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Aging of collagen: Comparative rates in four mammalian species
Exp. Geront. Vol.15,pp. 393-398. PergamonPressLtd. 1980.Printedin GreatBritain.
AGING OF COLLAGEN:
COMPARATIVE
FOUR MAMMALIAN
RATES IN
SPECIES
CLIVE R. HAMLIN, JEANNE H . LUSCHIN a n d ROBERT R . KOHN Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, U.S.A. (Received 12 October 1979; in revised f o r m 14 December 1979)
INTRODUCTION COLLAGEN comprises about 30°10 of total body protein and undergoes very marked changes in properties during aging. These changes suggest a progressive stabilization and stiffening through intermolecular cross linking, but few specific details are known about the chemistry of the changes with age. It has been argued that such aging of extracellular macromolecules is capable of explaining many debilities characterizing the aging syndrome. Aging of collagen and its possible significance have been reviewed (Kohn, 1978). It would be useful to know if aging of collagen is accelerated in species that have short lifespans and are presumably aging faster than species with longer lifespans. This question has received little attention despite the frequent use of short-lived species in studies ultimately aimed at understanding human aging. One study showed only small and variable differences in the amount of insoluble collagen that could be solubilized by heat and urea between a 2-yr-old dog, rat, cat and human being (Deyl et al., 1971). The significance of this observation is not clear because no distinction was made between maturation and aging (Vogel, 1978); the human being would be growing rapidly at that age and synthesizing large amounts of young, soluble collagen, while the other species would have stopped growing at different ages and would have undergone aging for different fractions of the 2-year period. Furthermore, the procedure used actually showed a perplexing decrease of insoluble collagen between 2- and 80-yr-old tendons in human beings. Another study has indicated that stabilization of aldimine bonds in collagen does not differ significantly in 6- and 30-month-old rats, cats and golden hamsters (Vancikovfi and Deyl, 1973). In a third study the force of thermal contraction of collagen fibers has been shown to increase with age, and similar forces were developed in fibers from a 3-yr-old rat and a 3-yr-old cat (Verz~ir, 1968). One other study could neither prove nor disprove the hypothesis that changes in tail tendon characteristics with age are an accurate reflection of biological aging when mice of many different genotypes and two species were used (Harrison et ai., 1978). The rates of enzymatic digestion of purified insoluble human tendon collagen, with both collagenase and trypsin, have been found to decrease in a precise fashion with increasing age after maturity (Hamlin and Kohn, 1971; Hamlin et al., 1978a). This resistance to digestion is thought to reflect a stabilization or cross-linking of the collagen. When the collagenase procedure was applied to collagen samples, their chronological ages were determined with considerable precision (Hamlin et al., 1975). This procedure has now been used to determine apparent rates of collagen aging after maturity in rat, dog, macaque and man. 393
394
(_LIVE R. HAMLIN,JEANNE H. LUSCHINAND ROBERTR. KOHN
M A T E R I A L S AND M E T H O D S Tissues H u m a n material was obtained from the A u t o p s y Service of the University Hospitals of Cleveland. Macaca mulatta, M. nemestrina, and M. fascicularis tissues were obtained from G. Knitter of the Regional Primate Research Center, University of Washington and from H. M. McClure of the Yerkes Regional Primate Center, Emory University. Canisfamiliaris (Beagle dog) tissues were provided by M. Goldman, W. L. Spangler, and R. Sullivan of the Radiobiological Laboratory, University of California, Davis, and by the Animal Facilities, Case Western Reserve University. Rattus norvegicus (Fischer 344) material was obtained through the courtesy of J. Roberts and S. I. Baskin of the Pharmacology Department, Medical College of Pennsylvania. Tissues were frozen immediately after removal and kept in the frozen state until used. None of the tissues were directly involved in any disease process. Dura mater was the source of collagen in all cases except for rats, in which the a m o u n t s of dura were insufficient. Pooled fascia and tendon from jaw and neck muscles of rats were used. This substitution was judged legitimate because of the observation that h u m a n diaphragm tendon, dura, and myocardial collagen all showed similar absolute values for collagenase digestion and similar changes with age (Hamlin et al., 1978b; Zwolinski et al., 1976). The purpose of this preliminary study was to compare the relative resistance of collagen to digestion during the aging period of the different species, as distinguished from the period of growth and maturation. The most definitive period of aging was judged to extend from the age of growth cessation, meaning no further lengthening of long bones, to the end of the m a x i m u m reported lifespan. Required ages were obtained from published tables (Rockstein et al., 1977; Manville et al., 1972; van Wagenen, 1962). The age of maturation and of m a x i m u m lifespan respectively, that were used were: man, 20 and 115 yrs; macaque, 5 and 29 yrs; dog, 1 and 20 yrs; rat, 0.55 and 4.7 yrs. Preparation o f insoluble collagen The method used to prepare insoluble collagen from both tendon and dura mater has been described previously (Hamlin and Kohn, 1971; Hamlin et al., 1975). The collagen contents of the samples were assessed by performing hydroxyproline determinations and assuming the hydroxyproline content to be 14°Y0for collagen. Enzyme Purified collagenase EC 3.4.4.19 (Worthington Biochemicals, C L S P A , lot 58N432Y, 485 units/mg) prepared from cultures of CIostridium histolyticum, was used without further purification. pH-S T A T measurements The p H - S T A T measurements were performed on a Radiometer TTA3 titration apparatus by using 5 mg of collagen dispersed in 14 ml 0.1 M CaCl~ and maintained at pH 7.8. A coUagenase : collagen ratio of 1 : 50 was used, and the addition of 0.01 M N a O H was monitored continuously for 1 h. The extent of collagen digestion was measured by determining the volume of alkali dispensed during 35 min o f reaction.
RESULTS Figure 1 shows the extent of sample digestion by collagenase, plotted against age. Figure 2 shows the same data plotted against percent of lifespan after maturity. The data used to plot Figs. 1 and 2 are listed in Tables 1 and 2. The human samples show the expected increased stability with age. Dog and macaque samples also show evidence of increased stabilization with age, but less predictably. The rat samples failed to show any clear change. It must be borne in mind that the assay system has been optimized for human samples, the relevant factors being calcium ion concentration, collagenase concentration, and time of reaction. Changes to the assay conditions, optimizing for dog, macaque or rat specimens, might lead to similar precision as that found with human specimens. This has not been attempted primarily because it would negate the possibility of direct comparison between species. It has previously been shown that the increasing stabilization with increasing age of human collagen to collagenase digestion disappears at 1.0 M calcium concentration but is rapidly re-established on dilution to 0.1 M calcium (Hamlin et al., 1978b). This effect is completely reversible. The possibility of a similar phenomenon occurring with the rat samples prompted running the collagenase at lower calcium ion concentrations. No obvious changes with age could be seen even at 0.005 M calcium.
AGING
OFCOLLAGEN:
COMPARATIVE
RATES IN FOUR MAMMALIAN
395
SPECIES
Human
75-
5025-
0
10 20 30
I
I
8
1
40
50
00
70
1
00
’
’
90 100
AGE (years)
FIG. 1. Rates of collagen digestion vs age for indicated species. NaOH required (d, 0.01 M) to maintain pH 7.8 at 37°C during the initial 35 min of collagenase digestion. o = rat, A = dog, + = macaque, 0 = human.
I I
-10
!
I
I
I
0
10
20
30
I
40
I
I
I
I
t
I
50
60
70
80
90
100
% of Life Span After Maturity FIG. 2. Same data shown in Fig. 1. Plotted vs percent of lifespan after maturity. 0
n = human.
= rat, A = dog, 4=
DISCUSSION Collagen from a 2.3-yr-old rat does not behave, under the conditions used in this experiment, in the same manner as a 9%yr-old dog, as a 14.2-yr-old macaque, or a 56yr-old human, all of whom would have aged to the same relative proportion of their maximum lifespans. On the other hand, both the dog and the macaque samples do show
396
CI.IVF R. HAMLIN, JEANNE H. LUSCHIN AND ROBERT R. KOHN TABLE 1. VOLUME N a O H REQUIRED (/A, 0.01 M) TO MAINTAIN pH 7.8 DURING THE INITIAl 35 MIN OF COI~I AGENASE DIGESTION FOR VARIOUS AGED RAT SAMPLES (INITIAl. COLLAGEN CONCENTRATIONS: 5 m g iN 14 ml 0.1 M SPECIFIED SALT SOLUTION AT
37°C, 0.1 mg COLLAGENASEADDEDIN 1.0 ml SALTSOt.UTIONAT t = 0 min) Age (yrs)
0.005 M CaCI2 0.025 M CaCI2 0.095 M NaCI 0.075 M NaCI
0.5 0.7 1.0 1.5 1.6 2.3
234 264 276 296 259 242
0.1 M CaCI2
295 285 283 295 252 275
260 270 252 267 244 258
TABLE 2. VOLUME N a O H REQUIRED (~l, 0.01 M) TO MAINTAIN pH 7.8 DURING THE INITIAL 35 MIN OF COLLAGENASE DIGESTION FOR SAMPLES OF THE INDICATED SPECIES. (INITIAL COLLAGEN
CONCENTRATIONS: 5 mg IN 14 ml 0.1 M CaC12 AT 37°C, 0.1 mg COLLAGENASEADDEDIN 1.0 ml CaC12 AT t = 0 min) Species Dog
Age (yrs)
Vol
Age (yrs)
2.0 282 8.4 142 11.4 195 12.4 113 Estimated regression equation:y =
Primate: M. fascicularis Primate: M. mulatta
3.0* 18.0
250 215
13.6 14.3 14.7 15.0 258 - 1.5x
Vol 173 193 106 194
7.0
269
22.0 25.0
175 183
Primate: M. nemestrina
4.0* 238 12.0 201 6.5 222 14.0 250 Estimated regression equation: y = 252 - 0.85x Human 22 194 63 126 32 174 71 111 43 158 83 103 53 135 93 82 Estimated regression equation: y = 193 - 1.45x *Omitted when computing regression equation owing to subject not having reached maturity.
evidence of a progressive, age-related, increased stabilization to collagenase digestion, as d o t h e h u m a n s p e c i m e n s . In a d d i t i o n , s o m e o l d e r d o g s p e c i m e n s h a v e b e c o m e stabilized to a much greater extent than human specimens of the same chronological age, but to a similar e x t e n t if relative l i f e s p a n s are used. T h e i n c r e a s e d s t a b i l i z a t i o n t o c o l l a g e n a s e d i g e s t i o n w i t h a g i n g in h u m a n s a m p l e s c l e a r l y is closely a s s o c i a t e d w i t h i n c r e a s e d age a f t e r m a t u r i t y . H o w e v e r , t h e i n c r e a s e d s t a b i l i z a t i o n is p r o b a b l y n o t t h e r e s u l t o f all t h e c h a n g e s t h a t h a v e o c c u r r e d in t h e c o l l a g e n m a t r i x .
AGING OF COLLAGEN: COMPARATIVE RATES IN FOUR MAMMALIAN SPECIES
397
Rather, it probably reflects a certain specific constellation of structural parameters, both basic to the collagen matrix and specific aging changes superimposed upon that structure. A demonstration of this has been shown by differences in stabilization with collagenase and trypsin at various calcium concentrations (Hamlin et al., 1978a, b). At a 0.1 M calcium concentration and using collagenase to digest the collagen, a well-defined increased stabilization occurred with increasing age, but with trypsin the collagen samples were uniformly resistant to digestion. When the calcium concentration was increased to 1.0 M, the collagen became more rapidly digested by collagenase and without apparent age differences, but trypsin digestion yielded a clearly defined increased stabilization with increasingly aged samples. The stability effects were completely reversible in that upon dilution of the collagen suspensions from 1.0 to 0.1 M calcium, the age effect with collagenase was re-established as was resistance to trypsin digestion. With this experience, we do not interpret the lack of increased stabilization to collagenase digestion in rats with age to mean that rat collagen has not changed with age. Other treatments are sensitive to age changes in rat collagen. For example, the solubility and breaking time in urea of rat-tail collagen fibers have been shown to undergo dramatic changes with increasing age after maturity (Delbridge and Everitt, 1972; Everitt and Delbridge, 1972). Furthermore, we suspect that in rats altered conditions of collagenase digestion might reveal an age-related increased stabilization. Another unknown factor, with species of varying metabolic rates, is the potential effect of specific changes in the collagen matrices. For example, a specific change may have little effect in man with a heart rate of 70/min, but the same change may have serious effects in a rat whose heart beats 500 times/min coupled with a different physiological demand placed on tissues and the associated higher frequencies of tissue movement. In other words, a rat must maintain its collagen properties within much narrower limits than must man in order to survive. Also, although the most fundamental molecular aging processes may be similar, though occurring at different rates in different mammals, and physiological decline appears in all species, different disease patterns between species most probably play some role in lifespan. As an example, atherosclerosis is the major age-related disease and cause of death in man, but does not occur to any significant degree in the rat. Again, though it might be expected that basic aging processes are similar in different species, one must exercise extreme caution when extrapolating an age-related observation from a short-lived species such as rat to a long-lived species such as human. From our perspective, we believe studies aimed at understanding human aging will avoid conceptual problems and controversy if they are performed with human tissues, rather than tissues from short-lived species. SUMMARY 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.
398
CLIVER. HAMLIN.JEANNEH. LUSCHINANDROBERTR. KOHN
Acknowledgements--We are very grateful to the individuals noted under 'Materials and Methods' who provided us with samples. This study was supported in part by grant AG 00361 from the National Institute on Aging.
REFERENCES KOHN, R. R. (1978) Principles o f Mammalian Aging, 2nd Ed. Prentice-Hall, N.J. DEYL, Z., JURICOVA,M., ROSMUS,J. and ADAM, M. (1971)Exp. Geront. 6,227. VOGEL, H. G. (1978) Conn. Tiss. Res. 6, 161. VANCIKOVA,O. and DEYL, Z. (1973)Exp. Geront. 8, 297. VERZAR, F. (1968) Exp. Geront. 3, 69. HARRISON, D. E., ARCHER, J. R., SACHER,G. A. and BOYCE, F. M. (1978)Exp. Geront. 13,63. HAMLm, C. R. and KOHN, R. R. (1971)Biochim. biophys. Acta 236, 458. HAMLIN, C. R., LUSCHIN,J. H. and KOHN, R. R. (1978a) Exp. Geront. 13,415. HAMLIN, C. R., KOHN, R. R. and LUSCHIN,J. H. (1975) Diabetes 24, 902. HAMLIN, C. R., LUSCHIN,J. H. and KOHN, R. R. (1978b)Exp. Geront. 13,403. ZWOLINSKI, R. J., HAMLIN,C. R. and KOHN, R. R. (1976)Proc. Soc. exp. BioL Med. 152, 362. ROCKSTEIN, M., CHESKY, J. A. and SUSSMAN,M. L. (1977) In: Handbook o f The Biology o f Aging (Edited by C. E. FINCH and L. HAYFLICK),p. 3. Van Nostrand Reinhold, New York. MANVILLE, R. H., JONES, C., SACHER, G. A., JONES, M. L., CARLANDER,K. D. and COMFORT, A. (1972) In: Biology Data Book, 2nd Ed. (Edited by P. L. ALTMANand D. S. DITTMER), VOI~ 1, p. 229. Fed. Am. Soc. Exptl. Biol., Bethesda. VAN WAGENEN, G. (1962) In: Growth (Edited by P. L. ALTMAN and D. S. DITTMER), p. 349. Fed. Am. Soc. Exptl. Biol., Washington, D.C. DELBRIDGE, L. and EVERITT, A. V. (1972)Exp. Geront. 7,413. EVERITT, A. V. and DELBRIDGE, L. (1972) Exp. Geront. 7, 45.
AGING OF COLLAGEN:
COMPARATIVE
FOUR MAMMALIAN
RATES IN
SPECIES
CLIVE R. HAMLIN, JEANNE H . LUSCHIN a n d ROBERT R . KOHN Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, U.S.A. (Received 12 October 1979; in revised f o r m 14 December 1979)
INTRODUCTION COLLAGEN comprises about 30°10 of total body protein and undergoes very marked changes in properties during aging. These changes suggest a progressive stabilization and stiffening through intermolecular cross linking, but few specific details are known about the chemistry of the changes with age. It has been argued that such aging of extracellular macromolecules is capable of explaining many debilities characterizing the aging syndrome. Aging of collagen and its possible significance have been reviewed (Kohn, 1978). It would be useful to know if aging of collagen is accelerated in species that have short lifespans and are presumably aging faster than species with longer lifespans. This question has received little attention despite the frequent use of short-lived species in studies ultimately aimed at understanding human aging. One study showed only small and variable differences in the amount of insoluble collagen that could be solubilized by heat and urea between a 2-yr-old dog, rat, cat and human being (Deyl et al., 1971). The significance of this observation is not clear because no distinction was made between maturation and aging (Vogel, 1978); the human being would be growing rapidly at that age and synthesizing large amounts of young, soluble collagen, while the other species would have stopped growing at different ages and would have undergone aging for different fractions of the 2-year period. Furthermore, the procedure used actually showed a perplexing decrease of insoluble collagen between 2- and 80-yr-old tendons in human beings. Another study has indicated that stabilization of aldimine bonds in collagen does not differ significantly in 6- and 30-month-old rats, cats and golden hamsters (Vancikovfi and Deyl, 1973). In a third study the force of thermal contraction of collagen fibers has been shown to increase with age, and similar forces were developed in fibers from a 3-yr-old rat and a 3-yr-old cat (Verz~ir, 1968). One other study could neither prove nor disprove the hypothesis that changes in tail tendon characteristics with age are an accurate reflection of biological aging when mice of many different genotypes and two species were used (Harrison et ai., 1978). The rates of enzymatic digestion of purified insoluble human tendon collagen, with both collagenase and trypsin, have been found to decrease in a precise fashion with increasing age after maturity (Hamlin and Kohn, 1971; Hamlin et al., 1978a). This resistance to digestion is thought to reflect a stabilization or cross-linking of the collagen. When the collagenase procedure was applied to collagen samples, their chronological ages were determined with considerable precision (Hamlin et al., 1975). This procedure has now been used to determine apparent rates of collagen aging after maturity in rat, dog, macaque and man. 393
394
(_LIVE R. HAMLIN,JEANNE H. LUSCHINAND ROBERTR. KOHN
M A T E R I A L S AND M E T H O D S Tissues H u m a n material was obtained from the A u t o p s y Service of the University Hospitals of Cleveland. Macaca mulatta, M. nemestrina, and M. fascicularis tissues were obtained from G. Knitter of the Regional Primate Research Center, University of Washington and from H. M. McClure of the Yerkes Regional Primate Center, Emory University. Canisfamiliaris (Beagle dog) tissues were provided by M. Goldman, W. L. Spangler, and R. Sullivan of the Radiobiological Laboratory, University of California, Davis, and by the Animal Facilities, Case Western Reserve University. Rattus norvegicus (Fischer 344) material was obtained through the courtesy of J. Roberts and S. I. Baskin of the Pharmacology Department, Medical College of Pennsylvania. Tissues were frozen immediately after removal and kept in the frozen state until used. None of the tissues were directly involved in any disease process. Dura mater was the source of collagen in all cases except for rats, in which the a m o u n t s of dura were insufficient. Pooled fascia and tendon from jaw and neck muscles of rats were used. This substitution was judged legitimate because of the observation that h u m a n diaphragm tendon, dura, and myocardial collagen all showed similar absolute values for collagenase digestion and similar changes with age (Hamlin et al., 1978b; Zwolinski et al., 1976). The purpose of this preliminary study was to compare the relative resistance of collagen to digestion during the aging period of the different species, as distinguished from the period of growth and maturation. The most definitive period of aging was judged to extend from the age of growth cessation, meaning no further lengthening of long bones, to the end of the m a x i m u m reported lifespan. Required ages were obtained from published tables (Rockstein et al., 1977; Manville et al., 1972; van Wagenen, 1962). The age of maturation and of m a x i m u m lifespan respectively, that were used were: man, 20 and 115 yrs; macaque, 5 and 29 yrs; dog, 1 and 20 yrs; rat, 0.55 and 4.7 yrs. Preparation o f insoluble collagen The method used to prepare insoluble collagen from both tendon and dura mater has been described previously (Hamlin and Kohn, 1971; Hamlin et al., 1975). The collagen contents of the samples were assessed by performing hydroxyproline determinations and assuming the hydroxyproline content to be 14°Y0for collagen. Enzyme Purified collagenase EC 3.4.4.19 (Worthington Biochemicals, C L S P A , lot 58N432Y, 485 units/mg) prepared from cultures of CIostridium histolyticum, was used without further purification. pH-S T A T measurements The p H - S T A T measurements were performed on a Radiometer TTA3 titration apparatus by using 5 mg of collagen dispersed in 14 ml 0.1 M CaCl~ and maintained at pH 7.8. A coUagenase : collagen ratio of 1 : 50 was used, and the addition of 0.01 M N a O H was monitored continuously for 1 h. The extent of collagen digestion was measured by determining the volume of alkali dispensed during 35 min o f reaction.
RESULTS Figure 1 shows the extent of sample digestion by collagenase, plotted against age. Figure 2 shows the same data plotted against percent of lifespan after maturity. The data used to plot Figs. 1 and 2 are listed in Tables 1 and 2. The human samples show the expected increased stability with age. Dog and macaque samples also show evidence of increased stabilization with age, but less predictably. The rat samples failed to show any clear change. It must be borne in mind that the assay system has been optimized for human samples, the relevant factors being calcium ion concentration, collagenase concentration, and time of reaction. Changes to the assay conditions, optimizing for dog, macaque or rat specimens, might lead to similar precision as that found with human specimens. This has not been attempted primarily because it would negate the possibility of direct comparison between species. It has previously been shown that the increasing stabilization with increasing age of human collagen to collagenase digestion disappears at 1.0 M calcium concentration but is rapidly re-established on dilution to 0.1 M calcium (Hamlin et al., 1978b). This effect is completely reversible. The possibility of a similar phenomenon occurring with the rat samples prompted running the collagenase at lower calcium ion concentrations. No obvious changes with age could be seen even at 0.005 M calcium.
AGING
OFCOLLAGEN:
COMPARATIVE
RATES IN FOUR MAMMALIAN
395
SPECIES
Human
75-
5025-
0
10 20 30
I
I
8
1
40
50
00
70
1
00
’
’
90 100
AGE (years)
FIG. 1. Rates of collagen digestion vs age for indicated species. NaOH required (d, 0.01 M) to maintain pH 7.8 at 37°C during the initial 35 min of collagenase digestion. o = rat, A = dog, + = macaque, 0 = human.
I I
-10
!
I
I
I
0
10
20
30
I
40
I
I
I
I
t
I
50
60
70
80
90
100
% of Life Span After Maturity FIG. 2. Same data shown in Fig. 1. Plotted vs percent of lifespan after maturity. 0
n = human.
= rat, A = dog, 4=
DISCUSSION Collagen from a 2.3-yr-old rat does not behave, under the conditions used in this experiment, in the same manner as a 9%yr-old dog, as a 14.2-yr-old macaque, or a 56yr-old human, all of whom would have aged to the same relative proportion of their maximum lifespans. On the other hand, both the dog and the macaque samples do show
396
CI.IVF R. HAMLIN, JEANNE H. LUSCHIN AND ROBERT R. KOHN TABLE 1. VOLUME N a O H REQUIRED (/A, 0.01 M) TO MAINTAIN pH 7.8 DURING THE INITIAl 35 MIN OF COI~I AGENASE DIGESTION FOR VARIOUS AGED RAT SAMPLES (INITIAl. COLLAGEN CONCENTRATIONS: 5 m g iN 14 ml 0.1 M SPECIFIED SALT SOLUTION AT
37°C, 0.1 mg COLLAGENASEADDEDIN 1.0 ml SALTSOt.UTIONAT t = 0 min) Age (yrs)
0.005 M CaCI2 0.025 M CaCI2 0.095 M NaCI 0.075 M NaCI
0.5 0.7 1.0 1.5 1.6 2.3
234 264 276 296 259 242
0.1 M CaCI2
295 285 283 295 252 275
260 270 252 267 244 258
TABLE 2. VOLUME N a O H REQUIRED (~l, 0.01 M) TO MAINTAIN pH 7.8 DURING THE INITIAL 35 MIN OF COLLAGENASE DIGESTION FOR SAMPLES OF THE INDICATED SPECIES. (INITIAL COLLAGEN
CONCENTRATIONS: 5 mg IN 14 ml 0.1 M CaC12 AT 37°C, 0.1 mg COLLAGENASEADDEDIN 1.0 ml CaC12 AT t = 0 min) Species Dog
Age (yrs)
Vol
Age (yrs)
2.0 282 8.4 142 11.4 195 12.4 113 Estimated regression equation:y =
Primate: M. fascicularis Primate: M. mulatta
3.0* 18.0
250 215
13.6 14.3 14.7 15.0 258 - 1.5x
Vol 173 193 106 194
7.0
269
22.0 25.0
175 183
Primate: M. nemestrina
4.0* 238 12.0 201 6.5 222 14.0 250 Estimated regression equation: y = 252 - 0.85x Human 22 194 63 126 32 174 71 111 43 158 83 103 53 135 93 82 Estimated regression equation: y = 193 - 1.45x *Omitted when computing regression equation owing to subject not having reached maturity.
evidence of a progressive, age-related, increased stabilization to collagenase digestion, as d o t h e h u m a n s p e c i m e n s . In a d d i t i o n , s o m e o l d e r d o g s p e c i m e n s h a v e b e c o m e stabilized to a much greater extent than human specimens of the same chronological age, but to a similar e x t e n t if relative l i f e s p a n s are used. T h e i n c r e a s e d s t a b i l i z a t i o n t o c o l l a g e n a s e d i g e s t i o n w i t h a g i n g in h u m a n s a m p l e s c l e a r l y is closely a s s o c i a t e d w i t h i n c r e a s e d age a f t e r m a t u r i t y . H o w e v e r , t h e i n c r e a s e d s t a b i l i z a t i o n is p r o b a b l y n o t t h e r e s u l t o f all t h e c h a n g e s t h a t h a v e o c c u r r e d in t h e c o l l a g e n m a t r i x .
AGING OF COLLAGEN: COMPARATIVE RATES IN FOUR MAMMALIAN SPECIES
397
Rather, it probably reflects a certain specific constellation of structural parameters, both basic to the collagen matrix and specific aging changes superimposed upon that structure. A demonstration of this has been shown by differences in stabilization with collagenase and trypsin at various calcium concentrations (Hamlin et al., 1978a, b). At a 0.1 M calcium concentration and using collagenase to digest the collagen, a well-defined increased stabilization occurred with increasing age, but with trypsin the collagen samples were uniformly resistant to digestion. When the calcium concentration was increased to 1.0 M, the collagen became more rapidly digested by collagenase and without apparent age differences, but trypsin digestion yielded a clearly defined increased stabilization with increasingly aged samples. The stability effects were completely reversible in that upon dilution of the collagen suspensions from 1.0 to 0.1 M calcium, the age effect with collagenase was re-established as was resistance to trypsin digestion. With this experience, we do not interpret the lack of increased stabilization to collagenase digestion in rats with age to mean that rat collagen has not changed with age. Other treatments are sensitive to age changes in rat collagen. For example, the solubility and breaking time in urea of rat-tail collagen fibers have been shown to undergo dramatic changes with increasing age after maturity (Delbridge and Everitt, 1972; Everitt and Delbridge, 1972). Furthermore, we suspect that in rats altered conditions of collagenase digestion might reveal an age-related increased stabilization. Another unknown factor, with species of varying metabolic rates, is the potential effect of specific changes in the collagen matrices. For example, a specific change may have little effect in man with a heart rate of 70/min, but the same change may have serious effects in a rat whose heart beats 500 times/min coupled with a different physiological demand placed on tissues and the associated higher frequencies of tissue movement. In other words, a rat must maintain its collagen properties within much narrower limits than must man in order to survive. Also, although the most fundamental molecular aging processes may be similar, though occurring at different rates in different mammals, and physiological decline appears in all species, different disease patterns between species most probably play some role in lifespan. As an example, atherosclerosis is the major age-related disease and cause of death in man, but does not occur to any significant degree in the rat. Again, though it might be expected that basic aging processes are similar in different species, one must exercise extreme caution when extrapolating an age-related observation from a short-lived species such as rat to a long-lived species such as human. From our perspective, we believe studies aimed at understanding human aging will avoid conceptual problems and controversy if they are performed with human tissues, rather than tissues from short-lived species. SUMMARY 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.
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CLIVER. HAMLIN.JEANNEH. LUSCHINANDROBERTR. KOHN
Acknowledgements--We are very grateful to the individuals noted under 'Materials and Methods' who provided us with samples. This study was supported in part by grant AG 00361 from the National Institute on Aging.
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