PMC

Correlation between %C-Alb and blood urea concentrations. (A) Correlation between blood urea and %C-Alb in ArMORR study subjects with ESRD (n = 187). (B) Correlation between blood urea and %C-Alb in 4D study subjects with ESRD (n = 1,161). (C) Correlation between %C-Alb and blood urea in non-hemodialysis CKD subjects (n = 122). Pearson correlation coefficients (r) and P-values are shown.

Kaplan-Meier curve estimates of the incidence of all-cause mortality in ESRD patients. Subjects were categorized into upper, middle, and lower tertiles according to serum %C-Alb values measured at the outset of the study. (A) 12 month survival rates in ArMORR study subjects. (B) 12 month survival rates in 4D study subjects. Numbers of subjects at risk at different time points during each study shown in the tables at bottom.

Average carbamylated albumin values in uremic and non-uremic patients. (A) Average %C-Alb values in non-uremic subjects (n = 20) and in patients with stage 3 or 4 chronic kidney disease (CKD) (n = 122) and ArMORR HD subjects (n = 187). (B) Average %C-Alb in ArMORR survivors who lived longer than 12 months (n = 106) and in ArMORR cases who died during the 12-month study period (n = 81) . Individual %C-Alb values for each group are shown in . Data are expressed as average carbamylated albumin as a % of total; error bars, 95% confidence intervals of the mean; Student’s t-test P-values shown.

Effects in mice of low protein diet on albumin carbamylation by urea or cyanate. (A) Serum amino acid concentrations after 15 days of low- or normal-protein diets. Values are normalized to average concentrations of amino acid in animals on a normal protein diet. *P < 0.05 using unpaired T test, comparing average amino acids in animals on low and normal protein diets. (B) %C-Alb values in low- or normal-protein fed animals before and 30 minutes after cyanate injection (100 mg/kg body weight). (C) %C-Alb values in animals fed low or normal protein diets supplemented with or without urea (67 mg per gram of feed). Bars indicate mean ± SD, n = 6 animals per group. P-values were calculated using unpaired T test.

Identification of carbamylation on Lys-549 of cyanate-treated albumin. Cyanate-treated and untreated purified human albumin from a commercial source was digested with glutamyl endoproteinase and analyzed by LC-MS/MS. Peptides were identified by matching fragmentation spectra to sequences from the human SwissProt proteome with the AB Sciex Protein Pilot software programmed to search for both carbamylated and non-carbamylated peptide forms. (A) Summary reports for the carbamylated and non-carbamylated forms of the peptide encompassing lysine residue 549 (sequence RQIKXQTALVE where X = N-ε-carbamoyl-L-lysine). Contrib, contribution of identified peptide (in ProtScore units) to protein identification; Conf, percent confidence of peptide identification; z, peptide charge; Spectrum, within-run identifier for MS/MS spectra used for peptide identification. (B) MS/MS spectra for carbamylated and non-carbamlyated digested albumin peptides encompassing Lys-549; the modification state-revealing fragment ions are shown in red. (C) Predicted ion fragments of carbamylated and non-carbamylated peptide forms; detected fragment ions matching the predicted fragmentation spectra are highlighted in red. Both peptides were identified with 99+% confidence using the Paragon algorithm, which compares the confidence of the match between the observed spectra and the identified peptide sequence to the combined confidence for all other possible peptide matches.().