Chemical Ionization-Mass Spectrometry

We describe a method for quantifying diacylglycerols as their 1-pentafluorobenzoyl-2-acyl-3-acetyl-glycerol derivatives by capillary gas chromatography-negative ion chemical ionization-mass spectrometry. The basis of the method resides in the sequential treatment of diacylglycerols with acetic anhydride, pancreatic lipase, and pentafluorobenzoyl chloride. Cultured rat mesenteric artery vascular smooth muscle cells (VSMC) were incubated for 20 min in the presence of vehicle or vasopressin (10 −7 M). The incubations were stopped by aspirating the medium and adding 2 ml of methanol containing 790 pmol of internal standard 1-stearoyl-2-(10,13)-nonadecadienoyl-glycerol. After extraction, diacylglycerols were isolated by thin-layer chromatography, acetylated, and treated with pancreatic lipase. The resulting 2-acyl-3-acetylglycerols were then purified by thin-layer chromatography, transformed into their 1-pentafluorobenzoyl-derivatives, and monitored by capillary gas chromatography-negative ion chemical ionization-mass spectrometry on the selected ion-monitoring mode ( m/ z 614 and 604 for 2-arachidonoyl and 2-nonadecadienoyl species, respectively). The levels of diacylglycerols bearing an arachidonoyl moiety were 128 ± 26 pmol/100 nmol lipid phosphorus in resting cells and 333 ± 28 in stimulated cells (mean ± SD, n = 3, P < 0.01). The presence of diacylglycerol species bearing an oleoyl or a linoleoyl group at the second position could also be detected in VSMC preparations by this approach. This new method can he applied to quantitate various diacylglycerol species bearing distinct acyl moieties at the second position of the glycerol molecule.