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COLLAGEN AND OF HUMAN * GERALD D. WEINSTEIN, B.A. ANI) ROBERT J. BOUCEK, M.D.

As connective ages, the following changes The washed residue was dried, weighed and ex- have been observed: (1) a reduction in its swellingposed to purified elastasef in a buffer of pH 8.8 properties as noted by Banfield (1) and Eldencontaining 0.2 M Tris for 24 hours at 37° C. After (2); (2) an alteration in the contraction and re-centrifugation for 30 minutes at 40,000 rpm (105,- 000 g.), the supernatant fluid was hydrolyzed with laxation of described by Banga etat. HC1 as above for the analysis. (3)as well as by Verzär (4); (3) an increase in the Amino acid analysis consisted of the determina- size of collagen reported by Jackson (5);tion of using the method of and (4) a reduction in the ratio of hexosamine toNeuman and Logan (9) or a modification of the collagen reported by Sobel (6). In order to de-Stegemann method (10). were termine any alteration in the quantity of collagenconverted to collagen values using the standard and elastin in human age, thefactor of 7.46 and to elastin values using the factor following study was made. Corium from non-of 50. Both collagen and elastin were expressed exposed areas of the body was studied in humanas the percentage of FFDW. beings of both sexes and of varying ages. This The resistant residue was suspended in water and analyzed for hexoses by the addition of report presents the results of these studies. 2 volumes of anthrone reagent (11). Glucose was METHOD used as the standard and the anthrone value ex- pressed on the basis of the FFDW. Contaminants, Caucasian abdominal , obtained from freshsuch as hair and elastase, were found to be an- autopsy material and frozen until used, was takenthrone-negative. from a uniform location along the midline autopsy incision. In the determination of collagen, 83 hu- RESULTS man subjects were used: 47 males, ranging in age The percentage of collagen in the corium from from 25 to 90 years; and 36 females, ranging fromhuman males and females was approximately 21 to 78 years. All these subjects had died sud- 75.9 denly. In determining elastin, 17 males from 25 to77% of the FFDW (77.7 5.9% , 96 years and 10 females from 21 to 74 years were6.4% c?) (Table I). Plotting the percentage of used. collagen against the chronological age of the Samples in the range of 1 to 2 gms. wet weightdonor by the method of least squares revealed no were prepared by removing the hair and epidermissignificant slope in either sex. with heat after the Baumberger technic (7) and Total elastin in 17 males was 4.5 1.4% and then homogenizing in distilled water using ain 10 females was 4.0 0.9%. Elastase treatment VirTis homogenizer. An alcohol-ether mixture in a of 17 preparations caused 99% of elastin to be 3:1 ratio was used for fat extraction and, afterdigested. No sex or age difference was observed. drying to constant weight at 100° C. in a vacuum A hexose-positive residue remained following oven, the samples were weighed to obtain the fat- free dry weight (FFDW). the alkali extraction, repeated autoclaving and The dried sample was treated with 0.1 N NaOH,elastase incubation which was 0.044% 0.016% then neutralized to pH 7.0 prior to autoclavingin 15 individuals and showed no sex or age effect. after the technic of Lowry (8). Two 3-hour periods of autoclaving at 25 lbs. were used to con- DISCUSSION vert the collagen to soluble . The super- The observation of a constant quantity of natant fluids obtained by centrifugation at 3,000g.collagen and elastin in the unexposed skin sug- were hydrolyzed in 6 N HC1 for 16 hours at 110° C.gests a lack of uniformity of connective tissue for the amino acid analysis. alterations with age. For example, the connective From the Divisions of Dermatology and Car-tissue of definitely reflects the effect of diology of the Department of Medicine, University of Miami School of Medicine and Howard Hughes age. There the intima increases in its thickness, Medical Institute, Miami, Florida. collagen accumulates in the media, and the in- This investigation was supported in part byternal elastic membrane becomes frayed. In Grants A-2586 and 2G-224, from the N.I.H., U. S. Public Health Service. tElastase,2 X Crystalline, Worthington Bio- Received for publication February 20, 1960. chemical Corporation, Freehold, N. J. 227 228 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE I union having tinctorial properties indistinguish- Fibrousproteins of human corium able from elastin but in reality not elastin as determined by biochemical criteria. Male Female According to Hall's study, a cellulose-like sub- stance was observed to develop in the corium 75.9 6.4* 77.7 5.9 Collagen with age (17). If this were a general phenomenon, (47) (36) then the anthrone-positive material present in Elastin 4.45 1.4 3.95 0.9 this study should have increased with time. This (17) (10) was not found and it suggests that the anthrone- positive material could have represented bound * Meanand standard deviation of the mean. hexoses not liberated by the multiple extraction procedures, or perhaps the presence of other sub- addition, a loss of elastic properties of the vesselstances giving a positive reaction with the occurs and Roach and Burton have suggestedanthrone in the residue. In either event, this ma- that this is related to the extent of internal cross-terial did not increase with tissue age. linking of collagen (12). Also, histologists have observed an increase of a substance in the corium SUMMARY from exposed areas of the body which they term (1) The collagen of human corium was ap- 'senile elastosis'. McMfflan and Lev have ob-proximately 77 per cent and elastin approxi- served an increase in the thickness of the endo-mately 4 per cent (fat-free dry weight). These cardium with increasing age (13). On the otherpercentages were not influenced by the age or sex hand, advancing years seem to cause no increaseof the donor. in the amount of collagen in the myocardium (2) Elastin at all tissue ages was readily di- (14). gested by elastase. Tissue when injured is stimulated to produce (3) No evidence was obtained to suggest that connective tissue. This is a universal reaction ofage increased a cellulose-like substance in the the body and is responsible for the proc-corium. ess. The sex of the injured may be of im- portance in determining the amount of collagen ACKNOWLEDGMENT which is present in the healing process. For ex- The authors acknowledge the support and ample, when reproducible injury is induced inencouragement of Dr. Harvey Blank. by the implantation of a polyvinyl in the subcutaneous tissues, the male produces REFERENCES a greater amount of collagen than the female 1.BANFIELD,W. G.: The and swelling (15, 16). excited by injury may thus ofcollagen in dilute acid withage variations be affected in the degree of collagen synthesis in man. Anat. Rec., 114: 157—171, 1952. 2.ELDEN, H. R., NOBLE, N. L. AND BOUCEK, and accumulation by the sex of the host. R. J.: The swelling and extractability of The study of elastin of human corium reveals humansponge-biopsy connective tissue. that the quantity and quality as reflected by the Gerontologia 3: 204—212, 1959. 3. BANGA,I.,BAL6, J. ANDSZAEO,D.: Contrac- reaction to the elastase was not influ- tion and relaxation of collagen. , 174: 788—789, 1954. enced by age. Yet pathologists and histologists 4.VEEzAR, F.: The of collagen. In Con- have observed the development of senile elastosis nective Tissue, edited by Tunbridge, R. E., as an aging effect. It should be pointed out that Keech, M., Delafresnaye, J. F. and Wood, senile elastosis has been observed only in the G.C., pages 208—221. BlackwellScientific Publications, Oxford, 1957. exposed skin areas. Again, the possibility of in- 5. JAcKson, S. F.:Structural problems associated jury and response of the corium to injury under- withthe formation of collagen in vivo. In Connective Tissue, edited by Tunbridge, lying these changes is entirely possible, the injury R. E., Keech, M., Delafresnaye, J. F. and in the exposed areas presumably being from the Wood, G. C., pages 77—85. Blackwell Scien- light. Furthermore, it is not certain tific Publications, Oxford, 1957. 6. SOBEL, H., BAGAY, S., WEIGHT, E. T., LIcH- that senile elastosis represents a true increase TENSTEIN, I. AND NELSON, N. H.: The influ- in the elastic fibers or whether it is representative ence of age upon the hexosamine-collagen of an increase in the collagen which may be in- ratio of dermal biopsies from men. J. Geront., 13: 128—131, 1958. timately bound to the pre-existing elastin, this 7. BAUMBERGER, J. P., SUNTZEFF, V. AND Cow- COLLAGEN AND ELASTIN OF HUMAN DERMIS 229

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