Quantification of Short-Chain Chlorinated Paraffins by Deuterodechlorination Combined with Gas Chromatography–Mass Spectrometry

Analysis of short-chain chlorinated paraffins (SCCPs) is extremely difficult because of their complex compositions with thousands of isomers and homologues. A novel analytical method, deuterodechlorination combined with high resolution gas chromatography–high resolution mass spectrometry (HRGC–HRMS), was developed. A protocol is applied in the deuterodechlorination of SCCPs with LiAlD₄, and the formed deuterated <i>n</i>-alkanes of different alkane chains can be distinguished readily from each other on the basis of their retention time and fragment mass ([M]⁺) by HRGC–HRMS. An internal standard quantification of individual SCCP congeners was achieved, in which branched C₁₀-CPs and branched C₁₂-CPs were used as the extraction and reaction internal standards, respectively. A maximum factor of 1.26 of the target SCCP concentrations were determined by this method, and the relative standard deviations for quantification of total SCCPs were within 10%. This method was applied to determine the congener compositions of SCCPs in commercial chlorinated paraffins and environmental and biota samples after method validation. Low-chlorinated SCCP congeners (Cl_1–4) were found to account for 32.4%–62.4% of the total SCCPs. The present method provides an attractive perspective for further studies on the toxicological and environmental characteristics of SCCPs

Stepwise addition of DMSO.

<p><b>(A)</b> Comparison of the mortality rate of cells between stepwise and single-step addition. <b>(B)</b> Bleb index in the stepwise addition method. HeLa cells were treated with 20% DMSO for 30 minutes, and the solution was removed quickly and changed to 40% DMSO for 30 minutes. It was then changed to 60% DMSO for 30 minutes and, finally, to 80% DMSO. The inverted fluorescence microscope was used to observe dead cells labeled by PI and Hoechst. For <b>(A)</b>, the number of cells used was approximately 500 and the experiment was repeated 5 times. For <b>(B)</b>, the number of cells used was approximately 40. **p<0.01 was considered statistically significant.</p

The autophagy induced by the addition of CPAs.

<p><b>(A)</b> GFP-LC3/HeLa cells were treated with various concentrations of DMSO, and GFP green fluorescence dots appeared in cells. <b>(B)</b> LC3 conversion was determined by western blot in HeLa cells treated with different concentrations of DMSO. <b>(C)</b> Effect of DMSO on the autophagy rate. <b>(D)</b> GFP-LC3 /HeLa cells were inhibited by 3-MA, and then stimulated by 30% DMSO. Shrinkage of cell nuclei is a hallmark of apoptosis. <b>(E)</b> Autophagy reduced the apoptosis in the presence of 30% DMSO. **p<0.01 was considered statistically significant. The experiments were repeated 5 times. The number of cells used was approximately 500.</p

Effect of the concentration of CPAs: (A) number of cell blebs; (B) total area of cell blebs; (C) bleb index; (D) mortality rate of cells; (E) schematic of A_lip and A_cyto.

<p>HeLa cells were treated with a series of solutions containing different amounts of DMSO or glycerol, as well as the fluorochromes Hoechst and PI. After 30 minutes, when cells were stable, an inverted fluorescence microscope was used to observe cell death. For <b>(A)</b>, <b>(B)</b> and <b>(C)</b>, the cell number was approximately 40. For <b>(D)</b>, the cell number was approximately 500 and the experiment was repeated 5 times. For <b>(E)</b>, the red boundary denotes the lipid bilayer and the green boundary denotes the cortical cytoskeleton.</p

Cell blebs and cytoskeleton under different DMSO concentrations.

<p><b>(A)</b> The bleb and cytoskeleton were observed by an inverted fluorescence microscope (membrane: red; cytoskeleton: green). <b>(B)</b> The bleb and cytoskeleton were observed by a confocal microscope (cytoskeleton: green; nucleus: blue). <b>(C)</b> The fluid flows in the formation of blebs under a hypoosmotic condition (0.1×PBS) and a hyperosmotic condition (25% DMSO in PBS). The experiments were repeated 3 times.</p

Cell blebs induced by the addition of CPAs.

<p>Various concentrations of <b>(A)</b> DMSO and <b>(B)</b> glycerol were applied to HeLa cells for 30 minutes. The development of cell blebs during the first 3 minutes was observed as the initial state and after 30 minutes as the stable state. Initiate: 3 minutes, and Stabilized: 30 minutes. The experiments were repeated 3 times.</p

Life cycle of a dynamic bleb.

<p><b>(A)</b> the inflation and retraction of one bleb (black arrows); <b>(B)</b> the actin microfilament reorganization during the bleb inflation and retraction; <b>(C)</b> the comparison of the inflation and retraction time between DMSO and glycerol. For <b>(A)</b> and <b>(B)</b>, the experiments were repeated 3 times. For <b>(C)</b>, the number of cells used was approximately 20.</p

A Proteomic Analysis of Individual and Gender Variations in Normal Human Urine and Cerebrospinal Fluid Using iTRAQ Quantification

<div><p>Urine and cerebrospinal fluid (CSF) are two important biofluids used for disease biomarker discovery. For differential proteomic analysis, it is essential to evaluate individual and gender variations. In this study, we characterized urinary and CSF proteomes of 14 healthy volunteers with regard to individual and gender variations using 2DLC-MS/MS analysis and 8-plex iTRAQ quantification. A total of 968/512 urinary/CSF proteins were identified, with 406/280 quantified in all individuals. The median inter-individual coefficients of variation (CVs) were 0.262 and 0.183 for urinary and CSF proteomes, respectively. Cluster analysis showed that male and female urinary proteomes exhibited different patterns, though CSF proteome showed no remarkable gender differences. In comparison with CSF proteome, urinary proteome showed higher individual variation. Further analysis revealed that individual variation was not correlated with protein abundance. The minimum sample size for proteomic analysis with a 2-fold change was 10 (4/5 for males/females using iTRAQ quantification) for urinary or 8 for CSF proteome. Intracellular proteins leaked from exfoliative cells tended to have higher CVs, and extracellular proteins secreted from urinary tract or originating from plasma tended to have lower CVs. The above results might be beneficial for differential proteomic analysis and biomarker discovery.</p></div

Comparison of the inter-individual variation of urinary and CSF proteomes.

<p>A. Distribution of inter-individual variations of CSF and urine are shown in a box plot. The inter-individual variation of CSF was much lower than that of urine. B. Scatter plots of the inter-individual variation for urine and CSF proteins. The CV determined for the inter-individual variation of CSF is plotted against the inter-individual variation of urine. The spots above the 45° line show a higher CV for urine than CSF, which is true for the vast majority of the spots.</p

The time to complete each step of sample preparation.

<p>The time to complete each step of sample preparation.</p