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Background The main functional property of collagen is to provide a supporting 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 evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the 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 before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL\WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC. Results Incubated fibrils revealed modifications in radius (228±5 nm) and gap depth (3.65±0.10 nm) that closely reproduce diabetes-induced damage (236±3 and 3.20±0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151±1 and 2.06±0.03 nm; p

This study investigated age-related changes in collagen solubility and collagen-linked fluorescence, and their relationship with the Maillard reaction. As a result of the collagen purification of rat lung samples, we obtained two pools of collagen with different degrees of solubility. The relative distribution of collagen between these two fractions was time-dependent, and the proportion of the smaller and less soluble fraction increased with time (r = 0.73, P < 0.0001). In this fraction, the intensity of fluorescence at Exc 335 nm/Em 385 nm, and the total amount of pentosidine increased with age ( r = 0.66, P < 0.002, and r = 0.69, P < 0.01, respectively). The mean values for fluorescence and pentosidine per milligram of collagen were, respectively, six and ten times greater in the less soluble fraction. In this fraction the pentosidine per milligram of collagen increased with age (r = 0.59, P < 0.03). Our results demonstrated the presence of pentosidine in rat lung collagen. Moreover, its accumulation in the less soluble fraction suggested a relationship between Maillard reaction products, physico-chemical changes in collagen solubility, and the ageing process in rat lungs.

Nonenzymatic glycation produces compounds with characteristic fluorescence in long-lived proteins. We recently described the influence of age in rat collagen-linked fluorescence. To examine the effect of age in humans, we studied the subcutaneous collagen-linked fluorescence in samples from 26 subjects of both sexes (age range, 42 to 78 years) who were undergoing vascular surgery. Intensity of fluorescence at 385 nm (upon excitation at 335 nm) and 440 nm (upon excitation at 370 nm) increased exponentially with age (r = .827, y = 114 + e0.038x, P less than .001; and r = .905, y = 36 + e0.039x, P less than 0.001, respectively). The two sets of data exhibited a high degree of correlation (r = .980, P less than .001, n = 26). Age-adjusted fluorescence data did not correlate with sex, body weight, or type of vascular pathology. The collagen fluorescence accumulation rate was 3.7% per year, and the characteristic time (CT) was 26 to 27 years. We conclude that the fluorescence measurement is a reliable methodology that can be used as a marker for biological age until new, more-specific tools are available.