A CHARACTERIZATION of BASOPHILIC DEGENERATION of COLLAGEN by HISTOCHEMICAL and MICROSPECTROSCOPIC PROCEDURES*Jprocedures*T P

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A CHARACTERIZATION OF BASOPHILIC DEGENERATION OF COLLAGEN BY HISTOCHEMICAL AND MICROSPECTROSCOPIC PROCEDURES*jPROCEDURES*t P. O'B. MONTGOMERY, M.D. Basophilic degeneration of dermal collagen is a histologic feature of senile atrophy of the skin. It is frequently seen in association with premalignant and malignant skin lesions (1). The purpose of this paper is to compare normal dermal collagen with dermal collagen exhibiting basophilic degeneration by means of a battery of histochemical procedures and by a microspectroscopic procedure. MATERIALS AND METHODS Normal skin and dermal collagen were obtained from two patients above 45 years of age. Collagen showing basophilic degeneration was obtained from the dermis adjacent to squamous cell carcinomas of the back of the hands of two patients. The histochemical procedures employed are given in A. G. E. Pearse's book, or they were performed as given by reference (2). The microspectroscopic data was obtained by the study of frozen sections of the collagen in question utilizing a B & L Grating Monochromator with hydrogen arc light source and B & L reflecting optics. The negative image of the collagen at selected wave lengths was recorded on Eastman S. A. No. 1 film. The background density of the film was kept constant by altering the exposure time as necessary atat thethe selectedselected wavewave length.length. DirectDirect filmfilm densitometrydensitometry was then accomplished with a photovolt densitometer. The resulting values were plotted on a graph. RESULTS Collagen showing basophilic degeneration contained large amounts of lipid material as demonstrated by positive Oil Red 0, Nile Blue Sulphate, and Sudan Black B stains and by the positive Schultz reaction. Normal collagen contained only a trace of these lipid materials. Normal collagen and degenerated collagen both gave positive results with the Periodic acid Schiff reaction, Congo Red, thethe MillonMillon reactionreaction forfor tyrosine,tyrosine, thethe methodmethod ofof AshbelAshbel andand SeligmanSeligman forfor freefree carbonyl groups (3), and Terry's polychrome methylene blue. This stain was unaltered by ribonuclease digestion. With all of these procedures the degenerated collagen gave a more intense reactionreaction thanthan thethe normalnormal collagen.colltgen. The normal collagen gave a moderately positive reaction for nonspecific esterase (4), the degenerated collagen gave a considerably reduced reaction. *Fromthe Department of Pathology, Southwestern Medical School of The University of Texas. Supported by U.SP.II.S. Grant 'A-643 through the National Advisory Arthritis and Metabolic Diseases Council. Received for publication September 27, 1954. 107 108 THE JOURNAL JOURNAL OF OF INVESTIGATIVE INVESTIGATIVE DERMATOLOGY DERMATOLOGY

FIG.Fio. 1. 1. U. U. V. V. absorption absorption curves curves of of normal normal collagen collagen and and collagen collagen showing showing hasophilic hasophilic de- de- generation.

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FIG. 2. U. V. absorption ofof normalnormal collagencollagen atat 290290 m,m The normal collagen gave an intensely positive alizarin red reaction for calcium while the degenerated collagen gave a negative reaction. The normal collagen and the degenerated collagen gave the same positive reaction for potassium (Macallum's method), vitamin C, acetal lipids as demonstrated by the plasmal reaction, and protein bound suiphydrylsulphydryl groups (5). BASOPHILIC DEGENERATION OF COLLAGEN 109

FIG. 3. U. V. absorption of basophilic degeneration of collagen at 290 m

The normal collagen and the degenerated collagen gave negative reactioisreactions for alkaline phosphatase, acid phosphatase, indole, and glycogen. Mast cells were demonstrated in increased numbers in the skin sections showing basophilic degeneration of collagen. These cells were easily identified by the presence of their basophilic cytoplasmic granules as stained with Terry's polychrome methylene blue. The results of the microspectrographic analyses are given in the graph in Fig. 1. The film density is plotted in arbitary units along the ordinate. One hundred units represents complete absorption of the light energy, and 0.3 units represents complete transmission of the light energy or background density. The wave length of light is plotted on the abcissa at 5 mi intervals. Normal collagen did not absorb significant amountsamounts ofof lightlight fromfrom 250250 mim to 355 mgi. Collagen displaying basophilic degeneration absorbed the majority of the light from 250 mim toto 310310 mji,mgi, and and very very little little lightlight fromfrom 320320 mimi toto 355 mjz.mgi.

DISCUSSION Basophilic degeneration of collagen is well described in the standard works on the histopathology of the skin (1). Since the epidermis overlying such collagen is frequently the site of neoplasia, it is natural to consider whether the two lesions are more than casually related. Alterations in the dermal collagen have been described in association with 110 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY experimental skin cancer by Orr and Ma (6, 7). These workers agree that dermal collagen is decreased in amount during the course of chemically induced skin cancer in mice. They do not, however, point out the feature of collagen studied here; namely basophilic degeneration. Cramer and Simpson have described an increase in mast cells in the dermis of the mouse skin subjected to chemical carcinogenesis, and they report similar findings in human senile keratoses (8). The data presented herein confirm this finding. These studies indicate that normal collagen and collagen showing basophilic degeneration are different in their histochemical and microspectrophotographic configurations. The relation between these differences and the frequent develop- ment of neoplasia in the overlying epidermis remain to be seen.

SUMMARY 1. Differences in normal collagen and collagen displaying basophilic degeneration have been described by means of histochemical and microspectrophotographic procedures. 2. Collagen displaying basophilic degeneration contains increased amounts of lipids, free aldehyde groups, tyrosine, free carbonyl groups and basophilic material, when compared with normal collagen. 3. Collagen displaying basophilic degeneration contained less non-specific esterase than normal collagen. 4. Collagen displaying basophilic degeneration gave a negative reaction for calcium while normal collagen gave an intense positive reaction. 5. Collagen displaying basophilic degeneration absorbed the majority of light energy from 250250 toto 310310 mp.mp. NormalNorma' collagen diddid notnot absorbabsorb significantsignificant amounts of light at these wave lengths. I wish to thank Mrs. Ovia Walker for her technical assistance. REFERENCES 1. LEVER,LEvER, W.W. F.: Histopathology of the Skin.Skin. LippincottLippincott 124, 124, 1949. 1949. 2. PEARSE,PEAR5E, A. G.C. E.: Histochemistry Theoretical aridand Applied. Boston, Little., 1953. 3. AsITBELL,ASITBELL, R.R. ANDAND SELIOMAN,SELIGMAN, A.A. M.:M.: AA newnew reagentreagent forfor thethe histochemicalhistochemical demonstrationdemonstration of active carbonylcarbonyl groups; a newnew methodmethod for for staining staining ketonicketonic steroids.steroids. Endocrinology.Endocrinology. 44: 565—583,565—583, 1949. 4. NACHLAS,NACHLAS, M. M. M. M. AND AND SELIGMAN, SELTOMAN, A.M.: A. EvidenceM.: Evidence for thefor thespecificity specificity of of esterase esterase andand lipaselipase by the use of three chromogenic substances. J. Biol. Chem.,Cliem., 181:181: 343—355,343—355, 1949.1949. 5. BARNETT, R. J. AND SELIOMAN,SELIGMAN, A. M.: Histochemical demonstration of protein bound sulphydryl groups. Science., 116: 323—327, 1952. 6. ORB,Orm, J.J. W.:W.: TheThe changeschanges antecedentantecedent to tumor formation during the treatment ofof mousemouse skin with carcinogenic hydrocarbons. J. Path. & Bact., 46: 495—515, 1938. 7. MA, CHUNG K.: Morphological andand chemicalchemical investigationinvestigation of of dermal dermal elastic, elastic, and and col- col lagenic tissue during epidermal carcinogenesis. Cancer Research., 9: 481—487, 1949. 8. CRAMER, W. AND SIMPSON, W. L.: Mast cells in experimental skin carcinogenesis. Cancer Research., 4:4: 601—616, 601—616, 1944.