Correlation of day7 changes in plasma and urinary levels of urea with changes in fat-free mass after bedrest.

<p>Plot of the after-bedrest final change in fat-free mass over the day7 change in plasma urea (R = 0.566, P = 0.009) and in urinary urea (R = 0.715, P<0.001). The correlation coefficient for day7 change in plasma and urinary urea were significant also after exclusion of the case with fat-free mass change <4 kg (R = 0.554 and 0.640, P<0.01) and with control for baseline values of the variables (partial R = 0.608 and 0.684, P<0.01).</p

Dietary data, anthropometrics, and erythrocyte-related indices during bedrest.

<p>On the left, changes over baseline in anthropometrical indices and dietary data in the insert. On the right: changes over baseline in erythrocyte-related indices and estimated changes in plasma volume in the insert. Data are shown as mean±SEM. Differences versus day0 were statistically significant at day7 only for fat mass (P = 0.009), at all time-points from day7 to day35 for weight, fat-free mass, muscle mass, hemoglobin, and hematocrit (P≤0.022).</p

Descriptive statistics at baseline and at bedrest termination (day0 and day35).

<p>Descriptive statistics at baseline and at bedrest termination (day0 and day35).</p

Urea and creatinine during bedrest.

<p>Mean±SEM pre-bedrest and during bedrest of plasma and urinary levels of urea and creatinine. Stars above the x-axis lines indicate time-points which were statistically different versus day0 (P<0.05).</p

Time-dependent, increased expression of ADAM17, MCP1 and Hsp60 in endothelial cells upon homocysteinylated albumin treatment.

<p>Panel A: Real time PCR evaluation during time course of <i>ADAM17</i>, <i>MCP1</i> and <i>Hsp60</i> mRNA. Panel B: ELISA assay of MCP1 released in the culture medium of treated cells. Panel C: Western blotting analysis of intracellular levels of ADAM17, and Hsp60, and analysis of Hsp60 released in the medium by immunoprecipitation and western blotting (Hsp60 IP). A: unmodified albumin control; AH: homocysteinylated albumin treatment. Levels of transcripts or proteins in the homocysteinylated albumin sample group were significantly increased compared to control (p<0.001).</p

ICAM1 expression in endothelial cells treated with N-homocysteinylated albumin.

<p>Panel A: expression levels of ICAM1 transcripts quantitated by real time PCR (treated: 1 µmol/L homocysteinylated albumin; control: unmodified albumin); (p<0.001). Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin; (p<0.001).</p

Characterization of homocysteinylated albumin by mass spectrometry.

<p>Panel A: ESI-MS of human serum albumin. Panel B: ESI-MS of homocysteinylated albumin. Inset: magnification on expanded scale of the signal at Da = 67805. The family of molecular ions is compatible with the structures shown in the panel.</p

Validation by Q-PCR of transcriptome results relevant to upregulated gene involved in endothelial dysfunction.

<p>A: unmodified albumin; AH: homocysteinylated albumin. Gene expression in the AH sample group was significantly increased with respect to the corresponding genes in the A sample group (p<0.001).</p

Effects of homocysteinylated albumin on monocyte adhesion.

<p>U937 monocytoid cells adhesion onto an endothelial monolayer (EAhy926) expressed as adherent cells (number/field; panel A) and percentage adherent cells compared to positive control (panel B). Counts are the mean of ten independent experiments, each carried out by counting five different fields/sample of triplicate samples. Examples of microscopic fields are shown on the right. C: negative control (untreated cells); A: unmodified albumin; AH: homocysteinylated albumin; AC: carboxymethylated albumin; T: positive control (Tnf-α). 0.3 or 1: homocysteine micromolar concentration present in the assay in the form of <i>N</i>-homocysteinyl groups bound to albumin, as comparable to the in vivo situation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031388#pone.0031388-Perna1" target="_blank">[14]</a>; p<0.0001.</p

Upregulated genes from transcriptional analysis of EAhy926 cells treated with homocysteinylated albumin compared to control.

<p>Homocysteinylated albumin concentration was comparable to that detected <i>in vivo</i> in hyperhomocysteinemic uremic patients compared to control.</p