Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation

Background The main functional property of collagen is to provide a support ing framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide eviden ce by scanning force microscopy of modifications in collagen structure caus ed by high glucose concentration, in vivo and in vitro, and to correlate th e data with markers of non-enzymatic glycation. Methods Tendon fibrils were obtained from the tails of 8-month-old rats (BB /WOR/MOL\BB) which developed diabetes spontaneously at least 12 weeks befor e they were killed, and from diabetes-resistant rats of the same strain (BB /WOR/MOL\WB). A scanning force microscope (SFM; Nanoscope III) equipped wit h a Contact Mode Head was used for imaging. Band interval, diameter and dep th of D-band gap were measured in non-diabetic and diabetic tail tendon fib rils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamin e was determined in the tendon fibrils by a colorimetric method and pentosi dine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ioni c exchange column HPLC. Results Incubated fibrils revealed modifications in radius (228 +/- 5 nm) a nd gap depth (3.65 +/- 0.10 nm) that closely reproduce diabetes-induced dam age (236 +/- 3 and 3.20 +/- 0.04 nm respectively) and were significantly di fferent from the pattern seen in non-diabetic fibrils (151 +/- 1 and 2.06 /- 0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diab etic (3.82 +/- 1.43 nmol/mg and 2.23 +/- 0.24 pmol/mg collagen respectively ) and in glucose-incubated fibrils (9.27 +/- 0.55 nmol/mg and 5.15 +/- 0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29 +/- 0.08 nmol /mg and 0.88 +/- 0.11 pmol/mg collagen respectively; p < 0.01); during the time course of incubation, an early increase in fructosamine was seen, wher eas pentosidine increased later. The D-band parameter was similar in all th ree groups, indicating that axial organization is not modified by non-enzym atic glycation. Conclusion This is the first description obtained with SFM of diabetes-indu ced ultrastructural changes in collagen fibrils. Moreover, the data present ed are consistent with the concept that chronic exposure of collagen to glu cose in vivo or in vitro leads to similar structural modifications in colla gen fibrils, probably through crosslinks. The correlation between morpholog ic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modification. Copyright (C) 2000 John Wi ley & Sons, Ltd.