Non-enzymic modification of tissue proteins by reducing sugars, the so-called Maillard reaction, is a prominent feature of aging. In articular cartilage, relatively high levels of the advanced glycation end product (AGE) pentosidine accumulate with age. Higher pentosidine levels have been associated with a stiffer collagen network in cartilage. However, even in cartilage, pentosidine levels themselves represent 1 cross-link per 20 collagen molecules, and as such cannot be expected to contribute substantially to the increase in collagen network stiffness. In the present study, we investigated a broad range of Maillard reaction products in cartilage collagen in order to determine whether pentosidine serves as an adequate marker for AGE levels. Not only did the well-characterized AGEs pentosidine, N ε -(carboxymethyl)lysine, and N ε -(carboxyethyl)lysine increase with age in cartilage collagen (all P 0.0001), but also general measures of AGE cross-linking, such as browning and fluorescence (both P 0.0001), increased. The levels of these AGEs are all higher in cartilage collagen than in skin collagen. INTRODUCTIONDuring aging, long-lived proteins such as collagen and eye lens proteins are non-enzymically modified by reducing sugars. The major initial product is fructose-lysine (FL) [1,2], which results from glycation of ε-amino groups on lysine residues. In subsequent Maillard or browning reactions, products known as advanced glycation end products (AGEs) are formed from FL [3,4], and accumulate with age in long-lived proteins [1,2,[5][6][7][8]. These AGEs include structurally characterized adducts, such as N ε -(carboxymethyl)lysine (CML) [1][2][3] and N ε -(carboxyethyl)-lysine (CEL) [9], fluorescent cross-links, such as pentosidine formed between lysine and arginine residues [5,10], as well as chemically unidentified compounds which result in proteinbound browning or fluorescence, and cross-linking [11][12][13][14].In comparison with other collagen-rich tissues such as skin, cartilage contains relatively large amounts of pentosidine [5,15]. Pentosidine levels in articular cartilage increase linearly with age [6-8], as was previously described for skin collagen [16] and lens proteins [10]. Pentosidine is also present in the proteoglycans in articular cartilage [17], but appears to be localized predominantly in the collagen component of the tissue [7]. Age-related accumulation of AGE cross-links in articular cartilage collagen may result in increased stiffening of the collagen network, as was shown after in itro ribosylation of cartilage [7]. Thus AGE cross-linking in i o may contribute to the age-related impairment of the ability of articular collagen to resist mechanically induced damage and eventually cartilage degeneration [18][19][20].Abbreviations used : AGE, advanced glycation end product ; CEL, N ε -(carboxyethyl)lysine ; CML, N ε -(carboxymethyl)lysine ; FL, fructose-lysine. 1 To whom correspondence should be addressed (e-mail JM.TeKoppele!pg.tno.nl).As a functional measure of glycation the digestibility o...