A validated method has been developed for the simultaneous measurement of reduced and oxidized glutathione in de-proteinised cellular extracts. This has been used to compare models of malignant and non-malignant human prostate cell lines. Analysis of LNCaP and DU145 cells showed a glutathione to glutathione disulfide ratio of 8:1 and 32:1 respectively, whilst the control cell line, PZ-HPV7 displayed a ratio of 93:1. Results indicate that the more aggressive phenotype displays adaptation to increased oxidative stress via up regulation of glutathione turnover. It was also noted that in the LNCaP and DU145 cell line, glutathione was only responsible for ca. 60% and 79% respectively, of the total cellular reduced thiol; indicating the presence of other biological thiols.
The low molecular weight thiols present in the deproteinized extract of a prostate cancer cell line (LNCaP-FGC) were analysed after derivatization with the Ellman reagent (ESSE). The mixed disulphides formed (RSSE) were fractionated, characterized and quantified by liquid chromatography on a C-18 column using UV detection. This revealed the presence, in femtomoles per cell, of glutathione (8.30 ± 0.73), cysteine (2.71 ± 0.04) and cysteinylglycine (0.83 ± 0.10), accounting for the bulk of the thiol present. Further analysis of the cell extracts using a novel and sensitive mass spectrometry technique allowed the detection of low level of an additional derivative which was identified as cysteinylglycerate using NMRspectroscopy.
Cosmetic skincare products often consist of residual chemical ingredients which are by-products of the manufacturing process that may cause side effects such as skin irritation or allergic dermatitis; investigating the safety of these products to protect the consumer is an important part in the quality control of formulations intended for skin application. Acrylates are a type of polymer widely used in pharmaceutical and cosmetic applications as copolymers, emulsifiers, and cross-linkers. Due to the acrylates being strong skin irritants and sensitizers, it is essential to quantify the levels of residual acrylate monomers in the formulation; these levels must be within the accepted value to be safe. Our previously reported novel hyaluronic acid (HA) hydrogel films were formulated using pentaerythritol tetraacrylate (PT) as the crosslinker. Therefore, it was crucial to analyze the residual PT in these hydrogel films. Gas chromatography (GC) and nuclear magnetic resonance (NMR) spectroscopy were used as analytical methods to detect the residual PT monomers in the HA hydrogel samples. Scanning electron microscopy (SEM) was conducted to investigate structural changes due to the PT monomers leaching out from the HA hydrogel films. The results from the GC method validation (linearity R² > 0.99, RSD for intra-day precision = 1.78%, inter-day precision = 2.52%, %recovery = 101.73%, %RSD = 1.59% for robustness, LOD, LOQ values 0.000032% m/m, 0.00013% m/m for sensitivity) revealed its suitability for such studies. NMR analysis results agreed with the GC results confirming the correct quantification of the extracted residual acrylate monomer. The maximum safe concentration of PT crosslinker in the formulation was determined to be 2.55% m/m.
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