Optimized LC-MS/MS Method for the Detection of ppCCK(21-44): A Surrogate to Monitor Human Cholecystokinin Secretion.

The hormone cholecystokinin (CCK) is secreted postprandially from duodenal enteroendocrine cells and circulates in the low picomolar range. Detection of this digestion and appetite-regulating hormone currently relies on the use of immunoassays, many of which suffer from insufficient sensitivity in the physiological range and cross-reactivity problems with gastrin, which circulates at higher plasma concentrations. As an alternative to existing techniques, a liquid chromatography and mass spectrometry-based method was developed to measure CCK-derived peptides in cell culture supernatants. The method was initially applied to organoid studies and was capable of detecting both CCK8 and an N-terminal peptide fragment (prepro) ppCCK(21-44) in supernatants following stimulation. Extraction optimization was performed using statistical modeling software, enabling a quantitative LC-MS/MS method for ppCCK(21-44) capable of detecting this peptide in the low pM range in human plasma and secretion buffer solutions. Plasma samples from healthy individuals receiving a standardized meal (Ensure) after an overnight fast were analyzed; however, the method only had sensitivity to detect ppCCK(21-44). Secretion studies employing human intestinal organoids and meal studies in healthy volunteers confirmed that ppCCK(21-44) is a suitable surrogate analyte for measuring the release of CCK in vitro and in vivo.

Lipidomes of Icelandic bryophytes and screening of high contents of polyunsaturated fatty acids by using lipidomics approach.

Bryophytes (mosses, liverworts, and hornworts) have interested researchers because of their high chemical diversity and their potential uses in pharmaceutical, food, and cosmetic industries. Specifically, long-chain polyunsaturated fatty acids (l-PUFA) such as arachidonic acid (AA) and eicosapentaenoic acid (EPA) are commonly found in bryophytes, but not in vascular plants. Bryophytes accumulate PUFAs in cold or even freezing temperature to keep the cell fluidity. Iceland has a long history of bryophyte vegetation. These bryophytes are highly adapted to the harsh environment in Iceland and therefore are expected to produce high amounts of PUFAs. However, despite the fact that hundreds of mosses and liverworts have been found in Iceland, their lipid profiles largely remain unknown. In this study, we performed untargeted lipidomics by using UPLC-ESI-QTOF-MS as a rapid screening strategy to examine the lipid compositions of 39 local bryophyte species in Iceland and aimed to find high AA and EPA producers. A total of 280 lipid molecular species from 15 lipid classes were quantified with isotope-labeled internal standards. AA and EPA were abundantly distributed in the phospholipids (mainly PC and PE) and glycerolipids (MGDG and DGDG) in six moss species, namely Racomotrium lanuginosum, R. ericoides, Bryum psedotriquetrium, Plagiomnium ellipticum, Hylocomium splendens, and Rhytidiadelphus triquetrus. Two of the six species (B. psedotriquetrium and H. splendens) also accumulated high concentrations of PUFA-containing-triacylglycerols.

Lipidomic analysis of moss species Bryum pseudotriquetrum and Physcomitrium patens under cold stress.

Bryophytes, which lack lignin for protection, support themselves in harsh environments by producing various chemicals. In response to cold stress, lipids play a crucial role in cell adaptation and energy storage. Specifically, bryophytes survive at low temperatures by producing very long-chain polyunsaturated fatty acids (vl-PUFAs). The in-depth understanding of the lipid response to cold stress of bryophytes was studied by performing lipid profiling using ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). Two moss species (Bryum pseudotriquetrum and Physcomitrium patens) cultivated at 23°C and at 10°C were included in this study. Relative quantitative lipid concentrations were compared and the potential lipid biomarkers were identified by multivariate statistical analysis in each species. In B. pseudotriquetrum, it was observed that the phospholipids and glycolipids increased under cold stress, while storage lipids decreased. The accumulation of the lipids with high unsaturation degrees mostly appears in phospholipids and glycolipids for both mosses. The results also indicate that two unusual lipid classes in plants, sulfonolipids and phosphatidylmethanol are biosynthesized by the bryophytes. This has not been seen previously and show that bryophytes have a very diverse chemistry and substantially different from other plant groups.

Effects of extraction parameters on lipid profiling of mosses using UPLC-ESI-QTOF-MS and multivariate data analysis.

INTRODUCTION: Non-target lipid profiling by using ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) has been used extensively in the past decades in plant studies. However, the lipidomes of bryophytes have only been scarcely studied, although they are the second largest group in plant kingdom. OBJECTIVES: We evaluated the effects of different cell disruption methods (no disruption, shake, ultrasound, and bead beating), and storage conditions (air-dried, freeze-dried, and fresh frozen) of five moss species (including Racomitrium lanuginosum B and D, Philonotis fontana, Sphagnum teres, and Hylocomium splendens). METHODS: The lipid profiling results of each extraction parameter were analyzed by using multivariate data analysis including unsupervised principal component analysis and supervised orthogonal projections to latent structures discriminant analysis. RESULTS: The results showed that extraction with bead beating resulted in the highest lipid content and the most detected features, but these were caused by the contamination from plastic tubes. Minor lipid metabolite changes were found in shaking and ultrasonication methods when compared with no disruption method. Significant amounts of phosphatidylcholine, diacylglyceryltrimethylhomoserine and their lyso lipids were observed in air-dried moss tissues, whereas diacylglycerol, triacylglycerol and ceramide were mostly exclusively detected when fresh frozen tissues were used for extraction. CONCLUSION: We concluded that lipid extraction using fresh frozen samples with ultrasound assistance provide the most original lipid composition and gave a relatively high lipid content.

Stability characterization, kinetics and mechanism of tacrolimus degradation in cyclodextrin solutions.

Tacrolimus is a macrolide lactone and potent immunosuppressant. It is highly lipophilic and has very limited aqueous solubility. Tacrolimus is highly susceptible to hydrolysis which results in very limited stability in aqueous solutions. Besides this, tacrolimus also undergoes dehydration and epimerization. Cyclodextrin (CD) complexation can increase the solubility and stability of hydrophobic drugs in aqueous solutions through the formation of drug/CD complexes. The aim of this study was to investigate degradation kinetics, mechanism and stability of tacrolimus in aqueous CD solutions, with the ultimate goal of developing an aqueous vehicle for ophthalmic delivery. For this, phase-solubility and kinetic studies in aqueous solutions containing different CDs at different pH values were performed. Mass spectrometry studies were also performed to elucidate the degradation mechanism of the drug in aqueous CD solution. The study showed that the drug has maximum stability between pH 4 and 6 and hydrolysis was the main cause of tacrolimus degradation in aqueous 2-hydroxypropyl-ßCD (HPßCD) solutions. ßCD and its derivatives were the better CD solubilizers for tacrolimus. The solubility and stability studies were further conducted with CD and surfactants, which is tyloxapol, tween 80 and poloxamer 407, where the combination provided better results compared to individual components.

Lipidomic study of cell lines reveals differences between breast cancer subtypes.

Breast cancer (BC) is the most prevalent type of cancer in women in western countries. BC mortality has not declined despite early detection by screening, indicating the need for better informed treatment decisions. Therefore, a novel noninvasive diagnostic tool for BC would give the opportunity of subtype-specific treatment and improved prospects for the patients. Heterogeneity of BC tumor subtypes is reflected in the expression levels of enzymes in lipid metabolism. The aim of the study was to investigate whether the subtype defined by the transcriptome is reflected in the lipidome of BC cell lines. A liquid chromatography mass spectrometry (LC-MS) platform was applied to analyze the lipidome of six cell lines derived from human BC cell lines representing different BC subtypes. We identified an increased abundance of triacylglycerols (TG) ≥ C-48 with moderate or multiple unsaturation in fatty acyl chains and down-regulated ether-phosphatidylethanolamines (PE) (C-34 to C-38) in cell lines representing estrogen receptor and progesterone receptor positive tumor subtypes. In a cell line representing HER2-overexpressing tumor subtype an elevated expression of TG (≤ C-46), phosphatidylcholines (PC) and PE containing short-chained (≤ C-16) saturated or monounsaturated fatty acids were observed. Increased abundance of PC ≥ C-40 was found in cell lines of triple negative BC subtype. In addition, differences were detected in lipidomes within these previously defined subtypes. We conclude that subtypes defined by the transcriptome are indeed reflected in differences in the lipidome and, furthermore, potentially biologically relevant differences may exist within these defined subtypes.

Valuable Fatty Acids in Bryophytes-Production, Biosynthesis, Analysis and Applications.

Bryophytes (mosses, liverworts and hornworts) often produce high amounts of very long-chain polyunsaturated fatty acids (vl-PUFAs) including arachidonic acid (AA, 20:4 △5,8,11,14) and eicosapentaenoic acid (EPA, 20:5 △5,8,11,14,17). The presence of vl-PUFAs is common for marine organisms such as algae, but rarely found in higher plants. This could indicate that bryophytes did not lose their marine origin completely when they landed into the non-aqueous environment. Vl-PUFA, especially the omega-3 fatty acid EPA, is essential in human diet for its benefits on healthy brain development and inflammation modulation. Recent studies are committed to finding new sources of vl-PUFAs instead of fish and algae oil. In this review, we summarize the fatty acid compositions and contents in the previous studies, as well as the approaches for qualification and quantification. We also conclude different approaches to enhance AA and EPA productions including biotic and abiotic stresses.

Determining gradient conditions for peptide purification in RPLC with machine-learning-based retention time predictions.

A strategy for determining a suitable solvent gradient in silico in preparative peptide separations is presented. The strategy utilizes a machine-learning-based method, called ELUDE, for peptide retention time predictions based on the amino acid sequences of the peptides. A suitable gradient is calculated according to linear solvent strength theory by predicting the retention times of the peptides being purified at three different gradient slopes. The advantage of this strategy is that fewer experiments are needed to develop a purification method, making it useful for labs conducting many separations but with limited resources for method development. The preparative separation of met-enkephalin and leu-enkephalin was used as model solutes on two stationary phases: XBridge C18 and CSH C18. The ELUDE algorithm contains a support vector regression and is pre-trained, meaning that only 10-50 peptides are needed to calibrate a model for a certain stationary phase and gradient. The calibration is done once and the model can then be used for new peptides similar in size to those in the calibration set. We found that the accuracy of the retention time predictions is good enough to usefully estimate a suitable gradient and that it was possible to compare the selectivity on different stationary phases in silico. The absolute relative errors in retention time for the predicted gradients were 4.2% and 3.7% for met-enkephalin and leu-enkephalin, respectively, on the XBridge C18 column and 2.0% and 2.8% on the CSH C18 column. The predicted retention times were also used as initial values for adsorption isotherm parameter determination, facilitating the numerical calculation of overloaded elution profiles. Changing the trifluoroacetic acid (TFA) concentration from 0.05% to 0.15% in the eluent did not seriously affect the error in the retention time predictions for the XBridge C18 column, an increase of 1.0 min (in retention factor, 1.3). For the CSH C18 column the error was, on average, 2.6 times larger. This indicates that the model needs to be recalibrated when changing the TFA concentration for the CSH column. Studying possible scale-up complications from UHPLC to HPLC such as pressure, viscous heating (i.e., temperature gradients), and stationary-phase properties (e.g., packing heterogeneity and surface chemistry) revealed that all these factors were minor to negligible. The pressure effect had the largest effect on the retention, but increased retention by only 3%. In the presented case, method development can therefore proceed using UHPLC and then be robustly transferred to HPLC.

Altered plasmalogen content and fatty acid saturation following epithelial to mesenchymal transition in breast epithelial cell lines.

Epithelial to mesenchymal transition (EMT) is a developmental event characterized by phenotypic switching from a polarized epithelial phenotype to an unpolarized mesenchymal phenotype. Changes to plasma membrane function accompany EMT yet the differences in lipid composition of cells that have undergone EMT are relatively unexplored. To address this the lipidome of two cell models of EMT in breast epithelial tissue, D492 and HMLE, were analyzed by untargeted LC-MS. Detected masses were identified and their abundance was compared through multivariate statistical analysis. Considerable concordance was observed in eight lipid components between epithelial and mesenchymal cells in both cell models. Specifically, an increase in phosphatidylcholine and triacylglycerol were found to accompany EMT while phosphatidylcholine- and phosphatidylethanolamine plasmalogens, as well as diacylglycerols decreased. The most abundant fatty acid lengths were C16 and C18 but mesenchymal cells had on average shorter and more unsaturated fatty acids. The results are consistent with enhanced cell mobility post EMT and reflect a consequence of oxidative stress pre- and post EMT in breast epithelial tissue.