Effect of collagen turnover on the accumulation of advanced glycation end products

Collagen molecules in articular cartilage have an exceptionally long lifetime, which makes them susceptible to the accumulation of advanced glycation end products (AGEs). In fact, in comparison to other collagen-rich tissues, articular cartilage contains relatively high amounts of the AGE pentosidine. To test the hypothesis that this higher AGE accumulation is primarily the result of the slow turnover of cartilage collagen, AGE levels in cartilage and skin collagen were compared with the degree of racemization of aspartic acid (% d-Asp, a measure of the residence time of a protein). AGE (N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, and pentosidine) and % d-Asp concentrations increased linearly with age in both cartilage and skin collagen (p <0.0001). The rate of increase in AGEs was greater in cartilage collagen than in skin collagen (p <0.0001). % d-Asp was also higher in cartilage collagen than in skin collagen (p <0.0001), indicating that cartilage collagen has a longer residence time in the tissue, and thus a slower turnover, than skin collagen. In both types of collagen, AGE concentrations increased linearly with % d-Asp (p <0.0005). Interestingly, the slopes of the curves of AGEs versus % d-Asp, i.e. the rates of accumulation of AGEs corrected for turnover, were identical for cartilage and skin collagen. The present study thus provides the first experimental evidence that protein turnover is a major determinant in AGE accumulation in different collagen types. From the age-related increases in % d-Asp the half-life of cartilage collagen was calculated to be 117 years and that of skin collagen 15 years, thereby providing the first reasonable estimates of the half-lives of these collagens.

Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes. Evidence against a generalized increase in oxidative stress in diabetes

The glycoxidation products Nepsilon-(carboxymethyl)lysine and pentosidine increase in skin collagen with age and at an accelerated rate in diabetes. Their age-adjusted concentrations in skin collagen are correlated with the severity of diabetic complications. To determine the relative roles of increased glycation and/or oxidation in the accelerated formation of glycoxidation products in diabetes, we measured levels of amino acid oxidation products, distinct from glycoxidative modifications of amino acids, as independent indicators of oxidative stress and damage to collagen in aging and diabetes. We show that ortho-tyrosine and methionine sulfoxide are formed in concert with Nepsilon-(carboxymethyl)lysine and pentosidine during glycoxidation of collagen in vitro, and that they also increase with age in human skin collagen. The age-adjusted levels of these oxidized amino acids in collagen was the same in diabetic and nondiabetic subjects, arguing that diabetes per se does not cause an increase in oxidative stress or damage to extracellular matrix proteins. These results provide evidence for an age-dependent increase in oxidative damage to collagen and support previous conclusions that the increase in glycoxidation products in skin collagen in diabetes can be explained by the increase in glycemia alone, without invoking a generalized, diabetes-dependent increase in oxidative stress.

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

Accumulation of Maillard reaction products in skin collagen in diabetes and aging

To investigate the contribution of glycation and oxidation reactions to the modification of insoluble collagen in aging and diabetes, Maillard reaction products were measured in skin collagen from 39 type 1 diabetic patients and 52 nondiabetic control subjects. Compounds studied included fructoselysine (FL), the initial glycation product, and the glycoxidation products, N epsilon-(carboxymethyl) lysine (CML) and pentosidine, formed during later Maillard reactions. Collagen-linked fluorescence was also studied. In nondiabetic subjects, glycation of collagen (FL content) increased only 33% between 20 and 85 yr of age. In contrast, CML, pentosidine and fluorescence increased five-fold, correlating strongly with age. In diabetic patients, collagen FL was increased threefold compared with nondiabetic subjects, correlating strongly with glycated hemoglobin but not with age. Collagen CML, pentosidine and fluorescence were increased up to twofold in diabetic compared with control patients: this could be explained by the increase in glycation alone, without invoking increased oxidative stress. There were strong correlations among CML, pentosidine and fluorescence in both groups, providing evidence for age-dependent chemical modification of collagen via the Maillard reaction, and acceleration of this process in diabetes. These results support the description of diabetes as a disease characterized by accelerated chemical aging of long-lived tissue proteins.

Decrease in skin collagen glycation with improved glycemic control in patients with insulin-dependent diabetes mellitus

Glycation, oxidation, and nonenzymatic browning of protein have all been implicated in the development of diabetic complications. The initial product of glycation of protein, fructoselysine (FL), undergoes further reactions, yielding a complex mixture of browning products, including the fluorescent lysine-arginine cross-link, pentosidine. Alternatively, FL may be cleaved oxidatively to form N(epsilon)-(carboxymethyl)lysine (CML), while glycated hydroxylysine, an amino-acid unique to collagen, may yield N(epsilon)-(carboxymethyl)hydroxylysine (CMhL). We have measured FL, pentosidine, fluorescence (excitation = 328 nm, emission = 378 nm), CML, and CMhL in insoluble skin collagen from 14 insulin-dependent diabetic patients before and after a 4-mo period of intensive therapy to improve glycemic control. Mean home blood glucose fell from 8.7 +/- 2.5 (mean +/- 1 SD) to 6.8 +/- 1.4 mM (P less than 0.005), and mean glycated hemoglobin (HbA1) from 11.6 +/- 2.3% to 8.3 +/- 1.1% (P less than 0.001). These changes were accompanied by a significant decrease in glycation of skin collagen, from 13.2 +/- 4.3 to 10.6 +/- 2.3 mmol FL/mol lysine (P less than 0.002). However, levels of browning and oxidation products (pentosidine, CML, and CMhL) and fluorescence were unchanged. These results show that the glycation of long-lived proteins can be decreased by improved glycemic control, but suggest that once cumulative damage to collagen by browning and oxidation reactions has occurred, it may not be readily reversed. Thus, in diabetic patients, institution and maintenance of good glycemic control at any time could potentially limit the extent of subsequent long-term damage to proteins by glycation and oxidation reactions.

Age-dependent accumulation of N epsilon-(carboxymethyl)lysine and N epsilon-(carboxymethyl)hydroxylysine in human skin collagen

N epsilon-(Carboxymethyl)lysine (CML) is formed on oxidative cleavage of carbohydrate adducts to lysine residues in glycated proteins in vitro [Ahmed et al. (1988) J. Biol. Chem. 263, 8816-8821; Dunn et al. (1990) Biochemistry 29, 10964-10970]. We have shown that, in human lens proteins in vivo, the concentration of fructose-lysine (FL), the Amadori adduct of glucose to lysine, is constant with age, while the concentration of the oxidation product, CML, increases significantly with age [Dunn et al. (1989) Biochemistry 28, 9464-9468]. In this work we extend our studies to the analysis of human skin collagen. The extent of glycation of insoluble skin collagen was greater than that of lens proteins (4-6 mmol of FL/mol of lysine in collagen versus 1-2 mmol of FL/mol of lysine in lens proteins), consistent with the lower concentration of glucose in lens, compared to plasma. In contrast to lens, there was a slight but significant age-dependent increase in glycation of skin collagen, 33% between ages 20 and 80. As in lens protein, CML, present at only trace levels in neonatal collagen, increased significantly with age, although the amount of CML in collagen at 80 years of age, approximately 1.5 mmol of CML/mol of lysine, was less than that found in lens protein, approximately 7 mmol of CML/mol of lysine. The concentration of N epsilon-(carboxymethyl)hydroxylysine (CMhL), the product of oxidation of glycated hydroxylysine, also increased with age in collagen, in parallel with the increase in CML, from trace levels at infancy to approximately 5 mmol of CMhL/mol of hydroxylysine at age 80.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of in vitro non-enzymatic glycosylation of human skin collagen on susceptibility to collagenase digestion

The effect of glycosylation on susceptibility of skin collagen to collagenase digestion was studied in a skin sample obtained at autopsy from the interscapular region of a 24 year old white male who had died of an acute illness and who had no history of diabetes. Homogeneous suspensions of insoluble collagen were prepared, and were incubated in 50 mmol l-1 dextrose at pH 7.35 and 37 degrees C for 7 days. Non-enzymatic glycosylation measured by the weak acid hydrolysis/thiobarbituric acid method increased from 13.1 +/- 1.0 (n = 5) to 45.2 +/- 5.5 (n = 8) nmol fructose per 10 mg collagen (P less than 0.001). Digestion of collagen using clostridial collagenase was monitored by measuring (a) hydroxyproline content and (b) absorption at 206 nm of the supernatant after centrifugation to remove substrate. The rate of digestion was similar in glycosylated and control collagen. We conclude that the ketoamine link formed in non-enzymatic glycosylation does not increase the resistance of collagen to enzymatic digestion. The possibility remains that subsequent rearrangement of this link could be important in this respect.

Non-enzymatic glycosylation of skin collagen in patients with type 1 (insulin-dependent) diabetes mellitus and limited joint mobility

Forearm skin biopsies were obtained from diabetic subjects with and without limited joint mobility, and from non-diabetic control subjects. Collagen purified from these samples was assayed for non-enzymatic glycosylation. The level in all diabetic patients was significantly greater than that in control subjects (p less than 0.001), but those diabetic patients with limited joint mobility had a level of collagen glycosylation similar to that in those with normal joints (15.3 +/- 1.3 and 16.5 +/- 1.3 nmol fructose/10 mg protein, respectively; mean +/- SEM). Glycosylation of collagen in the diabetic patients correlated with glycosylated haemoglobin measured at the time of skin biopsy (r = 0.60). These results do not support the hypothesis that non-enzymatic glycosylation of collagen, as reflected by the ketoamine link, plays an important role in the development of limited joint mobility in diabetes.

Identification of a collagen type I adhesin of Bacteroides fragilis

Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼31 and ∼34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼31 kDa and the ∼34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein. © 2014 Galvão et al.

Marine Collagen Reinforcement of Calcium Phosphate Bone Cements: A Biological Assessment

Induction of in vivo responses by implanted biomaterials is of great interest in the medical device field. Calcium phosphate bone cements (CPCs) can potentially promote natural bone remodelling and ingrowth in vivo and, as such are becoming more common place in a range of orthopaedic procedures. However, concerns remain regarding their mechanical and handling properties. Compressive modulus and fracture toughness of CPCs can be improved, without compromising injectability and setting time, through the incorporation of bovine collagen fibres1. Incorporation of marine derived collagen fibres has also yielded similar improvements2. It is hypothesised that, due to its role in bone formation and function, that incorporation of collagen in CPCs will also result in biological benefits.
The biological properties of α-TCP-CPC were largely unchanged by the incorporation of marine derived collagen. However, as a result of significant improvements to the mechanical properties, its incorporation may still result in a suitable alternative to some commercially available bone cements.