Arch. histol. jap. Vol. 23, n. 2 (December 1962). P. 143-151.

Dept. of Dermatol. (Head: Prof. Y. TAKAHASHI), Tohoku Univ. Sch. of Med., Sendai.

Histochemical Study of Acid Mucopolysaccharides in Mucinoses.

皮 膚ムチ ン症 におけ る酸性 粘液多糖類の組織化学的研究.

Yoichiro SASAI 笹 井 陽 一 郎.

(Received November 11, 1962.)

Mucinoses comprise a group of conditions characterized by deposition of a ma- terial known as 'mucin' in the cutis. However, no agreement occurs concerning the exact nature of mucin (TROTTER and EDEN 1942, WATSON and PEARCE 1947, PALITZ and BRUNNER 1950, KEINING and BRAUN-FALCO 1956). The present study was designed to determine histochemical properties of acid mucopolysacchari- des of mucin in mucinoses.

I. Materials and Methods.

Biopsy skin specimens were removed from two cases with pretibial myxedema and one case with scleromyxedema. Each specimen was cut into two parts; one was placed in 10% aqueous calcium acetate formalin (LILLIE 1954), and the other in alcoholic lead nitrate formalin (LILLIE 1954). Then, they were dehydrated, and

were embedded in paraffin. Sections cut at a thickness of 6μ were stained 30min. with 0.05% toluidine blue either in aqueous 0.1M phosphate-citrate buffer of a se- lected pH (OHNO et al. 1951) or in 70% ethanol (LISON 1935) at room temperature.

Adjacent sections were stained with buffered 3% toluidine blue at 70℃ as described by LARSEN (1958). Alternatively sections were stained 30min. in a 1% solution of alcian blue in 3% acetic acid followed by periodic acid-SCHIFF stain as described by MOWRY (MOWRY and WINKLER 1956). Adjacent sections were prepared with only the 0.1% solution of alcian blue at varied pH, and with the PAS-procedure uncombined with alcian blue. And also, some sections were stained with aldehyde fuchsin as described by HALMI (HALMI and DAVIS 1953), and occasionally fol- lowed by alcian blue stain as developed by SPICER (SPICER and MEYER 1960).

Methylation was accomplished before staining by variable exposure at 60℃ to the preheated methanol made 0.1N with hydrochloric acid after LILLIE's (LILLIE 1954) modification of the procedure of FISHER and LILLIE (1954). Saponification involved a subsequent collodionization and 30min. immersion of the slides at 25℃. in a 1% potassium hydroxide solution in 70% ethanol (LILLIE 1958). Sulfation was performed according to the method described by LEWIS and GRILLO (1959).

II. Results.

The results are indicated in Table 1. The salient features in each disease are summarized as follows.

143 144 Y. SASAI:

Table1. Staining properties of mucin in myxedema and scleromyxedema.

Pretibial myxedema. In the middle to the lower dermis there was clearly delineated the area in which a large amount of mucin was present (Fig. 1). In that area the collagen fibres were frayed and were separated widely, but they stained normally with a variety of collagen stains. Mucin was stained deeply with alcian blue at pH 1.5 and somewhat lightly with aldehyde fuchsin, and showed strong metachromasia with 3% toluidine blue buffered to pH 4.1. With 0.05% toluidine blue buffered to 4.1, however, metachromasia was limited to some area (Fig. 2). After methylation, these properties disappeared (Fig. 3). When saponification was performed after methylation, both alcian blue affinity and metachromasia were restored. Scleromyxedema. In contrast with myxedema the mucin-deposition was found only in the deli- neated areas of the upper dermis (Fig. 4). An increase of collagen in amount and numerous fibroblasts were noticed in the areas of the mucin-deposition. Mucin stained deeply with alcian blue at pH 1.0 (Fig. 5) was metachromasic with 0.05% toluidine blue buffered to pH 2.0, and manifested a strong affinity to aldehyde fuchsin (Fig. 6). Methylation blocked these three results. When saponification was caught out after methylation, the alcian blue-affinity was restored, but it was found to become somewhat weaker. Histochemical Study of Acid Mucopolysaccharides in Mucinoses. 145

Fig. 1. Pretibial myxedema. DELAFIELD's hematoxylin-eosin stain. ×100

Fig. 2. Pretibial myxedema. Toluidine blue (0.05%) stain at pH 4.1. Mucin showing metachromasia. ×400 146 Y. SASAI:

Fig. 3. Pretibial myxedema. Toluidine blue (0.05%) stain at pH 4.1, following methyla-

tion. Mucin showing blockade of metachromasia. ×400

Fig. 4. Scleromyxedema. DELAFIELD's hemato- xylin-eosin stain. ×100 Histochemical study of Acid Mucopolysaccharides in Mucinoses. 147

Fig. 5. Scleromyxedema. Alcian blue stain at pH 1.0. Mucin

showing strong alcian blue affinity. ×400

III. Comment.

Histochemical classification of mammalian mucopolysaccharides rests on their comparative staining characteristics with the specific histochemical methods for mucin. The staining of acid tissue components with basic dyes in metachromatic color, 'metachromasia', was observed by EHRLICH (1876-1877). LISON (1935) explained metachromasia as a specific property of high molecular weight sulfated compounds. However, some workers (WISLOCKI et al. 1947) favor the possibility that hyaluronic acid, lacking sulfate esters, imparts metachromasia to body fluids or tissue sections, whereas other question (MEYER 1947, SYLVEN and MALMG- REN 1952, HAMERMAN and SCHUBERT 1953). The application of basic dyes at controlled pH levels was developed by DEMPSEY et al. (DEMPSEY, BUNTING, SINGER and WISLOCKI 1947) to characterize mucopolysaccharides according to their pH signature. It seems now to be agreed that staining with toluid itie blue pH 4.0 indicates nucleic acids or sulfated mucopolysaccharides. The alcian blue stain developed by STEEDMAN (1950) and MOWRY (1956). is useful in identifying acid 148 Y. SASAI:

Fig. 6. Scleromyxedema. Aldehyde fuchsin-alcian blue (pH 1.5) sequence.

With some exception mucin colors with aldehyde fuchsin. ×400

mucopolysaccharides. LISON (1954) found that there was aclose agreement between staining with alcian blue and that with metachromatic dyes. On the other hand, an enzyme applied to a tissue section should, at least theoretically, remove only its specific substrate. If this manoevure renders a previously positive-staining reaction negative, the chemical nature of the stainable substance is apparently more specifi- cally identified. But, because of the relatively low specificity of the enzyme prepara- tions, there are a number of possible fallacies. In the present study, many definite and certain speculative interpretations of the results are warranted. It seems reasonable to conclude that mucins in the above- mentioned diseases contain two carbohydrate components, manifesting independent histochemical characteristics. One of these is a PAS-positive component. The other is an acid mucopolysaccharide which yields metachromasia with toluidine blue, and reacts with alcian blue. According to the mechanism suggested by FRENCH and BENDITT (1953). metachromasia in acid mucopolysaccharides may be inhibited by combining their anionic group with protein. LARSEN (1958) showed that the Histochemical Study of Acid Mucopolysaccharides in Mucinoses. 149 presence of free protein interferred with the demonstration of metachromasia in acid mucopolysaccharides when dilute solution of toluidine blue were utilized. At high temperature and low pH, according to him, highly concentrated dye cations can ex- change with the blocking protein, and metachromasia become manifest. Under the above conditions, alcian blue-reactive mucin manifested strong metachromasia. And also, with an alloxan-SCHIFF reaction, it was found to be rich in protein in the mucin-deposited. areas. KANTOR and SCHUBERT (1957) pointed out that methylation of acid muco- polysaccharides eliminated their metachromasia. In sulfated mucopolysaccharide, the sulfate group is removed by means of methylation, resulting in the substitution of hydroxyl group and the formation of free methyl sulfate ester. With carboxyl or phosphoryl groups, the acidic groups remain attached, and become esterified. In these latter cases, saponification restores the original structure and, therefore, the pro- perty of metachromasia. But, with sulfated mucopolysaccharides, restoration of the property of metachromasia by saponification is impossible. In the myxedema, this procedure caused the restitution both of the metachromasia and of the alcian blue staining. In the scleromyxedema, however, both the metachromasia at pH 4.1 and the alcian blue staining were partly restored. The histochemical application of aldehyde fuchsin was first described by GOMO- RI (1950). ABUL-HAJ and RINEHART (1952-1953), noting the polysaccharide character of aldehyde fuchsin-reactive substrate, concluded that the dye reacted with sulfated mucopolysaccharide. SPICER and MEYER (1960) observed that sulfated mucopolysaccharide usually colored intensely with aldehyde fuchsin, whereas non- sulfated relatively weakly. According to them, the former accepts the first stain in the aldehyde fuchsin-alcian blue as well as the alcian blue-aldehyde fuchsin procedure, whereas the latter stains with the second dye in aldehyde fuchsin-alcian blue pro- cedure and also in the reverse procedure. With some exceptions, the myxedema-mucin was colored by the second stain in aldehyde fuchsin-alcian blue sequence. However, the scleromyxedemamucin stained mostly with the first dye. Accordingly, it seems that the mucin in myxedema is mainly composed of non- sulfated mucopolysaccharide, and that in scleromyxedema it is composed of both non- sulfated and sulfated mucopolysaccharide.

IV. Summary.

1. The histochemical properties of acid mucopolysaccharides of mucin in pre- tibial myxedema and scleromyxedema were surveyed. 2. In myxedema, the mucin stained deeply with alcian blue, somewhat lightly with aldehyde fuchsin, and showed partly metachromasia. 3. In scleromyxedema, the mucin stained intensely with alcian blue, was meta- chromatic with toluidine blue, and manifested relatively strong affinity for aldehyde fuchsin.

内 容 自 抄.

脛 骨 前 に で き た ミ ク セ デ ー マ の2例 と ス ク レ ロ ミ ク セ デ ー マ の1例 よ り材 料 を 150 Y. SASAI:

採 取 し, そ れ ら に お け る ム チ ン に つ い て 組 織 化 学 的 に 検 索 した. 脛 骨 前 の ミ ク セ デ ー マ に あ っ て は, ム チ ン は ア ル シ ア ン 青 (pH1.5) に 強 染 し, ア ル デ ハ イ ド-フ ク シ ン に 淡 染 し, pH4.1に お い て トル イ ジ ン 青 に よ り メ タ ク ロ マ ジ ア を 示 した. ス ク レ ロ ミ ク セ デ ー マ に あ っ て は, ム チ ン はpH1.0に お い て ア ル シ ア ン 青 に 強

染 し, ア ル デ ハ イ ド-フ ク シ ン に 強 染 した。 ま た, pH2.0に お い て, トル イ ジ ン 青によりメタクロマジアをあらわ した. ミクセデーマのムチンはおもに非硫酸化 の ム コ 多 糖 類 を 持 ち, ス ク レ ロ ミ ク セ デ ー マ の も の は 非 硫 酸 化 と硫 酸 化 の ム コ 多

糖 類 を 持 つ よ う で あ る.

References.

Abul-Haj, S. K. and J. F. Rinehart: Fuchsin-aldehyde staining of sulfated mucopoly- saccharides and related substances. J. Nat. Canc. Inst. 13 (1952-1953). P. 232. - Dempsey, E. W., H. Bunting, M. Singer and G. B. Wislocki: The dye binding capacity and other chemo-histological properties of mammalian mucopolysaccharides. Anat. Rec. 98 (1947). P. 417. - Ehrlich, P.: Beitrage zur Kenntnis der Anilinfarbungen und ihrer Verwendung in der mikroskopischen Technik. Arch. mikr. Anat. 13 (1876-1877). S. 263. - Fisher, E. R. and R. D. Lillie: The effect of methylation on basophilia. J. Histochem. Cytochem. 2 (1954). P. 81. - French, J. E. and E. P. Benditt: The histochemistry of connective tissue, II. The effect of proteins on the selective staining of mucopolysaccharides by basic dyes. J. Histochem. Cytochem. 1 (1953). P. 321. - Gomori, G. L.: Aldehyde fuchsin: A new stain for elastic tissue. Amer. J. Clin. Path. 20 (1950). P. 665. - Halmi, N. S. and J. Davis: Comparison of aldehyde fuchsin staining, metachromasia and periodic acid-Schiff reactivity of various tissues. J. Histochem. Cytochem. 1 (1953). P. 447. - Hamerman, D. J. and M. Schubert: A quantitative study of the metachromasy in synovial fluid and mucin. J. gen. Physiol. 37 (1953). P. 291. - Kantor, T. G. and M. Schubert: A method for the desulfation of chondroitin sulfate. J. Amer. chem. Soc. 79 (1957). P. 152. - Keining, E. and O. Braun-Falco: zur Klinik und Pathogenese des Skleromyxoedems. Acta dermat. venereol. 36 (1956). S. 37. - Larsen, B.: Metachromasia inhibiting components in amyloid. J. Histochem. Cytochem. 6 (1958). P. 181. - Lewis, P. R. and T. A. L. Grillo: Histochemical demonstration of car- bohydrates by a convenient sulphation technique. Histochemie 1 (1959). P. 391. -Lillie, R. D.: Histopathologic Technic and Practical Histochemistry. New York, Blakiston Co., 1954. - Me- thylation and alkali demethylation. J. Histochem. Cytochem. 6 (1958). P. 131. - Lison, L.: Etudes sur la metachromasie. Colorants metachromatiques et substances chromatropes. Arch. de Biol. 46 (1935). P. 599. - Alcian blue 8G with chlorantine fast red 5B. A technique for selec- tive staining of mucopolysaccharides. Stain Technol. 29 (1954). P. 131. - Meyer, K.: The biological significance of hyaluronic acid and hyaluronidase. Physiol. Rev. 27 (1947). P. 335. - Mowry, R. W.: Alcian blue techniques for the histochemical study of acidic carbohydrates. J. Histochem. Cytochem. 4 (1956). P. 407. - Mowry, R. and C. H. Winkler: The coloration of acidic carbohydrates of bacteria and fungi in tissue sections with special reference to capsules of Cryptococcus neoformans, Pneumococcus and Staphylococcus. Amer. J. Path 36 (1956). P. 628. - Ohno, K., Notsuke, I. and T. Kawai: Histochemical study of acid mucopoly-saccharides. (Jap.) Igaku to Seibutsu-gaku. 19 (1951). P. 326. - Palitz, L. L. and M. J. Brunner: The mucinoses. A classification with histochemical studies on the nature of mucin. J. Invest. Dermat. 14 (1950). P. 159. - Spicer, S. S. and D. B. Meyer: Histochemical differentia- tion of acid mucopolysaccharides by means off combined aldehyde fuchsin-alcian blue staining. Histochemical Study of Acid Mucopolysaccharides in Mucinoses. 151

Amer. J. Clin. Path. 33 (1960). P. 453. - Steedman, H. F.: Alcian blue 8GS: A new stain for mucin. Quart. J. micr. Sci. 91 (1950). P. 477. - Sylven, B. and H. Malmgren: On the alleged metachromasia of hyaluronic acid. Lab. Invest. 1 (1952). P. 413. - Trotter, W. R. and K. C. Eden: Localized pretibial myxedema in association with toxic goitre. Quart. J. Med. 11 (1942). P. 229. - Watson, E. M. and R. H. Pearce: Mucopolysaccharide content of skin in localized (pretibial) myxedema. Amer. J. Clin. Path. 17 (1947). P. 507. - Wislocki, G. B., H. Bunting and E. W. Dempsey: Metachromasia in mammalian tissuess and its relation- ship to mucopolysaccharide. Amer. J. Anat. 81 (1947). P. 1.