Novel biomarkers for diabetic patients at risk of developing secondary complications.

Author: Orban, Thomas

Date: 2015

Publisher: University of Canterbury. School of Biological Sciences

Type: Thesis, Scholarly text

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Diabetes is a chronic, incurable disease characterized by chronic hyperglycemia. Patients who control their diabetes poorly are at risk of developing long-term complications, such as cardiovascular diseases, renal failure and retina damage. Glycated hemoglobin (HbA1c), formed by the reaction of glucose with hemoglobin, is considered the gold standard for the monitoring of glycemic control, but it does not discriminate between the different types of secondary complications associated with diabetes. Diabetes and its complications are linked to oxidative stress and the resulting lipid peroxidation produce reactive aldehydes including acrolein. Acrolein forms adducts with nucleophilic residues of proteins, which can be used as biomarkers of oxidative stress. It is hypothesized in the present study that specific adducts of acrolein with hemoglobin could be used as reliable biomarkers for secondary complications linked with diabetes. In this context, reactions of acrolein with the two nucleophilic amino acids cysteine and lysine have been studied. The formation of adducts was confirmed for both of these amino acids, and lead to the discovery of the novel compound FTT-cysteine. Studies on hemoglobin itself were conducted in order to understand how acrolein influences the structure and function of this protein and where the preferential sites of modification were located. It was concluded that acrolein does not significantly alter the structure of hemoglobin and actually tends to stabilize it due to the formation of internal cross-links. MS studies revealed that both the single addition of acrolein and the formation of the aromatic compound MP-lysine, arising from reaction with two molecules of acrolein, occur in hemoglobin samples. ELISA tests were developed that could successfully determine the presence of naturally forming hemoglobinacrolein adducts in vivo. X-ray crystallography could show the presence of extra electron density in the vicinity of Cys 93 on the β subunit. A proteomics search determined the sites of preferential reaction on hemoglobin, one of which is Cys 93. These results paved the way for the design of two novel synthetic hexapeptides, Ser-Ala-Gln-Val-Lys-Gly and Leu-His-Cys-Asp-Lys-Leu,whose reactivity towards acrolein was then studied. Both peptides were successfully modified by reaction with acrolein: MS studies revealed the formation of MP-lysine for Ser-Ala-Gln-Val-Lys-Gly and of a single cysteine Michael adduct for Leu-His-Cys-Asp-Lys-Leu. Future work will include the synthesis of 16-residue peptide chains as determined by the results from the proteomics search, against which antibodies will be raised. The antibodies will be used to develop highly specific ELISA assays that will hopefully correlate with secondary complications of diabetes. The outcome of this research could lead to the development of a convenient product that could be used to evaluate the risks that diabetic patients have of developing specific secondary complications.

Copyright: Copyright Thomas Orban