Allysine and α-Aminoadipic Acid as Markers of the Glyco-Oxidative Damage to Human Serum Albumin under Pathological Glucose Concentrations

Abstract

Understanding the molecular basis of the disease is of the utmost scientific interest as it contributes to the development of targeted strategies of prevention, diagnosis, and therapy. Protein carbonylation is a typical feature of glyco-oxidative stress and takes place in health disorders such as diabetes. Allysine as well as its oxidation product, the α-amino adipic acid (α-AA) have been found to be markers of diabetes risk whereas little is known about the chemistry involved in its formation under hyperglycemic conditions. To provide insight into this issue, human serum albumin was incubated in the presence of FeCl3 (25 μM) and increasing glucose concentrations for 32 h at 37 °C. These concentrations were selected to simulate (i) physiological fasting plasma concentration (4 mM), (ii) pathological pre-diabetes fasting plasma concentration (8 mM), and pathological diabetes fasting plasma concentration (12 mM) of glucose. While both allysine and α-AA were found to increase with increasing glucose concentrations, the carboxylic acid was only detected at pathological glucose concentrations and appeared to be a more reliable indicator of glyco-oxidative stress. The underlying chemical mechanisms of lysine glycation as well as of the depletion of tryptophan and formation of fluorescent and colored advanced glycation products are discussed.

Keywords: AGEs; Maillard reaction; allysine; diabetes; protein oxidation; α-aminoadipic acid.

Figure 1

Protein carbonylation (nmol allysine/mg protein) in HSA solution (5 mg/mL; 37 °C; 32 h) incubated with increasing concentrations of glucose. Different letters (a–c) at the same sampling time denote significant differences (p < 0.05) between treatments. Ns: no significant differences.

Figure 2

(A) Carbonylation of a protein-bound lysine residue in the presence of glucose and transition metals in accordance with the mechanisms proposed by Suyama et al. [22]. (B) Carbonylation of a protein-bound lysine residue in the presence of a hydroxyl-radical generating system as proposed by Akagawa and Suyama [23]. (C) Formation of α-AA from a protein-bound allysine residue in the presence of peroxide as proposed by Utrera and Estévez [14].

Figure 3

Glucose (mM) consumed during incubation of HSA solutions (5 mg/mL; 37 °C; 32 h) with increasing concentrations of the glucose. To calculate the glucose consumed in the reaction at each sampling point, the remaining glucose (quantified) was subtracted to the initial glucose concentrations of each experimental unit. Different letters (a–c) at the same sampling time denote significant differences (p < 0.05) between treatments.

Figure 4

α-Aminoadipic acid (α-AA) concentration (nmol α-AA/mg protein) in HSA solution (5 mg/mL; 37 °C; 32 h) incubated with increasing concentrations of glucose. Different letters (a–c) at the same sampling time denote significant differences (p < 0.05) between treatments. Ns: no significant differences.

Figure 5

Tryptophan concentration (µM) in HSA solution (5 mg/mL; 37 °C; 32 h) incubated with increasing concentrations of glucose. Different letters (a–c) at the same sampling time denote significant differences (p < 0.05) between treatments. Ns: no significant differences.

Figure 6

Advanced glycation end-product (AGE) concentration (fluorescent units) in HSA solution (5 mg/mL; 37 °C; 32 h) incubated with increasing concentrations of glucose. Ns: no significant differences between treatments at the same sampling time.

Figure 7

Yellowness (dimensionless) in HSA solution (5 mg/mL; 37 °C; 32 h) incubated with increasing concentrations of glucose. Different letters (a–c) at the same sampling time denote significant differences (p < 0.05) between treatments. Ns: no significant differences.