The effect of non-enzymatic glycation of keratins on the physical properties of plantar epidermis in Type II diabetic and non-diabetic individuals
As keratin is the principle structure of the plantar stratum corneum, it has been postulated that changes in this protein, associated with non-enzymatic glycation, may contribute to abnormalities in the development of plantar callus in diabetes consequently leading to ulceration of plantar skin. The clinical appearance of the skin in the diabetic state has been described as being thicker and less flexible than non-diabetic skin, particularly in the hands. A group of six Type II diabetic patients and six age and sex matched controls were initially selected for the verification of the methods of analysis to be used on a larger sample group. Epidermal keratin extraction protocols were tested and their validity confirmed using immunoblot analyses. The early glycation product, furosine and the advanced glycation end-products pentosidine and carboxymethyllysine were the glycation products chosen for quantification in plantar callus tissue. The quantification of furosine and pentosidine were successfully achieved using high performance liquid chromatography assays. The qualitative detection of CML was achieved using gas chromatography/mass spectrometry (GC/MS). In response to the lack of sensitivity of the GC/MS detection method for CML, polyclonal and monoclonal antibodies were developed. Screening of these antibodies identified a lack of specificity for CML detection and quantification. In addition to the quantification of the glycation compounds in plantar epidermal tissues; the flexibility and thickness of the skin on three different sites on the feet were measured. The epidermal thickness was measured using high frequency ultrasound imaging and the mechanical properties of the epidermis were tested using vertical negative pressure methods. The selected group of Type II diabetic subjects (n=103), in the final clinical study, showed a statistically significant increase in levels of pentosidine in plantar callus specimens compared with the control group (n=87). The quantity of furosine and pentosidine measured in the callus samples did not conrelate with those measured in blood serum. The HbA1c levels showed no association between glycation of plantar epidermal proteins and glycaemic control. The echogeneic images captured by ultrasonography were thickest on the plantar metatarsal sites, followed by the medial longitudinal arch and finally the dorsal skin sites. The epidermal thickness in the PMA region was greater in diabetics in comparison to controls and an association with neuropathy was suggested. Pedal skin exhibited viscoelastic properties. The magnitude of the elastic component, measured immediately after the negative displacing pressure was removed, was significantly greater in diabetic skin than controls on all three sites on the foot. Plantar diabetic skin was significantly less plastic than non-diabetic plantar skin.