Determination of Glycerophospholipids in Biological Material Using High-Performance Liquid Chromatography with Charged Aerosol Detector HPLC-CAD-A New Approach for Isolation and Quantification.

The method of using high-performance liquid chromatography with a charged aerosol detector method (HPLC-CAD) was developed for the separation and determination of phospholipids isolated from cell membranes. The established cell lines-normal and neoplastic prostate cells and normal skin fibroblasts and melanoma cells-were selected for the study. Chromatographic separation was performed in the diol stationary phase using a gradient elution based on a mixture of n-hexane, isopropanol and water with the addition of triethylamine and acetic acid as buffer additives. Taking the elements of the Folch and Bligh-Dyer methods, an improved procedure for lipid isolation from biological material was devised. Ultrasound-assisted extraction included three extraction steps and changed the composition of the extraction solvent, which led to higher recovery of the tested phospholipids. This method was validated by assessing the analytical range, precision, intermediate precision and accuracy. The analytical range was adjusted to the expected concentrations in cell extracts of various origins (from 40 µg/mL for PS up to 10 mg/mL for PC). Both precision and intermediate precision were at a similar level and ranged from 3.5% to 9.0%. The recovery for all determined phospholipids was found to be between 95% and 110%. The robustness of the method in terms of the use of equivalent columns was also confirmed. Due to the curvilinear response of CAD, the quantification was based on an internal standard method combined with a power function transformation of the normalized peak areas, allowing the linearization of the signal with an R2 greater than 0.996. The developed method was applied for the isolation and determination of glycerophospholipids from cell membranes, showing that the profile of the tested substances was characteristic of various types of cells. This method can be used to assess changes in metabolism between normal cells and neoplastic cells or cells with certain pathologies or genetic changes.

Persistent imbalance, anti-apoptotic, and anti-inflammatory signature of circulating C-C chemokines and cytokines in patients with paroxysmal nocturnal hemoglobinuria.

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal non-malignant disease in which hematopoietic cell apoptosis may play an important pathophysiological role. Previous studies of the content of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) indicated the possibility of remote transmission of anti-apoptotic signals between pathological and normal hematopoietic progenitors. METHODS: The study determined the plasma levels of beta chemokines and cytokines in N = 19 patients with PNH and 31 healthy controls. The research material was peripheral blood plasma (EDTA) stored at -80 °C until the test. Beta chemokine and cytokine concentrations were tested in duplicate with Bio-Plex Pro Human Cytokine Assay (Bio-Rad, Hercules, CA, USA) using a Luminex 200 flow cytometer and xPONENT software (Luminex Corporation, Austin, TX, USA). In peripheral blood CD34+ cells we tested the proportions of PI(3,4,5)P3+ and Annexin binding apoptotic phenotype using FC and phosflow. RESULTS: Compared to the control group, the PNH group showed a significant increase in the plasma concentration of some beta chemokines and cytokines, including MIP-1alpha/CCL3, eotaxin/CCL11, MCP1/CCL2, IL4 and G-CSF. In the group of PNH patients, a significant decrease in the concentration of some cytokines was also observed: RANTES/CCL5, MIP-1beta/CCL4, PDGF-BB and IL9. At the same time, the plasma concentrations of the chemokine IP-10/CXCL10 and the cytokines IFN-gamma, TNF, IL6 and IL10 showed no significant deviations from the values for the control group. Anti-apoptotic phenotype and phosphatidylinositol (3,4,5)-trisphosphate content in PNH clone of CD34+ cells were associated with the level of CCL3 and negatively associated with CCL5, CCL4, PDGF-BB and IL9. CONCLUSIONS: This data suggest the existence of apoptotic and PI(3,4,5)P3 imbalance in PNH CD34+ cells driven by anti-apoptotic cytokine biosignature in PNH. Plasma cytokines and intracellular enzymes that regulate the phosphoinositide pathways may become a therapeutic target in PNH.

Role of thiamine in Huntington's disease pathogenesis: In vitro studies.

BACKGROUND: Oxidative stress accompanies neurodegeneration and also causes abnormalities in thiaminedependent processes. These processes have been reported to be diminished in the brains of patients with several neurodegenerative diseases. OBJECTIVES: The aim of this work was to conduct a comparative analysis of the impact of supplemented thiamine on the viability of human B lymphocytes with CAG abnormal expanded huntingtin gene (mHTT) (GM13509) and control, B lymphocytes without mHTT (GM14467) through the following studies: determination of the supplemented thiamine concentrations, which are effective for cell growth stimulation after incubation in thiamine deficit conditions; determination of cell capability to intake the exogenous thiamine; evaluation of exogenous thiamine influence on the profile of the genes related to thiamine and energy metabolism; determination of ATP synthesis and activities of thiamine-dependent enzymes, KGDHC and BCKDHC in the intact cells and upon the exogenous thiamine. MATERIAL AND METHODS: The following methods were used: EZ4U test for cell growth analysis; HPLC for determination of thiamine intake and ATP synthesis, qRT-PCR for evaluation of the gene profiles and spectrophotometric method for KGDHC and BCKDHC activities determination. RESULTS: Maximal cell growth stimulation was observed at 2.5 mM in GM14467 up to 135% of the control culture and at 5.0 mM in GM13509 cells up to 165% of the control culture. Native levels of total ATP and KGDHC and BCKDHC activities in both cell types were comparable and did not changed upon thiamine deficit or supplementation. GM13509 cells showed more of an increase in growth stimulation upon thiamine supplementation than GM14467 cells and this effect was reflected in the increase of intracellular thiamine concentration. CONCLUSIONS: The above results and reported changes in expression of GAPDH, IDH1 and SLC19A3 genes observed upon thiamine deficit conditions suggest that intracellular thiamine status and energy metabolism can have a role in HD pathogenesis.

The influence of Selol on the expression of oxidative stress genes in normal and malignant prostate cells.

Selol is a mixture of selenitriglycerides, obtained by the chemical modification of sunflower oil, which contain selenium at the +4 oxidation state. The aim of the present study was to describe the changes in the expression of genes related to oxidative stress caused by Selol in prostate cells: both normal (PNT1A) and malignant (LNCaP). The changes in gene expression in PNT1A and LNCaP cell lines under the influence of Selol were measured using a 96-well RT(2) Profiler ™PCR Array: Human Oxidative Stress and Antioxidant Defense, which arrayed 84 genes functionally involved in the cellular oxidative stress response. Based on the obtained data, LNCaP cells exhibited a significantly lower potential for antioxidant defence when compared to PNT1A cells. The response of the malignant LNCaP cells to exposure to Selol was significantly different from that of the normal PNT1A cells, especially after 48 h of incubation. In the case of LNCaP cells, Selol causes down-regulation of the expression of many vital genes. Under in vitro conditions, the efficacy of Selol slightly changes with increasing concentration, but significantly increases when the incubation time is lengthened.

SEC ICP MS and CZE ICP MS investigation of medium and high molecular weight complexes formed by cadmium ions with phytochelatins.

Size-exclusion chromatography (SEC) and capillary zone electrophoresis (CZE) coupled with inductively coupled plasma mass spectrometry were applied to characterize low, medium, and high molecular weight cadmium complexes with glutathione and phytochelatins (PCs). The dominant stoichiometry of the complexes formed in vitro was established as 1:1 using electrospray ionization mass spectrometry. Calculated molecular masses of Cd1L1 complexes were used for calibration of the SEC and CZE methods. The results showed a lower (2 kDa) SEC column exclusion limit for cadmium complexes compared with free peptides (10 kDa), and most of the high molecular weight cadmium species were eluted in the void volume of the column. Moreover, the CZE method based on the semiempirical model of Offord to elucidate peptide migration allowed us to show a high propensity of Cd-PC complexes for polymorphism on complexation, which was also observed for extracts of Arabidopsis thaliana treated with cadmium. All the information presented is vital for understanding the mechanism of metal deactivation in plants.