The relationship between the extractability of collagen by enzymatic digestion and the degree of nonenzymatic glycation of collagen was examined in the aorta and skin from 38 subjects without diabetes mellitus (mean age: 62.3 +/- 20.2 years). Samples were obtained from the aortic media (M), lesion-free intima (I), atherosclerotic intima (A) and dermis of the skin (S). Collagen was extracted first by incubation with 1/50 (enzyme/substrate weight ratio) pepsin at 4 degrees C for 24 h (P-fraction) and then by incubation with 1/10 (enzyme/substrate weight ratio) pepsin at room temperature for 24 h (EP-fraction). The pepsin-insoluble precipitates were digested by incubation with 270 units of bacterial collagenase at 37 degrees C for 24 h (PIS-fraction). Collagen contents, ketoamines and collagen-linked fluorescence (CLF) were measured in each fraction. The amount of ketoamines and the level of CLF correlated inversely with the susceptibility of collagen to pepsin digestion in various tissues, including M, I, A and S. These values were highest in both the P- and EP-fractions of M, which contained the least amount of collagen extracted by pepsin digestion. In contrast, they were lowest in S, where the concentration of collagen extracted by pepsin digestion was greatest among all of the tissue samples. Atherosclerotic intima (A) and aortic media (M) showed an age-related increase in the total amount of collagen digested with pepsin and collagenase, which depended mainly on an increase in the content of pepsin-insoluble collagen. Although the total amount of collagen did not increase with advancing age in I or S, collagen in I and S became progressingly resistant to pepsin digestion. These results suggest that the age-related decrease in the susceptibility of collagen to pepsin digestion may be due to nonenzymatic glycation in atherosclerotic lesions as well as normal tissues, including the aortic media, lesion-free intima and skin. The level of CLF significantly increased with age in the P-fraction and/or EP fraction of M, I and S. However, there was no relationship between the level of CLF and the subject's age in A. Thus, the accumulation of advanced glycation endproducts (AGEs) on collagen fibers may be partially responsible for the increase in collagen matrix in atherosclerotic lesions of subjects without diabetes mellitus.