Date of Award
Chemistry and Biochemistry
Thibert, Roger Joseph,
Health Sciences, Pathology.
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The carbamylation reaction in vivo, involves the nonenzymatic, covalent attachment of isocyanic acid, the spontaneous dissociation product of urea, to proteins. Carbamylated proteins have been proposed as markers of uremia and indicators of uremic control. An enzyme-linked immunosorbant assay for carbamylated albumin, the major serum protein, was developed. Furthermore, an investigation was carried out to determine the relationship between carbamylated hemoglobin (CHb) and carbamylated total protein (CTP), and also their association with pre-dialysis urea and dialysis dose in hemodialyzed patients. Polyclonal antibodies were made against in vitro prepared heavily carbamylated albumin in rabbits. The antisera were purified using protein A affinity columns and specific affinity columns (albumin, carbamylated albumin, and carbamylated hemoglobin). The albumin-affinity purified polyclonal antibody was found to be specific for carbamylated albumin, and did not react with albumin, carbamylated hemoglobin, carbamylated fibrinogen, homocitrulline or carbamylaspartate. Using in vitro carbamylated albumin as the standard, the competitive assay had a detection limit of 25 pmol of carbamyl groups and had a detection range of about 3 orders of magnitude. The sandwich assay, however, had a greater sensitivity (1 pmol of carbamyl groups), and a wider linear range (at least 5 orders of magnitude). Despite the sensitivity of these assays, they could not be used for the determination of minor degrees of carbamylation, as occurs in vivo. A six-month longitudinal study of seven hemodialyzed patients showed that correlations of CHb and CTP concentrations with currently used uremic indices were not significant. These data suggest that measurement of CHb or CTP may not be meaningful for hemodialyzed patients on maintenance dialysis. Hemodialyzed patients were found to have significantly higher CHb $(157\pm40$ $\mu$g valine hydantoin/g Hb) and CTP $(0.117\pm0.011$ A/mg protein) concentrations as compared to normal individuals $(53\pm20$ $\mu$g valine hydantoin/g Hb and $0.08\pm0.01$ A/mg protein, respectively). A high correlation was found between CHb and CTP concentrations $(r=0.87,\ p<0.0001),$ demonstrating a strong relationship between these two different half-lived proteins. This study shows that the carbamylated proteins, CHb and CTP, are positively associated and reflect the degree of urea exposure in blood.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1996 .B33. Source: Dissertation Abstracts International, Volume: 57-07, Section: B, page: 4313. Adviser: Roger Joseph Thibert. Thesis (Ph.D.)--University of Windsor (Canada), 1996.
Balion, Cynthia Marie., "Development and evaluation of methods for the determination of carbamylated proteins in hemodialyzed patients." (1996). Electronic Theses and Dissertations. 1695.