Robert R. Kohn

Effects of age and diabetes mellitus on the solubility and nonenzymatic glucosylation of human skin collagen.

Collagen from human skin was fractionated into neutral salt-soluble, acid-soluble, pepsin-released, and insoluble fractions. No age-related changes were observed in the proportion of collagen extracted by neutral salt. A significant age-related decrease in the proportion of acid-soluble collagen was found. A highly significant (P less than 0.001) age-related decrease in the amount of collagen released by pepsin digestion was observed, with a concomitant age-related increase in the fraction of insoluble collagen. The amount of ketoamine-linked glucose bound to this insoluble collagen also increased significantly with age. Skin collagen from three juvenile onset diabetics (JOD) and one young maturity onset diabetic (MOD) appeared to have undergone accelerated aging. JOD and the young MOD had significantly less collagen released by pepsin digestion and significantly more insoluble collagen than would be predicted by their ages. The collagen released by pepsin digestion of the diabetic samples had more high molecular weight components than similar fractions obtained from age-matched nondiabetic controls. There was also more ketoamine-linked glucose bound to the insoluble collagen of JOD than to that fraction from comparably aged control subjects. The apparent acceleration of collagen aging in diabetes mellitus may play a role in complications of diabetes that occur in collagen-rich tissues.

Apparent Accelerated Aging of Human Collagen in Diabetes Mellitus

The chronologic ages of human subjects were determined experimentally by enzymatic digestion of tendon collagen samples. Determined age closely matched actual age for individuals dying with a variety of major diseases. Juvenile diabetics did not fit this pattern; their experimentally determined ages were significantly greater than their actual ages. This raises the possibility of relationships between diabetes mellitus, changes in connective tissue, and accelerated aging.

Effects of age and diabetes mellitus on the solubility of collagen from human skin, tracheal cartilage and dura mater.

Abstract The solubility of collagen from human skin, tracheal cartilage and dura mater was compared. Skin contained the greatest amount of soluble collagen followed by dura and cartilage. An age-related decrease in the proportion of neutral salt-soluble collagen was observed for cartilage and dura. The proportion of collagen soluble in acetic acid decreased as a function of age in skin and dura. An age-related decrease in the proportion of skin and dura collagen released by pepsin digestion was also observed, as was a concomitant increase in the proportion of insoluble collagen in these tissues. Individuals with juvenile onset diabetes had significantly less salt-soluble dura collagen, acid-soluble skin and dura collagen, pepsin-released skin and dura collagen, and significantly more insoluble skin and dura collagen than comparably aged nondiabetics. One young maturity onset diabetic had significantly less salt-soluble dura collagen, pepsin-released skin and dura collagen, and significantly more insoluble skin and dura collagen than similarly aged non-diabetics. The solubility of collagen from tissues of older maturity onset diabetics, however, did not differ significantly from other old nondiabetics. These data indicate that there is a direct effect of age on the solubility of collagen from human tissues, and that based on solubility, cartilage collagen ages faster than dura collagen, with skin undergoing the slowest relative aging. These data also support previous observations indicating that collagen from individuals with diabetes mellitus undergoes accelerated aging. Alterations in collagen may be responsible for some of the debilities of age that occur in collagen-rich tissues, and which appear at an earlier age and more severely in individuals with diabetes mellitus.

Human aging and disease

Abstract Human aging is characterized by a logarithmic increase in death rate due to a large number of diseases; by a decrease in efficiency of homeostatic mechanisms and by chemical and physical changes at tissue, cell, and molecular levels. Increasing debility and disease most likely result from decreased efficiency of homeostasis and from tissue changes responsible for the latter. The probable importance of basic age-related changes in connective tissue and the vascular system is emphasized. Reasons for using human tissue in the study of human aging and postulates useful in relating basic change to debility are presented.

Glucosylation of Human Collagen

Many of She debilities that characterize the aging syndrome can be explained by cross-linking of macromol-ecules such as collagen. And several of the characteristic complications of diabetes mellitus resemble age-like changes in collagen-rich tissues. In this laboratory, studies of human collagen that is not the site of lesions have revealed an age-related decrease in susceptibility to collagenase digestion and increased glucosylation of tendon collagen, both markedly increased in diabetes. An age-related decrease in solubility and an increase in glucosylation of skin collagen, both also markedly increased in diabetes, were also observed. Glucosylation of skin collagen was less than that of tendon, possibly because of the greater turnover of skin collagen. Collagen alterations in diabetes were most apparent in samples from younger adult subjects. Samples from older diabetics gave values within the range of nondiabetics, suggesting that the degree to which collagen can be cross-linked or glucosyiated is limited, and that limits may be attained at earlier ages in diabetes. The association between glucosylation and cross-linking of collagen suggests that glucosylation may cause cross-linking, as has been shown to occur in albumin and eye lens protein. Both the cross-linking of collagen occurring in maturation and glucosylation of protein involve the -amino group of lysine. If blood glucose is high during periods of collagen synthesis, namely during growth or fi-brosis, it is conceivable that lysine and hydroxylysine are glucosyiated rather than deaminated to form normal cross-links, and that an abnormal collagen forms that plays a role in the complications of diabetes. Control of diabetes during periods of collagen synthesis would then be a critical factor in preventing subsequent complications. Epidemiologie studies of collagen properties, bearing on questions about the roles of genetic factors and control of diabetes in alterations in collagen and in complications of diabetes, would be feasible by the use Of skin biopsies.

Determination of human chronological age by study of a collagen sample

Abstract The stabilization (‘cross-linking’) of insoluble human collagen has been shown to progress so predictably that it can form the basis of a method to determine an individuals age by study of a tissue specimen.

Abnormal collagen in cultures of fibroblasts from human beings with diabetes mellitus

Abstract Synthesis of collagen by fibroblasts from diabetic human beings was investigated to determine if abnormal synthesis might be responsible for an apparent acceleration of collagen aging observed previously in diabetics. Increased material was synthesized by cells from diabetics, and the material was abnormal in that it contained a very high molecular weight material and lacked a presumed procollagen dimer. Alpha chains did not appear altered. It is suggested that cultures of cells from diabetics yield a non-helical peptide of procollagen that contains abnormal reducible cross links and lacks normal non-reducible cross links.

Partial characterization of the age-related stabilizing factor of post-mature human collagen-I. by the use of bacterial collagenase

Abstract A stabilizing factor that causes resistance to digestion becomes increasingly important in collagen of post-mature individuals as they age. This is demonstrated with paired diaphragm tendon and the dura mater from single individuals. The factor is rapidly and irreversibly lost when purified collagen is heated to70°C. Contact with 70% formic acid quickly abolished any detectable age differences, yet strong acids and bases did not disrupt the stabilizing factor. Treatment with cyanogen bromide in 70% formic acid at 30°C failed to solubilize the collagen even when samples were first heated to 100°C in the presence of 0·01 M sodium hydroxide. Other treatments, including exposure to sodium borohydride or glycosidases, had no detectable effect on teh stabilizing factor. Increasing calcium concentration enhanced the rate of enzymatic digestion when using bacterial collagenase. Below 0·5 M calcium, age differences are readily observed with collagenase, but the differences are lost at higher concentrations. The age differences are regained when the calcium ion concentration is reduced. This rapid reversal in contrast to the irreversible loss of the age difference caused by 70% formic acid treatment or collagen denaturants. These two latter treatments may not abolish the stabilization factor but could instead modify the collagen structure to the point where it can no longer retard the rate of collagenase digestion.

Aging of collagen: comparative rates in four mammalian species.

Abstract Resistance to collagenase digestion was used to compare rates of collagen aging in rat, dog, macaque, and man. The apparent rates were different for each species. Rat collagen was uniformly and readily digested, even at low calcium ion concentration where human collagen is digested slowly. Dog, macaque and human collagens all showed evidence of a progressive, age-related, increased stabilization. Some older dog specimens showed a much greater extent of stabilization than human specimens of the same chronological age, but similar if relative lifespans are used. These results demonstrate the problems caused by uncritically extrapolating an age-related observation from a short-lived species such as rat to a long-lived species such as human.

Age-Related Alteration in Human Heart Collagen

Summary A technique for the purification of insoluble human myocardial collagen is described. The digestion rate of old collagen with collagenase is much less than that of mature collagen. These findings are similar to those in human tendon collagen. It is suggested that progressive stabilization of collagen with age may be a generalized phenomenon, and responsible, in part, for the decline of myocardial function with age. The authors gratefully acknowledge the assistance of Dr. Lester Adelson and Dr. Charles S. Hirsch of the Cuyahoga County Coroners Office for providing the heart tissue.