RBCs protect plasma proteins against CSE-induced carbonylation.

<p>Human plasma diluted 1∶10 (i.e., at the protein concentration of 4 mg/ml) with PBS/NaCl was incubated with 1–16% (v/v) CSE in the absence (filled circles) or presence of 2.5% (v/v; open circles) or 5% (v/v; open squares) erythrocytes. After a 60-min incubation at 37°C, samples were centrifuged and protein solutions were recovered from the supernatants. The extent of CSE-induced carbonylation of plasma proteins was then evaluated by spectrophotometric determination after protein carbonyl derivatization with DNPH, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. Data are presented as the mean ± SD of three independent determinations.</p

Effect of GSH, Cys-SH, or NAC on CSE-induced Cys34 oxidation.

<p>(<b>A</b>) HSA-SH solutions (60 µM) were incubated with vehicle (control; filled circles) or 0.3 µM (triangles), 3 µM (squares) or 30 µM (open circles) GSH (molar ratio [GSH]/[HSA] = 0.005–0.05–0.5, respectively), for 30 min, before exposing protein solutions to 0%, 1%, 4% and 16% (v/v) CSE, for 60 min. (<b>B</b>) HSA-SH solutions (60 µM) were incubated with vehicle (control; filled circles) or 1 µM (triangles), 10 µM (squares) or 100 µM (open circles) Cys-SH (molar ratio [Cys-SH]/[HSA] = 0.016–0.16–1.6, respectively), for 30 min, before exposing protein solutions to 0%, 1%, 4% and 16% (v/v) CSE, for 60 min. (<b>C</b>) HSA-SH solutions (60 µM) were incubated with vehicle (control; filled circles) or 1 µM (triangles), 10 µM (squares) or 100 µM (open circles) NAC (molar ratio [NAC]/[HSA] = 0.016–0.16–1.6, respectively), for 30 min, before exposing protein solutions to 0%, 1%, 4% and 16% (v/v) CSE, for 60 min. (<b>D</b>) Experiment was performed as in (<b>A</b>) except that HSA solutions were exposed to CSE immediately after GSH addition. (<b>E</b>) Experiment was performed as in (<b>B</b>) except that HSA solutions were exposed to CSE immediately after Cys-SH addition. (<b>F</b>) Experiment was performed as in (<b>C</b>) except that HSA solutions were exposed to CSE immediately after NAC addition. Conditions of incubation and estimation of HSA Cys34 free sulfhydryl group by biotin–HPDP binding and Western blot analysis are described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. Densitometric analyses of biotin-HPDP incorporation are shown. Data are presented as the mean ± SD of three independent determinations.</p

Prevention of CSE-induced oxidation of HSA Cys34 sulfhydryl group by means of supra-physiological concentrations of GSH, Cys-SH, or NAC.

<p>HSA-SH solutions (60 µM) were incubated for 30 min with different concentrations of (<b>A</b>) GSH (squares), (<b>B</b>) Cys-SH (triangles) or NAC (open circles) so as to obtain molar ratios of each aminothiol to HSA-SH equal to 25∶1, 50∶1, 100∶1, 200∶1, 400∶1, 800∶1 compared with the physiological plasma ratio, before exposing protein solutions to 16% (v/v) CSE, for 60 min. Results are shown as concentration of the Cys34 sulfhydryl group. Estimation of HSA Cys34 free sulfhydryl group by biotin–HPDP binding and Western blot analysis are described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. Densitometric analysis of biotin-HPDP incorporation is shown. Data are presented as the mean ± SD of three independent determinations.</p

Effect of CSE on HSA Cys34 free sulfhydryl group as determined by the Ellman assay.

<p>HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% (v/v) CSE and then exhaustively dialyzed. The concentration of Cys34 sulfhydryl groups in HSA samples was determined by the Ellman assay at 412 nm as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. Data are presented as the mean ± SD of three independent measurements. Inset: Effect of CSE on HSA Cys34 free sulfhydryl group as determined by biotin-HPDP binding and Western blot analysis. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% CSE, exhaustively dialyzed and then labeled at Cys34 with biotin-HPDP as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. Proteins (10 µg/lane) were separated by SDS-PAGE and biotin-HPDP binding was detected by Western blot analysis using streptavidin-HRP as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a> (immunoblot inset). Amido Black staining of the same PVDF membrane showed equal protein loading and transfer (not shown). Immunoblot shown is representative of three independent determinations. Bar-graph inset shows densitometric analysis of biotin-HPDP incorporation. Data are presented as the mean ± SD of three independent determinations.</p

Estimation of a possible non-specific biotin-HPDP binding to human plasma proteins.

<p>All the experiments were performed with control human plasma proteins, i.e. not exposed to CSE, in the absence of RBCs. (<b>A</b>) Human plasma was diluted at the protein concentration of 4 mg/ml with PBS/NaCl and the extent of free sulfhydryl groups was evaluated by biotin-HPDP, varying the time of incubation with biotin-HPDP between 5 and 60 min. All the other experimental conditions for the biotinylation reaction, non-reducing SDS-PAGE, Western blot analysis with streptavidin-HRP and enhanced chemiluminescence were the same as those described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. Molecular weight (MW) markers are indicated on the left. (<b>B</b>) Human plasma was diluted at the protein concentration of 4 mg/ml with PBS/NaCl and the extent of free sulfhydryl groups was evaluated by biotin-HPDP, varying its concentration between 10 and 200 µM. All the other experimental conditions for the biotinylation reaction, non-reducing SDS-PAGE, Western blot analysis with streptavidin-HRP and enhanced chemiluminescence were the same as those described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. (<b>C</b>) Human plasma was diluted at the protein concentration of 4 mg/ml with PBS/NaCl. Plasma proteins were incubated for 30 min at 50°C with 20 mM NEM, precipitated with 100% acetone and protein pellets were resuspended with PBS/NaCl containing 0.4 mM biotin–HPDP (for further details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and Methods</a>) (lanes 1); plasma proteins were incubated with 0.4 mM biotin instead of biotin-HPDP (the biotinylation reaction was performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and Methods</a>) (lanes 2); plasma proteins were incubated for 30 min at 50°C with 20 mM NEM before starting the biotinylation reaction with 0.4 mM biotin–HPDP (for further details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and Methods</a>) (lanes 3); plasma proteins were incubated for 30 min at 50°C with 20 mM NEM but were not subject to the biotinylation reaction (lanes 4); plasma proteins were not subject to the biotinylation reaction (lanes 5). In panels A and B, each electrophoretic lane was loaded with one-half of the amount of total proteins loaded in panel C and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#pone-0029930-g006" target="_blank">Fig. 6A</a>. After non-reducing SDS-PAGE, the Western blot was developed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and Methods</a>.</p

Reversibility of Cys34 oxidation as determined by biotin–HPDP binding and slot blot analysis.

<p>(<b>A</b>) HSA samples (60 µM) were treated with CSE and then incubated for 15 min with different concentrations of DTT as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. The biotinylation reaction was carried out as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. A series of control and CSE-treated HSA samples was added with 20 mM NEM and incubated for 30 min at 50°C before DTT addition (strips marked with NEM). In another series of control and CSE-treated HSA samples, reduction of HSA sulfhydryl modifications was obtained through a prolonged dialysis against 50 mM PBS, pH 7.4, added with 5 mM GSH (strips marked with GSH). After protein precipitation and resuspension in TBS, diluted protein solutions (3 µg total protein) were applied to each slot and the membrane was probed with HRP-conjugated streptavidin and developed by using enhanced chemiluminescence (strips a, c, e, g, i) as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. Strips marked with b, d, f, h, l show the corresponding duplicate slot-blot stained for proteins with Amido black. (<b>B</b>) Graph shows densitometric analysis of biotin-HPDP incorporation in HSA samples treated with CSE without further DTT, NEM or dialysis against GSH (filled circles) and in HSA samples treated with CSE and then dialyzed against GSH (open circles). Data are presented as the mean ± SD of three independent determinations.</p

RBCs protect plasma protein sulfhydryl groups against CSE oxidizing effects.

<p>Human plasma diluted 1∶10 (i.e., at the protein concentration of 4 mg/ml) with PBS/NaCl was incubated with 1–16% (v/v) CSE in the absence (filled circles) or presence of 2.5% (v/v; open circles) or 5% (v/v; open squares) erythrocytes. After a 60-min incubation at 37°C, samples were centrifuged and protein solutions were recovered from the supernatants. (<b>A</b>) The extent of CSE-induced oxidation of plasma protein free sulfhydryl groups was then evaluated by biotin-HPDP, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>, followed by Western blot analysis with streptavidin-HRP and enhanced chemiluminescence, after protein separation by non-reducing SDS-PAGE. Arrow indicates the HSA protein band. Molecular weight (MW) markers are indicated on the left. (<b>B</b>) Graph shows densitometric analysis of the HSA protein band, corresponding to biotin-HPDP incorporation in HSA Cys34.</p

RBCs protect the Cys34 sulfhydryl group of HSA against CSE oxidizing effects.

<p>HSA-SH solutions (60 µM) were exposed to 1–16% (v/v) CSE in the absence (filled circles) or presence of 2.5% (v/v; open circles) or 5% (v/v; open squares) erythrocytes in 200 mM PBS/NaCl, containing 5 mM glucose. After a 60-min incubation at 37°C, samples were centrifuged and HSA solutions were recovered from the supernatants. (<b>A</b>) The extent of CSE-induced oxidation of HSA Cys34 free sulfhydryl group was then evaluated by biotin-HPDP, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>, followed by Western blot analysis with streptavidin-HRP and enhanced chemiluminescence, after protein separation by non-reducing SDS-PAGE. (<b>B</b>) Graph shows densitometric analysis of the HSA protein band, corresponding to biotin-HPDP incorporation. Data are presented as the mean ± SD of three independent determinations.</p

SDS-PAGE gel (10% w/v) representing human (lane 1) and swallow (lanes 2–4) plasma protein migration pattern after Coomassie Brilliant Blue staining.

<p>The black arrow indicates serum albumin. Molecular Weight Standards (M.W.) migration is shown on the left.</p

Linear models of oxidative damage biomarkers in relation to sex, breeding stage and sampling date.

<p>Where appropriate, models were run while controlling for heterogeneity of variances between levels of the fixed factors (see Materials and Methods for details). Terms shown in italics were removed from the model (see Materials and Methods for details).</p>a<p>: estimated mean values (s.e.): males, before laying = 1.81 (0.33), after laying = 0.74 (0.11); females, before laying = 3.62 (0.30), after laying = 0.81 (0.10).</p>b<p>: estimated slopes (s.e.): before laying =  −0.06 (0.02), <i>t</i> =  −2.71, <i>P</i> = 0.009; after laying = 0.01 (0.01), <i>t</i> = 0.99, <i>P</i> = 0.33.</p>c<p>: estimated mean values (s.e.): males = 1.43 (0.04); females = 1.77 (0.06).</p>d<p>: estimated mean values (s.e.): before laying = 1.51 (0.05); after laying = 1.69 (0.05).</p