Aspartic acid racemization and collagen degradation markers reveal an accumulation of damage in tendon collagen that is enhanced with aging

Abstract

Little is known about the rate at which protein turnover occurs in living tendon and whether the rate differs between tendons with different physiological roles. In this study, we have quantified the racemization of aspartic acid to calculate the age of the collagenous and non-collagenous components of the high strain injury-prone superficial digital flexor tendon (SDFT) and low strain rarely injured common digital extensor tendon (CDET) in a group of horses with a wide age range. In addition, the turnover of collagen was assessed indirectly by measuring the levels of collagen degradation markers (collagenase-generated neoepitope and cross-linked telopeptide of type I collagen). The fractional increase in D-Asp was similar (p = 0.7) in the SDFT (5.87 x 10(-4)/year) and CDET (5.82 x 10(-4)/year) tissue, and D/L-Asp ratios showed a good correlation with pentosidine levels. We calculated a mean (+/-S.E.) collagen half-life of 197.53 (+/-18.23) years for the SDFT, which increased significantly with horse age (p = 0.03) and was significantly (p < 0.001) higher than that for the CDET (34.03 (+/-3.39) years). Using similar calculations, the half-life of non-collagenous protein was 2.18 (+/-0.41) years in the SDFT and was significantly (p = 0.04) lower than the value of 3.51 (+/-0.51) years for the CDET. Collagen degradation markers were higher in the CDET and suggested an accumulation of partially degraded collagen within the matrix with aging in the SDFT. We propose that increased susceptibility to injury in older individuals results from an inability to remove partially degraded collagen from the matrix leading to reduced mechanical competence.

FIGURE 1.

d/l-Asp ratio for the SDFT (♦) and CDET (×) as a function of horse age. A linear equation was found to give the best fit (n = 32, r = 0.69 and n = 32, r = 0.64 for the SDFT (solid line) and CDET (dashed line), respectively). No significant difference was found in the rate of accumulation of d-Asp with age in the SDFT compared with the CDET.

FIGURE 2.

Matrix half-life of the SDFT (♦) and CDET (×) as a function of horse age. A linear equation was found to give the best fit (n = 32, r = 0.67 and n = 32, r = 0.65 for the SDFT (solid line) and CDET (dashed line), respectively). No significant difference was found in the half-life of the matrix in the SDFT and CDET.

FIGURE 3.

Accumulation of the AGE pentosidine in the SDFT (♦) and CDET (×) as a function of horse age. A linear equation was found to give the best fit (n = 32, r = 0.85 and n = 32, r = 0.89 for the SDFT (solid line) and CDET (dashed line), respectively). Levels of pentosidine did not differ between tendons.

FIGURE 4.

Tissue fluorescence of the SDFT (♦) and CDET (×) as a function of horse age. A linear equation was found to give the best fit (n = 32, r = 0.88 and n = 32, r = 0.82 for the SDFT (solid line) and CDET (dashed line), respectively). Tissue fluorescence increased at a significantly lower rate in the CDET compared with the SDFT (p < 0.001).

FIGURE 5.

Concentration of the collagenase-generated neoepitope (C1,2C) in the SDFT (♦) and CDET (×) as a function of horse age. A linear equation gave the best fit (n = 32, r = 0.46 for the SDFT (solid line); C1,2C levels were not correlated with age in the CDET (dashed line)). C1,2C levels were significantly higher in the CDET compared with the SDFT (p = 0.007).

FIGURE 6.

A and B, concentration of ICTP extracted from the SDFT (♦) (A) and CDET (×) (B) as a function of horse age. A linear equation was found to give the best fit (n = 32, r = −0.64 and n = 32, r = −0.50 for the SDFT and CDET, respectively). The amount of ICTP extracted from the matrix was significantly greater in the CDET than in the SDFT (p < 0.001).

FIGURE 7.

Effect of temperature on predicted average collagen half-life in the SDFT. An exponential equation was found to give the best fit (r = 0.99).