In the investigation of the molecular basis for diabetic complications associated with diabetes mellitus, the most studied process is the nonenzymatic glycation (NEG) of human hemoglobin (HbA). In NEG, the initially-bound glucose is ring-closed and must ring open while bound to generate an electrophilic aldehyde that proceeds through the glycation process. The bound, transient, ring-opened glucose, serving as an electrophile, can react with a nucleophilic lysine or an N-terminal valine amine residue to form a covalently-bound Schiff base. The Schiff base can proceed to an Amadori intermediate, which then is able to generate an array of advanced glycation end products (AGE). This can result in structurally-modified proteins that may induce pathology. Inorganic phosphate (P<sub>i</sub>) is known to accelerate the overall process by facilitating ring opening of glucose while bound. However, this direct Pi effect is not the only role for the Pi anion. Pi can also react with ring-closed glucose in the erythrocyte <i>prior </i>to glucose binding and subsequently generate Pi-catalyzed glucose degradation products (GDP). These GDP can also potentially bind to HbA and contribute to the glycation process. The formation of Pi-mediated GDP relating to NEG is investigated here by model reactions, spectrophotometry, and Nuclear Magnetic Resonance spectroscopy (NMR). As many as 86 GDP can be formed. Of these, 61 are electrophilic. Within the electrophiles, 18 are transient and 43 are permanent, including three permanent dicarbonyls. Glyoxal was used as a model dicarbonyl.  Dicarbonyls can theoretically crosslink proteins and thus constitute an additional path towards pathology.  In reactions with glucose (followed by NMR), Pi induces time-dependent generation of GDP. Among the GDP formed, glyoxal was generated.  Incubation of glyoxal with bovine serum albumin (BSA) followed by spectrophotometric detection indicates that protein crosslinking does takes place. Thus in the assessment of the mechanisms for NEG, Pi-catalyzed GDP must be considered.

Towards an understanding of nonenzymatic glycation #3: The formation of phosphate-catalyzed glucose degradation products

251st National Meeting (2016)

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