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The Chemistry of the Human Sebaceous Gland*
THE CHEMISTRY OF THE HUMAN SEBACEOUS GLAND* I. HISTOCHEMICAL OBSERVATIONS
RAYMOND R. SUSKIND, M.D. WITH THE TECHNICAL ASSISTANCE OF CLARA KEENAN, ANN PRESNELL AND JEAN SEBASTIANI
The chemical processes which occur in the formation of normal sebum have been the concern of investigators for many years. The problems of determining the constituents of sebaceous secretion have been approached by various investigators from several directions. The first approach attempted was to study surface lipids. Attempts to analyze surface sebum were made as early as 1886 by Krukenberg (1), who collected lipids from the skin surface with blotting
paper. More recently Engman and Kooyman (2), Emanuel (3, 4), Rothman et al. (5, 6, 7), Weitkamp et al. (8), Butcher and Parnell (9), Kirk (10), Kvorning (11) and Kile, Snyder and Haefele (12) have collected surface lipids by various methods and determined several constituents quantitatively. A second method of approach has been that of studying the secretion or lipid material within the individual pilosebaceous structure and within the cells of the sebaceous acini. The investigators (13, 14-48) who have studied the individual gland have employed histochemical technics to localize and identify the different classes of lipid substances.
Both of the above methods of approach have apparent limitations. Surface lipids cannot be said to be derived exclusively from the sebaceous glands. It has
been known for a long time that normal epidermal cornification contributes cholesterol and perhaps other lipids to surface "sebum". There is still uncontradicted evidence that the sudoriparous glands may contribute fatty substances to the lipid covering of the cutaneous surface. The sum total of these fatty substances is probably acted upon by fat-splitting enzymes on the cutaneous surface and may account for the free fatty acids and higher alcohols which have been reported in surface lipid analyses. Hence, surface lipids are essentially chemically contaminated sebum. A third possible approach to the problems of the biochemistry of the human sebaceous gland is to conduct quantitative analyses of sebum collected from individual glands. The technical difficulties in the methods of collection and microanalysis are undoubtedly great, but not insurmountable. Histochemical methods for lipids have their own inherent limitations. Many
of the present methods lack absolute specificity. Several methods for lipids, *
From the Kettering Laboratory in the Department of Preventive Medicine and In-
dustrial Henith, and the Department of Dermatology and Syphilology, College of Medicine, University of Cincinnati. This work was made possible by n grant from the Bristol-Myers Company, New York,
N.Y. Read before the Society for Investigative Dermatology, San Francisco June 26, 1950. 37
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which still find their way into present day investigations, have been proved to have little or no histochemical validity (19). The conjoint application of several methods, each of which meets conditions which establish its histochemical validity (20), provides a series of scientific clues. When the set of clues is integrated, fundamental information may be obtained about the chemical processes in a cell, a gland or a skin appendage such as the pilosebaceous apparatus. Human sebaceous glands have been studied carefully with histochemical methods in one recent work by Montagna, who limited his observations to the glands of the human external auditory meatus (17). He and his co-workers previously demonstrated that considerable information may be obtained about the cytochemical constituents of the sebaceous glands of small mammals (l4-16, 18).
With these facts in mind, a series of experiments were undertaken to attempt to find out what chemical information concerning lipids and related enzymes of the human sebaceous gland might be obtained by means of histochemical methods. It was the purpose of these experiments to attempt to identify and localize different classes of lipids in the human sebaceous glands in apparently normal skin, and to compare this information with similar histochemical observations of altered pilosebaceous structures such as are seen in acne vulgaris, occupational acne and cutaneous cysts of possible sebaceous origin. MATERIALS
Sixty-one specimens of normal skin were obtained by biopsy or from post-mortem examinations from 24 persons ranging in age from new born to 92 years. This group of persons included 13 white males, 7 white females and 4 colored males. The areas of skin which were obtainable from post-mortem examinations included specimens from presternal, shoulder,
interscapular, and scalp regions. In one instance specimens of skin of the ala nasi were obtained. Four post mortem specimens from the pubic area were included in the series. Nine specimens of skin involved with early lesions of acne vulgaris were obtained from 4 patients: 2 white females and 2 white males, whose ages ranged from 13—22. They included
comedones, papules and nodules. Four comedones were examined for cholesterol. Two comedones were examined for alkaline phosphatase activity. Eleven biopsy specimens of early lesions of occupation acne (chloracne) were obtained from four patients who developed severe generalized acne as a result of exposure to tnchlorphenol or its polymer. The skin eruption was accompanied by systemic manifestations,
persistent increased prothrombin time, hypercholesteremia and hyperlipemia. These patients had clinical evidence of hepatic disease, recurrent peripheral neuritis and mild central nervous system symptoms. Six cutaneous cysts, diagnosed clinically as wens, were excised in toto from scalp, temporal area, back and chest of five adult persons. Histologically the cysts fulfilled the criteria for the diagnosis of epithelial cyst (31, 32). Two of the six cysts had, in addition, characteristics of sebaceous cysts (32). METHODS
All normal and abnormal specimens on which lipid detection methods were employed were fixed in Baker's formal-calcium and embedded in gelatin (21). In order to prevent loss of content of epithelial cysts, these six specimens were fixed in formal-calcium for 10 to 12 months. Prior to embedding, a portion of each specimen was treated with potassium dichromate (22) as a method of mordanting and rendering insoluble phospholipids for
CHEMISTRY OF HUMAN SEBACEOUS GLAND
39
staining with acid hematin. All of the specimens except the larger cysts were frozen and cut lOp in thickness. Cysts were cut iSp in thickness. Sections of each specimen were stained with Sudan IV and Sudan Black to demonstrate localization of all classes of lipids. Sections were also stained with Nile blue sulfate (20). The detection of phospholipids was attempted with Baker's acid hematin technic (21, 22, 25).
The technics employed to detect steroids were the Itomieu modification of the Liebermann-Burehard test (22), and treatment of sections with digitonin (22, 26). Unstained and untreated sections were examined under polarized light for birefringence. Alkaline phosphatase activity was studied in fresh biopsy specimens fixed in chilled acetone by the method of Gomori (27, 28). For purposes of histological identification and comparison, sections of all specimens were stained with hematoxylin and phloxine. OBSERYATIONS OF NORMAL TISSUES
Sudan Colorants
With few exceptions a rather distinct pattern of sudanophilic globule formation was noted in sebaceous gland acini in sections of skin from all age groups and all areas studied. At the fuadus of each acinus, the peripheral layer of sebaceous cells consisted generally of small cuboidal cells with relatively little cytoplasm, and an easily discernible centrally placed round nucleus. In the peripheral layer, or layers of fundic cells, sudanophilic globules were either absent, or were seen as small colored granules, few in number, and distributed throughout the cell (Fig. 4a, b, 7a). The more centrally located cells contained progressively greater numbers of sudanophilic globules which were larger in size. Subjacent to the acinar duct, their size was increased many fold and their cytoplasm filled with large sudanophilic globules (Fig. 4a, b). In the preductal portion of most active glands, large fat globules were admixed with shriveled nuclear particles. Lipid material commonly filled the entire duct and extended into the hair follicle. The sudanophilic substance extended from the duct, surrounded the hair shaft, covered the stratum corneum on the surface of the epithelium and was mixed with its keratin lamellae. In some of these sections, glands which had little or no sudanophilic material in the ducts and hair follicles, were frequently observed despite the usual pattern of lipid globule formation in the acini themselves. In two presternal specimens from persons in the older age group, the usual pattern of globule formation was not noted. The gland acini were small by comparison with the presternal sebaceous glands in the other subjects. The peripheral cells contained uniformly large globules, as did the central cells. This was noted in a 92 year old white man and a 70 year old white woman. When duplicate sections were treated with both Sudan IV and Sudan Black B, a difference in the staining capacity of the two colorants was noted. Sudan Black B not only stained the lipid globules much more intensely, hut stained globular material more uniformly than Sudan IV. Intracellular and extracellular globules which remained unstained with Sudan IV were frequently colored by Sudan Black B. A dynamic process of lipid globule formation is implied in the constant pat-
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tern which commences with the formation of small lipid granules in the peripheral cells of the fundus and ends with the extrusion of non-cellular lipid masses via the orifice of the hair follicle to cover the cutaneous surface.
Nile Blue Sulfate
The rose color imparted by Nile blue sulfate colorant revealed essentially the same pattern of lipid globule distribution in the sebaceous glands of all the areas studied as did the sudan colorants. A few globules of small size were stained in the peripheral cells of the acini. The globules were more numerous and larger in the centrally located cells. Masses of lipid substance stained a rose color in the
preductal part of the acini while in the duct, hair follicle and on the stratum corneum the color was light violet. The powdered Nile blue colorant is principally a blue oxazinc base. Oxidation
of the oxazine occurs in aqueous solution and is increased by the addition of sulfuric acid to the solution. The resultant staining solution is therefore a mixture of a blue oxazine and its oxidation product, an oxazone, which imparts a pink
color to certain of the lipids. Although this stain was originally believed to he specific for unsaturated triglycerides (pink) and fatty acids (blue) (29, 30), it has been demonstrated that the oxazone portion of the dye will stain unsaturated neutral fats, e.g. triglycerides, as well as hydrocarbons and cholesterol and its esters. The oxazine dye which imparts a blue color is not specific for fatty acids but also stains phospholipids and chromolipids (20, 23, 24). Birefringenee When unstained sections of all specimens were examined under polarized light,
optically active material was observed in the sebaceous gland of thirty of the forty-five normal specimens examined. Considerable numbers of crosses of polarization or spherocrystals were observed in the optically active sections (Fig. 4e, f). The occurrence of birefringence could not be correlated with age, or specific skin area. In most of the thirty specimens and in others in which no anisotropicity was demonstrable in the acini, birefringent masses were seen in the sebaceous gland ducts within the hair follicle and on the epidermal surface. The birefringence seen in the duct, follicle, and on the surface was commonly correlated with sudanophilic substance in the same areas. The property of birefringence varied from person to person and from gland to gland within the same section. When sebaceous acini from one cutaneous area of a given individual demonstrated marked anisotropicity, they were generally found to manifest the same intensity of birefringence in sections from different
areas, e.g. scalp, presternal, shoulder, interscapular areas. In a group of acini clustered about one follicle, marked birefringence might be observed throughout the acini and in the duct. In adjacent clusters of glands little or no birefringence might be observed. The sections in which marked anisotropicity was found throughout the sebaceous acini were from specimens which gave most intense reactions to the Lieber-
CHEMISTRY OF HUMAN SEBACEOUS GLAND
41
mann-Burchard test within the hair follicles and on the epidermal surface (Fig. 4e). This occurred in various cutaneous areas of four persons, ages 27—60, and included both sexes. Acid Hematin Baker's (22) technic, by which phospholipids are immobilized in situ and fixed,
afforded a method of detecting phospholipid collections in the sebaceous cell and in other parts of the pilosebaceous apparatus. Sections treated with the acid-hematin method revealed the following: 1) With several exceptions, the sudanophilic globules did not stain, but dispersed between the globules in no definite pattern, were minute black staining particles, round or rod-shaped (Fig. 5d). The interglobular particles were much more numerous in what appeared to be more active sebaceous acini than in the smaller, less active acini. These structures seem to correspond to those described by Montagna et a!. in the sebaceous glands of the external auditory meatus (17). In his opinion they fulfill the cytologic characteristics of Golgi bodies. The observation that they increase in number in hypersecreting acini supports this opinion (Fig. 5e). 2) In five out of twenty-four persons included in the present series there were blue masses throughout the sebaceous aiñi and more dense masses of the same
kind within the ducts and hair follicle. This is indicative of the presence of phosphatides in larger aggregates in the sebum of these persons (Fig. 5a, 7b).
3) Other structures which stained with this method were the erythrocytes, myelinated nerve fibres, the hair shaft, the eleidin-like granules of the Huxley layer of the inner root sheath and the lamellae of keratin immediately adjacent to the str atum granulosum. Tests for Steroids
1) Liebermann-Burchard Test
In twenty-two of the forty-five specimens of normal skin tested, the cells of the epidermis were colored light violet in the Liebermann-Burchard test. In the other specimens the epidermis did not color at all. In twenty-two specimens the lipid globules in the sebaceous acini developed the same light purple color. In no instance did the color ever progress to the green or blue-green stage which is usually accepted as being indicative of the presence of free or esterified cholesterol. In 13 specimens, a blue-green or green coloration developed on the skin surface. In skin specimens of three persons the same blue-green color developed in the sudanophilic material within the sebaceous ducts and hair follicles. The sebaceous glands of these persons showed marked anisotropicity. 2) Diqitonin The presence of free cholesterol in the sebaceous cells and acini as indicated
by its acetone insoluble digitonide could not be demonstrated in any of the specimens of normal skin which were tested with digitonin.
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Alkaline Phosphalase
Only biospy specimens of fresh skin were employed to determine alkaline phosphatase activity in the sebaceous glands. Repeated tests on six biopsies from
the iaterscapular area revealed no alkaline phosphatase activity within the sebaceous acini or their ducts. Positive reactions for alkaline phosphatase were demonstrable in the endothelium of arterioles adjacent to the gland acini, the hair shaft and hair papillae. THE CHEMICAL CONSTITUENTS OF THE HUMAN SEBACEOUS GLANDS HISTOCHEMICAL OBSERVATIONS
PATTERN OF SEBIJM FORMATION CONSTANT
PHOSPHOLIPIDS LARSER PHOSPHATIDE AGSRESATES in 21% 1PM
001.51 BODIES In oil specImens 1651
E
TRISLYCERIDES, WAXES,
FATTY ACIDS, ALCOHOLS
OHOLESTERYL ESTERS In SO%+ opectioeeo1Q5C I
No methods of identlflcotlon
FREE CHOLESTEROL Not identlf led
ALKALINE PHOSPHATASE
No octlolty
Fm. 1. The chemical constituents of the human sebaceous glands. Histochemical observations. OB5ERVATION5 IN ABNORMAL PILO5RBACEOU5 5TRUCTURE5
Acne Vulgaris
The same histochemical technics which were employed in an attempt to localize and identify lipids in normal skin were employed in nine biopsy specimens from acne vulgaris patients. Within the acini of the unplugged sebaceous glands which were found in some of the specimens, the same pattern of sudanophilic globule formation was observed in an exaggerated form. The cells close to the peripheral layer had large globules, and most of the central cells were bulging with large globules of lipid (Fig. 4c, d). The excretory ducts were filled with sudanophilic substance as was the hair follicle lumen and epidermal surface. Where the lumen of the follicular
orifice was dilated and plugged, the content of the ampulla-like structure was made up of an admixture of sudanophilic substance and concentrically arranged keratin-like material. Nile blue sulfate, as in the normal gland, stained most of the sudanophilic globules and revealed the same pattern as did the sudan dyes. When the glands whose cells were bulging with sudanophilic globules were stained
with the acid hematin, more numerous minute interglobular phospholipid particles were observed than in sections of normal skin (Fig. öe).
CHEMISTRY OF HUMAN SEBACEOIJS GLAND
43
In the limited number of sebaceous glands visualized in the biopsies of acne vulgaris lesions, birefringence within the acinar cells was observed only to a very limited extent, and in some acini not at all. However, the sebaceous ducts and follicles in the same sections were commonly filled with anisotropic material. In most sections of comedones, sudanophilic lipids made up the central mass of the plug content; in some, the lipids were distributed between the concentric
keratin laminations of the plug; in others the lipids were asymmetrically arranged in the plug. Irregular portions of the central sudanophilic mass stained intensely blue with acid hematin and were anisotropic. The sudanophihic lipids when present were stained pink or violet by Nile blue sulfate (Fig. 2, 5b), (6a, b). The innermost layer of the dilated follicle wall of comedones and early cysts was uniformly sudanophilic and markedly anisotropic. This layer, which stained intensely with acid hematin, was continuous with a layer of non-nucleated cells
of the epidermis which was superficial to the stratum granulosum. Located peripherally within the comedones at the proximal portion of the cyst-like structures and deeply staining with acid hematin, were collections of non-nucleated squamous structures. These were also observed within epithelial and sebaceous cysts. The sudanophilic masses in the comedones and early acne cysts were intensely
positive to both the Liebermann-Burchard and digitonin tests for cholesterol (Fig. 6a, h). Unlike the lipid in the normal sebaceous cell, the comedone mass contained considerable free cholesterol in addition to cholesteryl esters. Alkaline phosphatase activity was not observed within sebaceous glands of acne patients. In the comedones, moderate alkaline phosphatase activity was observed in the surface portion of the plug and at the site of some of the laminations (Fig. 5c). In the pustules of acne vulgaris, the area of liquefaction necrosis and dense infiltration of polymorphonuclear leukocytes contained few scattered sudanophilic droplets. In the deeper section of the necrotic area sudanophilic particles were more concentrated. No free phospholipids were observed in the neerotie area. Scattered tiny globules of neutral fats were observed in the necrotic area as well as in the wall of the pustules. Neither the contents of the pustules nor the surrounding walls were birefringent. Occupational Acne
rfhe sebaceous glands of the specimens taken from chloracne patients were hyperplastic. The pattern of sudanophilic globules in the acini was identical with that of the glands observed in sections of acne vulgaris. The globules stained
intensely with Nile blue sulfate and followed the sudan pattern in distribution within the acini. There were more acid hematin staining granules and rodlets in the sebaceous acini of these cases than in sebaceous glands of normal skin (Fig. 5e). Eight small cysts from these patients with occupational acne had the following histochemical characteristics: All the cysts were lined with a layer of sudanophilic material which stained
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intensely with acid hematin. Wherever a plugged orifice was demonstrated the lesion was histochemically similar to the comedo described in acne vulgaris. The chemical content of the intact cysts varied in composition. They contained variable amounts of keratin lamellae admixed with the identifiable lipids. Irregular masses which stained intensely with acid hematin were observed within the cysts. In the four specimens of cysts tested with the Liebermann-Burchard technic, strongly positive reactions were obtained. Th contents of two of four specimens, tested with digitonin were strongly positive. CONSTITUENTS IDENTIFIED HISTOCHEMICALLY IN COMEDONES
AND CYSTS OF ACNE VULARIS AND CHLORACNE
PHOS P H AlAS
PH OS PH OL IP ID
GHOLESTEROL
Frea and SIril'ed
SUDANOPHILIC LIPIDS
FIG. 2. Constituents indentified histochemically in comedones and cysts of acne vulgaris and chloracne.
Cutaneous Cysts
In cysts removed from the back and chest, portions of the wall and content had characteristics of sebaceous cysts as well as epithelial cysts. In a multiloculated cyst removed from the presternal area of a 70 year old white male, some locules contained lamellated masses of keratin interspersed with sudanophilic globules. Other locules were filled exclusively with intensely stained sudanophilic lipids. Much of the wall of this cyst had an hyperplastic keratohyalin layer. Upon staining with acid hematin, intensely stained cells which seemed to be desquamating into the cyst cavity were identified. The amorphous
and lamellated content also took the acid hematin stain. Large cholesterol crystals could be identified in the amorphous mass of the latter (Fig. 6c). In a second cyst from the chest wall of a 28 year old colored male, all of the cells of the cyst wall were compressed and, there were few intercellular bridges. The
cyst contained masses of sudanophilic lipid, but lamellated keratin was also
CHEMISTRY OF HUMAN SEBACEOLTS GLAND
45
present as well as a reticulated mass in which sudanophilic particles were disposed. The cyst content was digitonin positive. Only a small portion of the sudanophilic material within the cyst gave a positive reaction to the Liebermann-Burchard test. The unstained cyst content was markedly anisotropic. The other four cysts were unmistakeably epithelial cysts. Their cyst cavities were filled with concentric keratin lameilae. In the central portions of their contents were various sudanophilic masses, calcifications and large acicular crystals. Sudanophilic particles between lamellae and in central mass gave intensely positive reactions to the Liebermann-Burchard and digitonin tests. In two cysts the lamellations themselves reacted intensely to the LiebermannBurchard test. In unstained specimens of these cysts some of the concentric
laminations and most of amorphous material within the cyst content were intensely anisotropic. Concentric laminations were stained faintly by acid hematin (Fig. 3). CONSTITUENTS IDENTIFIED HISTOCHEMICALLY IN EPITHELIAL CYSTS
PHOSPHOLIPID .CHOLESTE AOL
Free arid Esterified SUDANOPHILIG LIPIDS
FiG. 3. Constituents identified histochemically in epithelial cysts DISCUSSION
The dynamics of sebum formation can be demonstrated morphologically with
the aid of the Sudan dyes. Sudan Black B, because of the uniformity and intensity with which it colors most lipids, is the most reliable of the sudan colorants. However, its opacity may obscure cytologic detail which may be retained with Sudan IV.
The increased secretory activity of the sebaceous gland in acne was easily demonstrated in the larger cells which were more generously filled with sudanophilic lipid. The observations made on many sections stained with Nile blue indicate that it provided no more information than did the general lipid colorants. It demon-
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strated the pattern of sebum formation but not as distinctly as did the Sudans. Nile blue sulfate consists, as has been stated heretofore, of a mixture of an oxazine dye and an oxazone colorant. According to Cain, one can distinguish with its
use "neutral" and "acidic" lipids (18). This investigator includes triglycerides, hydrocarbons, fat solvents except alcohol and acetone, higher alcohols such as cholesterol and its esters among the neutral lipids. The acidic lipids include fatty acids, phospholipids, chromolipoids. The blue oxazine colors the "acidic" lipids;
the rose oxazone colors the "neutral" lipids. Since Nile blue stains several classes of lipid in the same way it does not provide a means of differentiating one class of lipids from another in a tissue which apparently contains several. The minute rodlets and granules found in the cytoplasm of the sebaceous cell
and between the globules in the central portion of the acini seem to have the morphologic and histochemical characteristics of Golgi bodies or of mitochondna, as described previously by Bowen (33), Ludford (34), Nicolas (35) and Montagna (17). That their numbers were increased in the hyper secreting glands of acne vulgaris and occupational acne can be regarded as further evidence of their connection with the secretory process of the gland. The finding of larger amounts of phospholipid in the preductal portions of the normal acini and the excretory ducts of only five of sixteen persons tested with the Baker technic, is not inconsistent with the recent quantitative findings of Kvorning (lOd). Among 19 subjects whose surface lipids were analyzed for total lipids, triglycerides, unsaponifiable lipid, waxes, cholesterol and phosphatides, only six were found
to have more than 1 per cent phosphatide. Thirteen were found to have less than 1 per cent phosphatide. In the surface lipid extracted from five of the thirteen persons, no phosphatide was identifiable. The samples of surface lipid analyzed by Engman and Kooyman (2) for phospholipid were found to contain slightly more than 1 per cent of phospholipid. The conclusions drawn from the observations made for the presence of steroids depend significantly upon the interpretation of several findings. The only apparently definite results relating to cholesterol are those observed after treatment with digitonin. All of these tests were negative and, therefore, the probability of the presence of free cholesterol in the sebaceous acini, is small. Among the various lipids likely to be present in the sebaceous gland, only two may appear in ariisotropic form and produce crosses of polarization: cholesteryl esters and phosphatides. Since the number of sections of normal skin in which birefringence was observed was much larger than the number in which phospholipid was identified in any significant quantity, the spherocrystals are probably those of cholesteryl esters. An explanation of the methods employed to identify steroids may clarify the line of reasoning in connection with cholesterol and its esters at this point. The digitonin test will precipitate all 3 (3-hydroxy sterols and steroids of the same configuration. Cholesterol is the most significant of these in the human organism. The acetone insoluble cholesteryl digitonide crystals may be identified by their
anisotropicity and crosses of polarization. The Romieu modification of the Liebermann-Burchard color test requires the application of sulfuric acid and
.4
4 I. • •'
at' PL;
Pro. 4 a. Sebaceous glands from nose of newborn, Sudan Black B (225X). Demonstrates pattern of lipid distribution and formation in aeinus and duct.
b. Same area as (a) Sudan Black B, high power, (650X). Demonstrates pattern of lipid globule distribution in the peripheral and central cells of the acinus. c. Sebaceous glands from back of case of "Chloracne". Sudan Black B (225X). Demonstrates exaggerated pattern of lipid distribution. d. Same area as (e), Sudan Black B, high power, (650X). Demonstrates more numerous and larger lipid globules in peripheral aod central cells than in normal acinus. e. Marked birefringenee, showing spheroerystals in sebaceous glands and duets of skin of newborn. Unstained section photographed under polarized light (160X).
f. Moderate birefringence, showing spheroerystals in acini and duets of sebaceous glands. Unstained section photographed under polarized light (160X). 47
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B
FIG. 5 a. Sebaceous glands in skin of newborn, Bakers acid hematin stain (225X). Aggregates of phospholipid demonstrated, found in 21 per cent of normal persons. b. Acne vulgaris cyst, Bakers acid hematin stain (225x). Demonstrates phospholipid
in cyst content. c. Alkaline phospbatase activity in surface area and along laminations of comedo, acne vulgaris, Gomori technique (225X). d. Rodlets and granules in sebaceous acinus of normal scalp, phospholipid stain, Bakers acid hematin (1400X). They are tentatively identified as Golgi bodies.
e. Rodlets and granules in sebaceous acinus from back of acne patient. Increase in number of these structures observed over that seen in normal acinus.
CHEMISTRY OF HUMAN SEBACEOUS GLAND
49
acetic anhydride to give the required color reaction when either free or esterified cholesterol is preseat.
Fin. 6 a. Small cyst of acne vulgaris, uastaiocd and photographed under polarized light. Demonstrates mass of aoisotropic crystals in cyst content (160X). h. Small cyst of acne vulgaris, treated with digitonio and photographed under polarized light. The anisotropic mass is probably free cholesterol.
c. Cholesterol plates in epithelial cyst with sebaceous cyst features. Polarized light (650) .X Dark globule in center of crystal collection is stained by Sudan IV. Solid lipids or crystals do not stain with Sudan colorants.
Cholesteryl esters were thought to be present in the normal human sebaceous acini because: 1) Application of the Liebermann-Burchard test gave a light violet color to the lipid glohules in 22 of 45 normal specimens. This very mild reaction was no less intense than that given by the epidermis which is known to contain choles terol. This mild reaction may be significantly positive.
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2) The presence of spherocrystals or crosses of polarization in thirty of forty five specimens is evidence in support of the presence of cholesteryl esters.
3) The sections which, after treatment with the Liebermann-Burchard reagents, gave intensely positive reactions in the hair follicle and on the skin surface were from specimens which were also intensely anisotropic. 4) The presence of free cholesterol within the sebaceous acini may be ruled out by the universally negative reactions after treatment with digitonin.
FIG. 7a Sebaceous glands from nose of newborn, Sudan IV (650X). Note pattern of lipid globule distribution.
These are examples of four clues, each of which alone would not supply positive chemical information. When these clues are integrated, they give support to the presumption that the sebaceous acinus itself evolves cholesterol, which in all probability is present in an esterified form. The lipid constituents of acne vulgaris comedones and early cysts have not found to be qualitatively different from similar lesions of chloracne or from epithelial cysts. By histochemical methods one can demonstrate an admixture of phospholipid and free and combined cholesterol. The technies are not specific enough to identify other constituents such as triglycerides and fatty acids. The comedones and cysts of acne as well as epithelial cysts, are filled with varying
CHEMISTRY OF HUMAN SEJ3ACEOUS GLAND
51
amounts of keratin and lipid, and except for total size are indistinguishable histochemically frcm each other. Free and esterified cholesterol, as well as phospholipid, can be identified among the keratin laminations as well as in a
'I
t
4
S.
5
central mass of amorphous material in all three lesions. The information provided by the examination of this limited serles of normal and abnormal tissues, although revealing, is not conclusive enough to provide a pattern of metabolic synthesis such as that given by Melczer and Deme (13) for
Fie. 7b Sehaceous glands from nose of newborn, Bakers Acid-Hematin Technique (16OX). Note phospholipid material in excretory duets of sebaeeous glands.
human glands. Depending on numerous questionable assumptions regarding the
validity of their histochemical methods, Melczer and Deme conceived of a definite layering of specific lipids in the gland.
The metabolic pattern established by Montagna and Nohack (16c) for the rat was based upon much more valid methods. However, the localization of specific substances in the sebaceous glands of the rat differs from that indicated by our findings in human gland. The histochemical findings can best be supported by direct quantitative examination of lipids from individual glands. This Laboratory is now engaged in
the development of methods to attempt to make such examination possible
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TIlE JOURNAL OF INVESTIGATIVE DERMATOLOGY SUMMARY
1) A distinct pattern in the formation of globules of lipid is revealed by the use of the general fat colorants and Nile blue sulfate. The peripheral cells in the fundus of the acini contain little or no lipid. TLe more centrally located cells contain lipid globules which appear to become larger and/or more numerous in the central portion of the acini. In the preductAl region, the cells are disintegrated and the lipids coalesce and fill the excretory duct and hair follicle. In sebaceous glands of acne vulgaris and chloracne, the increased secretion of fat is visualized in the exaggerated pattern of the formation of lipid globule (Figs. 1, 4a—d and 7a). 2) The presence of phospholipids is limited in many glands to cytoplasmic granules and rodlets which are also found bet\veen the lipid globules. These
appear to be increased in number in the hyperactive glands of acne vulgaris and chloracnc. They are tentatively identified as Golgi bodies. In 21 per cent of persons studied, collections of phospholipid were observed in the excretory duct and follicle (Figs. 1, 5a, b, d, e and 7b). 3) ilistochemical methods give evidence of the presence of choleteryl esters in the sebaceous cell and in the sebum within the acini. There is no evidence of the presence of free cholesterol (Figs. 1, 2, Ga, b). 4) No alkaline phosphatase activity is observed within the sebaceous gland. 5) The uninfiamed plugged follicles and milia of acne vulgaris, the milia-like cysts of chloracne and epithelial cysts have comparable histological and histochemical features. Their lipids do not differ qualitatively from each other. In acne vulgaris there may be considerable or little sudanophilic lipid present with the keratin or the keratin may predominate. The same morphologic and histochemical variation is noted in the cysts of chloraene and in epithelial cysts. Each cyst contains identifiable phospholipids and free and combined cholesterol in varying amounts (Figs. 2, 3, Sb). 6) Nile blue sulfate has no value as a means of identifying a specific class of lipids in a tissue in which several classes of lipid are present. 7) ilistochemical identification of lipids in human sebaceous glands is, with present available methods, an indirect approach and requires corroboration by direct chemical analysis of sebaeeous secretion from individual glands.
The author is most grateful to Professor Joseph E. Homan, Director, Department of Medical Art, College of Medicine, University of Cincinnati, for his indefatigable efforts and sound advice in the preparation of the photographs of this report. REFERENCES
1. Quoted by Kvorning, S. A.: Investigations into the pharmacology of skin fats and ointments. I. The collection and quantitative determination of lipids on the skin. Aeta pharmaeol. 5: 248—261, 1949.
2. ENGMAN, M. F., AND KOOrMAN, D. J.: Lipids of the skin surface. Arch. Dermat. & Syph. 29: 12—19, 1934. 3. EMANUEL, S. V.: Quantitive determination of the sehaeeous glands' function, with
CHEMISTRY OF HUMAN SEBACEOUS GLAND
53
particular mention of the method employed. Aeta dermat.-venereol. 17: 444—456, 1936.
4. EMANUEL, S. V.: Mechanism of the sebum secretion. Acta dermat.-venereol. 19: 1—19, 1938.
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S., SMILJANIC, A. M., AND SHAPIRO, A. L.: Fungistatic action of hair fat on microsporon audouini. Proc. Soc. Exper. Biol. & Med. 60: 394—395, 1945.
ROTHMAN,
6. ROTHMAN, S., SHILJANIC, A. M.,AND WEITKAMP, A. W.: Mechanism of spontaneous cure
in puberty of ringworm of the scalp. Science. 104: 201—203, (Aug.) 1946. 7. ROTHMAN, S., SMILJANIC, A., SHAPIRO, A. L., AND WEITKAMP, A. W.: The spontaneous
cure ot tinea capitis in puberty. J. Invest. Dermat. 8: 81—98, 1947. 8. WEITKAHP, A. W., SMILJANIC, A. M., AND ROTHMAN, S.: The free fatty acids of human hair fat. J. Am. Chem. Soc. 69: 1936—1937, 1947.
9. BUTCHER, E. 0., AND PARNELL, J. P.: Sebaceous secretion on the human head. J. Invest. Dermat. 9: 67—74, 1947.
10. KIRK, E.: Quantitative determinations of the skin lipid secretion in middle-aged and old individuals. J. Gerontol. 3: 251—266, 1948.
11. KVORNING, S. A.: Investigations into the pharmacology of skin fats and ointments.
(a) The collection and quantitative determination of lipids on the skin. Acta pharmacol. 5: 248—261, 1949.
(b) On the occurrence and replenishment of fat on the skin in normal individuals. Ibid. 262—269, 1949.
(c) A method of determining the unsaponifiahle matter in lipid samples of less than 1 mg. Ibid. 375—382, 1949.
(d) Investigations into the composition of the lipids on the skin of normal individuals.
Ibid.
383—396, 1949.
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12. KILR, 11. L., SNYDER, H., AND HAEFRLE, Dermat. & Syph. 61: 792—799, 1950.
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W.: Nature of skin lipids in acne. Arch.
N. AND DaME, S.: Contribution to the activity of human sehaceous glands: Histologically demonstrable chemical changes during sehum formation. Derma-
13. MHLCZER, I.
tologica 86: 24—36, 1942.
14. MONTAGNA, W.: Histochemistry of the sehaceous gland of the rat. Anat. 11cc. 97: 356, 15.
1947.
MONTAGNA, W. AND HAMILTON, J. 13.: Histochemical analysis of the sebaceous glands
of the hamster. Federation Proc. 6: 166—167, 1947. 16. MONTAnNA, W. AND NORACK, C. R.: (a) The histology ot the preputial gland of the
rat.
Anat. 11cc. 96: 41—54, 1946.
preputial gland of the rat. Anat. 11cc. 96: 111—125, 1946. Histochemical observations on the sebaceous glands of the rat. Am. J. Anat. 81:
(b) The histochemistry of the (c)
39—GO, 1947. AND ZAK, F. G.: Pigment, lipids, and other substances in the glands of the external auditory meatus of man. Am. J. Anat. 83: 409—436, 1948.
17. M0NTAGNA, W., NORACK, C. 11.,
18. MONTAGNA, W. AND PAEK5, H. F.: A histochcmical study of the glands of the anal sac of the dog. Anat. 11cc. 100: 297—315, 1948. 19.
CAIN, A. J.: Histochemistry of lipoids in animals. Biol. Rev. Cambridge Philos. Soc. 25: 73—112, 1950.
20. LI50N, F.: Histochimi animale. Methodes et problemes. Paris, Gauthier-Villars, 1936. 21. BAKER, J. 11.: The structure and chemical composition of the Colgi element. Quart.
J. Micro. Sci. 85: 1—71, 1944. J. 11.: The histochemical
22. BAKER,
recognition of lipine. Quart. J. Micro. Sci. 87: 441—471,
1946.
23. CAIN,
A.
J.: The use of Nile blue in
the examination of lipoids. Quart. J. Micro.
Sci.
88: 383, 1947.
24. CAIN,
A.
25. BAKER,
J.: A further note on Nile blue. Quart. J. Micro. Sci. 89: 429, 1948.
J. 11.:
Further remarks on the histochemical recognition of lipine. Quart. J.
Micro. Sci. 88: 463—465, 1947.
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
26. CLICK. DAVID: Techniques of Histo- and Cytochemistry. New York, Interscience
Publishers, Inc., 1949. 27. GoMom, 0.: Microchemical demonstration of phosphatase in tissue sections. Proc. Soc. Exper. Biol. & Med. 50: 5—9, 1939.
28. C0M0EI, G.: Distribution of phosphatase in normal organs andtissues. J. Cell. & Comp. Physiol. 17: 71—83, 1941.
29. SMITH, J. L.: The simultaneous staining of neutral fat and fatty acid by oxazine dyes.
J. Path. 30.
SMITH, J.
& Bact. 12: 1—4, 1908. L.: The staining of fat by Nile blue sulfate. J. Path. & Bact. 15: 53—55, 1911.
31. Baonxas, A. C. AND
WILsoN, E.: Keratoms: A lesion often mistaken for sebaceous
cyst. S. Clin. North America. 10: 127—130, 1930.
32. Lova, W. H.
AND MONTGOMHRV, H.: Epithelial
cysts. Arch. Derraat. & Syph. 47: 185—
196, 1943. 33. BowxN, H. H.: The cytology of glandular secretion. Quart. ReV. Biol. 4: 484—519, 1929. 34: LUDFOHn, H. J.: The cytology of tsr tumors. Proc. Roy. Soc., London. 19: 557—577, 1925. HEGAIJn, C., FAVHE, M.: Sur les mitochondries des glandes sebacees de l'homme et sur la signification generale de ces organites du protoplasma. XVIIth.
35. NicoLAs, J.,
In. Congr. Med., Sec. 13, Dermat. & Syph. 101—104, 1914.
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linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
RAYMOND R. SUSKIND, M.D. WITH THE TECHNICAL ASSISTANCE OF CLARA KEENAN, ANN PRESNELL AND JEAN SEBASTIANI
The chemical processes which occur in the formation of normal sebum have been the concern of investigators for many years. The problems of determining the constituents of sebaceous secretion have been approached by various investigators from several directions. The first approach attempted was to study surface lipids. Attempts to analyze surface sebum were made as early as 1886 by Krukenberg (1), who collected lipids from the skin surface with blotting
paper. More recently Engman and Kooyman (2), Emanuel (3, 4), Rothman et al. (5, 6, 7), Weitkamp et al. (8), Butcher and Parnell (9), Kirk (10), Kvorning (11) and Kile, Snyder and Haefele (12) have collected surface lipids by various methods and determined several constituents quantitatively. A second method of approach has been that of studying the secretion or lipid material within the individual pilosebaceous structure and within the cells of the sebaceous acini. The investigators (13, 14-48) who have studied the individual gland have employed histochemical technics to localize and identify the different classes of lipid substances.
Both of the above methods of approach have apparent limitations. Surface lipids cannot be said to be derived exclusively from the sebaceous glands. It has
been known for a long time that normal epidermal cornification contributes cholesterol and perhaps other lipids to surface "sebum". There is still uncontradicted evidence that the sudoriparous glands may contribute fatty substances to the lipid covering of the cutaneous surface. The sum total of these fatty substances is probably acted upon by fat-splitting enzymes on the cutaneous surface and may account for the free fatty acids and higher alcohols which have been reported in surface lipid analyses. Hence, surface lipids are essentially chemically contaminated sebum. A third possible approach to the problems of the biochemistry of the human sebaceous gland is to conduct quantitative analyses of sebum collected from individual glands. The technical difficulties in the methods of collection and microanalysis are undoubtedly great, but not insurmountable. Histochemical methods for lipids have their own inherent limitations. Many
of the present methods lack absolute specificity. Several methods for lipids, *
From the Kettering Laboratory in the Department of Preventive Medicine and In-
dustrial Henith, and the Department of Dermatology and Syphilology, College of Medicine, University of Cincinnati. This work was made possible by n grant from the Bristol-Myers Company, New York,
N.Y. Read before the Society for Investigative Dermatology, San Francisco June 26, 1950. 37
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which still find their way into present day investigations, have been proved to have little or no histochemical validity (19). The conjoint application of several methods, each of which meets conditions which establish its histochemical validity (20), provides a series of scientific clues. When the set of clues is integrated, fundamental information may be obtained about the chemical processes in a cell, a gland or a skin appendage such as the pilosebaceous apparatus. Human sebaceous glands have been studied carefully with histochemical methods in one recent work by Montagna, who limited his observations to the glands of the human external auditory meatus (17). He and his co-workers previously demonstrated that considerable information may be obtained about the cytochemical constituents of the sebaceous glands of small mammals (l4-16, 18).
With these facts in mind, a series of experiments were undertaken to attempt to find out what chemical information concerning lipids and related enzymes of the human sebaceous gland might be obtained by means of histochemical methods. It was the purpose of these experiments to attempt to identify and localize different classes of lipids in the human sebaceous glands in apparently normal skin, and to compare this information with similar histochemical observations of altered pilosebaceous structures such as are seen in acne vulgaris, occupational acne and cutaneous cysts of possible sebaceous origin. MATERIALS
Sixty-one specimens of normal skin were obtained by biopsy or from post-mortem examinations from 24 persons ranging in age from new born to 92 years. This group of persons included 13 white males, 7 white females and 4 colored males. The areas of skin which were obtainable from post-mortem examinations included specimens from presternal, shoulder,
interscapular, and scalp regions. In one instance specimens of skin of the ala nasi were obtained. Four post mortem specimens from the pubic area were included in the series. Nine specimens of skin involved with early lesions of acne vulgaris were obtained from 4 patients: 2 white females and 2 white males, whose ages ranged from 13—22. They included
comedones, papules and nodules. Four comedones were examined for cholesterol. Two comedones were examined for alkaline phosphatase activity. Eleven biopsy specimens of early lesions of occupation acne (chloracne) were obtained from four patients who developed severe generalized acne as a result of exposure to tnchlorphenol or its polymer. The skin eruption was accompanied by systemic manifestations,
persistent increased prothrombin time, hypercholesteremia and hyperlipemia. These patients had clinical evidence of hepatic disease, recurrent peripheral neuritis and mild central nervous system symptoms. Six cutaneous cysts, diagnosed clinically as wens, were excised in toto from scalp, temporal area, back and chest of five adult persons. Histologically the cysts fulfilled the criteria for the diagnosis of epithelial cyst (31, 32). Two of the six cysts had, in addition, characteristics of sebaceous cysts (32). METHODS
All normal and abnormal specimens on which lipid detection methods were employed were fixed in Baker's formal-calcium and embedded in gelatin (21). In order to prevent loss of content of epithelial cysts, these six specimens were fixed in formal-calcium for 10 to 12 months. Prior to embedding, a portion of each specimen was treated with potassium dichromate (22) as a method of mordanting and rendering insoluble phospholipids for
CHEMISTRY OF HUMAN SEBACEOUS GLAND
39
staining with acid hematin. All of the specimens except the larger cysts were frozen and cut lOp in thickness. Cysts were cut iSp in thickness. Sections of each specimen were stained with Sudan IV and Sudan Black to demonstrate localization of all classes of lipids. Sections were also stained with Nile blue sulfate (20). The detection of phospholipids was attempted with Baker's acid hematin technic (21, 22, 25).
The technics employed to detect steroids were the Itomieu modification of the Liebermann-Burehard test (22), and treatment of sections with digitonin (22, 26). Unstained and untreated sections were examined under polarized light for birefringence. Alkaline phosphatase activity was studied in fresh biopsy specimens fixed in chilled acetone by the method of Gomori (27, 28). For purposes of histological identification and comparison, sections of all specimens were stained with hematoxylin and phloxine. OBSERYATIONS OF NORMAL TISSUES
Sudan Colorants
With few exceptions a rather distinct pattern of sudanophilic globule formation was noted in sebaceous gland acini in sections of skin from all age groups and all areas studied. At the fuadus of each acinus, the peripheral layer of sebaceous cells consisted generally of small cuboidal cells with relatively little cytoplasm, and an easily discernible centrally placed round nucleus. In the peripheral layer, or layers of fundic cells, sudanophilic globules were either absent, or were seen as small colored granules, few in number, and distributed throughout the cell (Fig. 4a, b, 7a). The more centrally located cells contained progressively greater numbers of sudanophilic globules which were larger in size. Subjacent to the acinar duct, their size was increased many fold and their cytoplasm filled with large sudanophilic globules (Fig. 4a, b). In the preductal portion of most active glands, large fat globules were admixed with shriveled nuclear particles. Lipid material commonly filled the entire duct and extended into the hair follicle. The sudanophilic substance extended from the duct, surrounded the hair shaft, covered the stratum corneum on the surface of the epithelium and was mixed with its keratin lamellae. In some of these sections, glands which had little or no sudanophilic material in the ducts and hair follicles, were frequently observed despite the usual pattern of lipid globule formation in the acini themselves. In two presternal specimens from persons in the older age group, the usual pattern of globule formation was not noted. The gland acini were small by comparison with the presternal sebaceous glands in the other subjects. The peripheral cells contained uniformly large globules, as did the central cells. This was noted in a 92 year old white man and a 70 year old white woman. When duplicate sections were treated with both Sudan IV and Sudan Black B, a difference in the staining capacity of the two colorants was noted. Sudan Black B not only stained the lipid globules much more intensely, hut stained globular material more uniformly than Sudan IV. Intracellular and extracellular globules which remained unstained with Sudan IV were frequently colored by Sudan Black B. A dynamic process of lipid globule formation is implied in the constant pat-
40
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
tern which commences with the formation of small lipid granules in the peripheral cells of the fundus and ends with the extrusion of non-cellular lipid masses via the orifice of the hair follicle to cover the cutaneous surface.
Nile Blue Sulfate
The rose color imparted by Nile blue sulfate colorant revealed essentially the same pattern of lipid globule distribution in the sebaceous glands of all the areas studied as did the sudan colorants. A few globules of small size were stained in the peripheral cells of the acini. The globules were more numerous and larger in the centrally located cells. Masses of lipid substance stained a rose color in the
preductal part of the acini while in the duct, hair follicle and on the stratum corneum the color was light violet. The powdered Nile blue colorant is principally a blue oxazinc base. Oxidation
of the oxazine occurs in aqueous solution and is increased by the addition of sulfuric acid to the solution. The resultant staining solution is therefore a mixture of a blue oxazine and its oxidation product, an oxazone, which imparts a pink
color to certain of the lipids. Although this stain was originally believed to he specific for unsaturated triglycerides (pink) and fatty acids (blue) (29, 30), it has been demonstrated that the oxazone portion of the dye will stain unsaturated neutral fats, e.g. triglycerides, as well as hydrocarbons and cholesterol and its esters. The oxazine dye which imparts a blue color is not specific for fatty acids but also stains phospholipids and chromolipids (20, 23, 24). Birefringenee When unstained sections of all specimens were examined under polarized light,
optically active material was observed in the sebaceous gland of thirty of the forty-five normal specimens examined. Considerable numbers of crosses of polarization or spherocrystals were observed in the optically active sections (Fig. 4e, f). The occurrence of birefringence could not be correlated with age, or specific skin area. In most of the thirty specimens and in others in which no anisotropicity was demonstrable in the acini, birefringent masses were seen in the sebaceous gland ducts within the hair follicle and on the epidermal surface. The birefringence seen in the duct, follicle, and on the surface was commonly correlated with sudanophilic substance in the same areas. The property of birefringence varied from person to person and from gland to gland within the same section. When sebaceous acini from one cutaneous area of a given individual demonstrated marked anisotropicity, they were generally found to manifest the same intensity of birefringence in sections from different
areas, e.g. scalp, presternal, shoulder, interscapular areas. In a group of acini clustered about one follicle, marked birefringence might be observed throughout the acini and in the duct. In adjacent clusters of glands little or no birefringence might be observed. The sections in which marked anisotropicity was found throughout the sebaceous acini were from specimens which gave most intense reactions to the Lieber-
CHEMISTRY OF HUMAN SEBACEOUS GLAND
41
mann-Burchard test within the hair follicles and on the epidermal surface (Fig. 4e). This occurred in various cutaneous areas of four persons, ages 27—60, and included both sexes. Acid Hematin Baker's (22) technic, by which phospholipids are immobilized in situ and fixed,
afforded a method of detecting phospholipid collections in the sebaceous cell and in other parts of the pilosebaceous apparatus. Sections treated with the acid-hematin method revealed the following: 1) With several exceptions, the sudanophilic globules did not stain, but dispersed between the globules in no definite pattern, were minute black staining particles, round or rod-shaped (Fig. 5d). The interglobular particles were much more numerous in what appeared to be more active sebaceous acini than in the smaller, less active acini. These structures seem to correspond to those described by Montagna et a!. in the sebaceous glands of the external auditory meatus (17). In his opinion they fulfill the cytologic characteristics of Golgi bodies. The observation that they increase in number in hypersecreting acini supports this opinion (Fig. 5e). 2) In five out of twenty-four persons included in the present series there were blue masses throughout the sebaceous aiñi and more dense masses of the same
kind within the ducts and hair follicle. This is indicative of the presence of phosphatides in larger aggregates in the sebum of these persons (Fig. 5a, 7b).
3) Other structures which stained with this method were the erythrocytes, myelinated nerve fibres, the hair shaft, the eleidin-like granules of the Huxley layer of the inner root sheath and the lamellae of keratin immediately adjacent to the str atum granulosum. Tests for Steroids
1) Liebermann-Burchard Test
In twenty-two of the forty-five specimens of normal skin tested, the cells of the epidermis were colored light violet in the Liebermann-Burchard test. In the other specimens the epidermis did not color at all. In twenty-two specimens the lipid globules in the sebaceous acini developed the same light purple color. In no instance did the color ever progress to the green or blue-green stage which is usually accepted as being indicative of the presence of free or esterified cholesterol. In 13 specimens, a blue-green or green coloration developed on the skin surface. In skin specimens of three persons the same blue-green color developed in the sudanophilic material within the sebaceous ducts and hair follicles. The sebaceous glands of these persons showed marked anisotropicity. 2) Diqitonin The presence of free cholesterol in the sebaceous cells and acini as indicated
by its acetone insoluble digitonide could not be demonstrated in any of the specimens of normal skin which were tested with digitonin.
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Alkaline Phosphalase
Only biospy specimens of fresh skin were employed to determine alkaline phosphatase activity in the sebaceous glands. Repeated tests on six biopsies from
the iaterscapular area revealed no alkaline phosphatase activity within the sebaceous acini or their ducts. Positive reactions for alkaline phosphatase were demonstrable in the endothelium of arterioles adjacent to the gland acini, the hair shaft and hair papillae. THE CHEMICAL CONSTITUENTS OF THE HUMAN SEBACEOUS GLANDS HISTOCHEMICAL OBSERVATIONS
PATTERN OF SEBIJM FORMATION CONSTANT
PHOSPHOLIPIDS LARSER PHOSPHATIDE AGSRESATES in 21% 1PM
001.51 BODIES In oil specImens 1651
E
TRISLYCERIDES, WAXES,
FATTY ACIDS, ALCOHOLS
OHOLESTERYL ESTERS In SO%+ opectioeeo1Q5C I
No methods of identlflcotlon
FREE CHOLESTEROL Not identlf led
ALKALINE PHOSPHATASE
No octlolty
Fm. 1. The chemical constituents of the human sebaceous glands. Histochemical observations. OB5ERVATION5 IN ABNORMAL PILO5RBACEOU5 5TRUCTURE5
Acne Vulgaris
The same histochemical technics which were employed in an attempt to localize and identify lipids in normal skin were employed in nine biopsy specimens from acne vulgaris patients. Within the acini of the unplugged sebaceous glands which were found in some of the specimens, the same pattern of sudanophilic globule formation was observed in an exaggerated form. The cells close to the peripheral layer had large globules, and most of the central cells were bulging with large globules of lipid (Fig. 4c, d). The excretory ducts were filled with sudanophilic substance as was the hair follicle lumen and epidermal surface. Where the lumen of the follicular
orifice was dilated and plugged, the content of the ampulla-like structure was made up of an admixture of sudanophilic substance and concentrically arranged keratin-like material. Nile blue sulfate, as in the normal gland, stained most of the sudanophilic globules and revealed the same pattern as did the sudan dyes. When the glands whose cells were bulging with sudanophilic globules were stained
with the acid hematin, more numerous minute interglobular phospholipid particles were observed than in sections of normal skin (Fig. öe).
CHEMISTRY OF HUMAN SEBACEOIJS GLAND
43
In the limited number of sebaceous glands visualized in the biopsies of acne vulgaris lesions, birefringence within the acinar cells was observed only to a very limited extent, and in some acini not at all. However, the sebaceous ducts and follicles in the same sections were commonly filled with anisotropic material. In most sections of comedones, sudanophilic lipids made up the central mass of the plug content; in some, the lipids were distributed between the concentric
keratin laminations of the plug; in others the lipids were asymmetrically arranged in the plug. Irregular portions of the central sudanophilic mass stained intensely blue with acid hematin and were anisotropic. The sudanophihic lipids when present were stained pink or violet by Nile blue sulfate (Fig. 2, 5b), (6a, b). The innermost layer of the dilated follicle wall of comedones and early cysts was uniformly sudanophilic and markedly anisotropic. This layer, which stained intensely with acid hematin, was continuous with a layer of non-nucleated cells
of the epidermis which was superficial to the stratum granulosum. Located peripherally within the comedones at the proximal portion of the cyst-like structures and deeply staining with acid hematin, were collections of non-nucleated squamous structures. These were also observed within epithelial and sebaceous cysts. The sudanophilic masses in the comedones and early acne cysts were intensely
positive to both the Liebermann-Burchard and digitonin tests for cholesterol (Fig. 6a, h). Unlike the lipid in the normal sebaceous cell, the comedone mass contained considerable free cholesterol in addition to cholesteryl esters. Alkaline phosphatase activity was not observed within sebaceous glands of acne patients. In the comedones, moderate alkaline phosphatase activity was observed in the surface portion of the plug and at the site of some of the laminations (Fig. 5c). In the pustules of acne vulgaris, the area of liquefaction necrosis and dense infiltration of polymorphonuclear leukocytes contained few scattered sudanophilic droplets. In the deeper section of the necrotic area sudanophilic particles were more concentrated. No free phospholipids were observed in the neerotie area. Scattered tiny globules of neutral fats were observed in the necrotic area as well as in the wall of the pustules. Neither the contents of the pustules nor the surrounding walls were birefringent. Occupational Acne
rfhe sebaceous glands of the specimens taken from chloracne patients were hyperplastic. The pattern of sudanophilic globules in the acini was identical with that of the glands observed in sections of acne vulgaris. The globules stained
intensely with Nile blue sulfate and followed the sudan pattern in distribution within the acini. There were more acid hematin staining granules and rodlets in the sebaceous acini of these cases than in sebaceous glands of normal skin (Fig. 5e). Eight small cysts from these patients with occupational acne had the following histochemical characteristics: All the cysts were lined with a layer of sudanophilic material which stained
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
intensely with acid hematin. Wherever a plugged orifice was demonstrated the lesion was histochemically similar to the comedo described in acne vulgaris. The chemical content of the intact cysts varied in composition. They contained variable amounts of keratin lamellae admixed with the identifiable lipids. Irregular masses which stained intensely with acid hematin were observed within the cysts. In the four specimens of cysts tested with the Liebermann-Burchard technic, strongly positive reactions were obtained. Th contents of two of four specimens, tested with digitonin were strongly positive. CONSTITUENTS IDENTIFIED HISTOCHEMICALLY IN COMEDONES
AND CYSTS OF ACNE VULARIS AND CHLORACNE
PHOS P H AlAS
PH OS PH OL IP ID
GHOLESTEROL
Frea and SIril'ed
SUDANOPHILIC LIPIDS
FIG. 2. Constituents indentified histochemically in comedones and cysts of acne vulgaris and chloracne.
Cutaneous Cysts
In cysts removed from the back and chest, portions of the wall and content had characteristics of sebaceous cysts as well as epithelial cysts. In a multiloculated cyst removed from the presternal area of a 70 year old white male, some locules contained lamellated masses of keratin interspersed with sudanophilic globules. Other locules were filled exclusively with intensely stained sudanophilic lipids. Much of the wall of this cyst had an hyperplastic keratohyalin layer. Upon staining with acid hematin, intensely stained cells which seemed to be desquamating into the cyst cavity were identified. The amorphous
and lamellated content also took the acid hematin stain. Large cholesterol crystals could be identified in the amorphous mass of the latter (Fig. 6c). In a second cyst from the chest wall of a 28 year old colored male, all of the cells of the cyst wall were compressed and, there were few intercellular bridges. The
cyst contained masses of sudanophilic lipid, but lamellated keratin was also
CHEMISTRY OF HUMAN SEBACEOLTS GLAND
45
present as well as a reticulated mass in which sudanophilic particles were disposed. The cyst content was digitonin positive. Only a small portion of the sudanophilic material within the cyst gave a positive reaction to the Liebermann-Burchard test. The unstained cyst content was markedly anisotropic. The other four cysts were unmistakeably epithelial cysts. Their cyst cavities were filled with concentric keratin lameilae. In the central portions of their contents were various sudanophilic masses, calcifications and large acicular crystals. Sudanophilic particles between lamellae and in central mass gave intensely positive reactions to the Liebermann-Burchard and digitonin tests. In two cysts the lamellations themselves reacted intensely to the LiebermannBurchard test. In unstained specimens of these cysts some of the concentric
laminations and most of amorphous material within the cyst content were intensely anisotropic. Concentric laminations were stained faintly by acid hematin (Fig. 3). CONSTITUENTS IDENTIFIED HISTOCHEMICALLY IN EPITHELIAL CYSTS
PHOSPHOLIPID .CHOLESTE AOL
Free arid Esterified SUDANOPHILIG LIPIDS
FiG. 3. Constituents identified histochemically in epithelial cysts DISCUSSION
The dynamics of sebum formation can be demonstrated morphologically with
the aid of the Sudan dyes. Sudan Black B, because of the uniformity and intensity with which it colors most lipids, is the most reliable of the sudan colorants. However, its opacity may obscure cytologic detail which may be retained with Sudan IV.
The increased secretory activity of the sebaceous gland in acne was easily demonstrated in the larger cells which were more generously filled with sudanophilic lipid. The observations made on many sections stained with Nile blue indicate that it provided no more information than did the general lipid colorants. It demon-
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
strated the pattern of sebum formation but not as distinctly as did the Sudans. Nile blue sulfate consists, as has been stated heretofore, of a mixture of an oxazine dye and an oxazone colorant. According to Cain, one can distinguish with its
use "neutral" and "acidic" lipids (18). This investigator includes triglycerides, hydrocarbons, fat solvents except alcohol and acetone, higher alcohols such as cholesterol and its esters among the neutral lipids. The acidic lipids include fatty acids, phospholipids, chromolipoids. The blue oxazine colors the "acidic" lipids;
the rose oxazone colors the "neutral" lipids. Since Nile blue stains several classes of lipid in the same way it does not provide a means of differentiating one class of lipids from another in a tissue which apparently contains several. The minute rodlets and granules found in the cytoplasm of the sebaceous cell
and between the globules in the central portion of the acini seem to have the morphologic and histochemical characteristics of Golgi bodies or of mitochondna, as described previously by Bowen (33), Ludford (34), Nicolas (35) and Montagna (17). That their numbers were increased in the hyper secreting glands of acne vulgaris and occupational acne can be regarded as further evidence of their connection with the secretory process of the gland. The finding of larger amounts of phospholipid in the preductal portions of the normal acini and the excretory ducts of only five of sixteen persons tested with the Baker technic, is not inconsistent with the recent quantitative findings of Kvorning (lOd). Among 19 subjects whose surface lipids were analyzed for total lipids, triglycerides, unsaponifiable lipid, waxes, cholesterol and phosphatides, only six were found
to have more than 1 per cent phosphatide. Thirteen were found to have less than 1 per cent phosphatide. In the surface lipid extracted from five of the thirteen persons, no phosphatide was identifiable. The samples of surface lipid analyzed by Engman and Kooyman (2) for phospholipid were found to contain slightly more than 1 per cent of phospholipid. The conclusions drawn from the observations made for the presence of steroids depend significantly upon the interpretation of several findings. The only apparently definite results relating to cholesterol are those observed after treatment with digitonin. All of these tests were negative and, therefore, the probability of the presence of free cholesterol in the sebaceous acini, is small. Among the various lipids likely to be present in the sebaceous gland, only two may appear in ariisotropic form and produce crosses of polarization: cholesteryl esters and phosphatides. Since the number of sections of normal skin in which birefringence was observed was much larger than the number in which phospholipid was identified in any significant quantity, the spherocrystals are probably those of cholesteryl esters. An explanation of the methods employed to identify steroids may clarify the line of reasoning in connection with cholesterol and its esters at this point. The digitonin test will precipitate all 3 (3-hydroxy sterols and steroids of the same configuration. Cholesterol is the most significant of these in the human organism. The acetone insoluble cholesteryl digitonide crystals may be identified by their
anisotropicity and crosses of polarization. The Romieu modification of the Liebermann-Burchard color test requires the application of sulfuric acid and
.4
4 I. • •'
at' PL;
Pro. 4 a. Sebaceous glands from nose of newborn, Sudan Black B (225X). Demonstrates pattern of lipid distribution and formation in aeinus and duct.
b. Same area as (a) Sudan Black B, high power, (650X). Demonstrates pattern of lipid globule distribution in the peripheral and central cells of the acinus. c. Sebaceous glands from back of case of "Chloracne". Sudan Black B (225X). Demonstrates exaggerated pattern of lipid distribution. d. Same area as (e), Sudan Black B, high power, (650X). Demonstrates more numerous and larger lipid globules in peripheral aod central cells than in normal acinus. e. Marked birefringenee, showing spheroerystals in sebaceous glands and duets of skin of newborn. Unstained section photographed under polarized light (160X).
f. Moderate birefringence, showing spheroerystals in acini and duets of sebaceous glands. Unstained section photographed under polarized light (160X). 47
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B
FIG. 5 a. Sebaceous glands in skin of newborn, Bakers acid hematin stain (225X). Aggregates of phospholipid demonstrated, found in 21 per cent of normal persons. b. Acne vulgaris cyst, Bakers acid hematin stain (225x). Demonstrates phospholipid
in cyst content. c. Alkaline phospbatase activity in surface area and along laminations of comedo, acne vulgaris, Gomori technique (225X). d. Rodlets and granules in sebaceous acinus of normal scalp, phospholipid stain, Bakers acid hematin (1400X). They are tentatively identified as Golgi bodies.
e. Rodlets and granules in sebaceous acinus from back of acne patient. Increase in number of these structures observed over that seen in normal acinus.
CHEMISTRY OF HUMAN SEBACEOUS GLAND
49
acetic anhydride to give the required color reaction when either free or esterified cholesterol is preseat.
Fin. 6 a. Small cyst of acne vulgaris, uastaiocd and photographed under polarized light. Demonstrates mass of aoisotropic crystals in cyst content (160X). h. Small cyst of acne vulgaris, treated with digitonio and photographed under polarized light. The anisotropic mass is probably free cholesterol.
c. Cholesterol plates in epithelial cyst with sebaceous cyst features. Polarized light (650) .X Dark globule in center of crystal collection is stained by Sudan IV. Solid lipids or crystals do not stain with Sudan colorants.
Cholesteryl esters were thought to be present in the normal human sebaceous acini because: 1) Application of the Liebermann-Burchard test gave a light violet color to the lipid glohules in 22 of 45 normal specimens. This very mild reaction was no less intense than that given by the epidermis which is known to contain choles terol. This mild reaction may be significantly positive.
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
2) The presence of spherocrystals or crosses of polarization in thirty of forty five specimens is evidence in support of the presence of cholesteryl esters.
3) The sections which, after treatment with the Liebermann-Burchard reagents, gave intensely positive reactions in the hair follicle and on the skin surface were from specimens which were also intensely anisotropic. 4) The presence of free cholesterol within the sebaceous acini may be ruled out by the universally negative reactions after treatment with digitonin.
FIG. 7a Sebaceous glands from nose of newborn, Sudan IV (650X). Note pattern of lipid globule distribution.
These are examples of four clues, each of which alone would not supply positive chemical information. When these clues are integrated, they give support to the presumption that the sebaceous acinus itself evolves cholesterol, which in all probability is present in an esterified form. The lipid constituents of acne vulgaris comedones and early cysts have not found to be qualitatively different from similar lesions of chloracne or from epithelial cysts. By histochemical methods one can demonstrate an admixture of phospholipid and free and combined cholesterol. The technies are not specific enough to identify other constituents such as triglycerides and fatty acids. The comedones and cysts of acne as well as epithelial cysts, are filled with varying
CHEMISTRY OF HUMAN SEJ3ACEOUS GLAND
51
amounts of keratin and lipid, and except for total size are indistinguishable histochemically frcm each other. Free and esterified cholesterol, as well as phospholipid, can be identified among the keratin laminations as well as in a
'I
t
4
S.
5
central mass of amorphous material in all three lesions. The information provided by the examination of this limited serles of normal and abnormal tissues, although revealing, is not conclusive enough to provide a pattern of metabolic synthesis such as that given by Melczer and Deme (13) for
Fie. 7b Sehaceous glands from nose of newborn, Bakers Acid-Hematin Technique (16OX). Note phospholipid material in excretory duets of sebaeeous glands.
human glands. Depending on numerous questionable assumptions regarding the
validity of their histochemical methods, Melczer and Deme conceived of a definite layering of specific lipids in the gland.
The metabolic pattern established by Montagna and Nohack (16c) for the rat was based upon much more valid methods. However, the localization of specific substances in the sebaceous glands of the rat differs from that indicated by our findings in human gland. The histochemical findings can best be supported by direct quantitative examination of lipids from individual glands. This Laboratory is now engaged in
the development of methods to attempt to make such examination possible
52
TIlE JOURNAL OF INVESTIGATIVE DERMATOLOGY SUMMARY
1) A distinct pattern in the formation of globules of lipid is revealed by the use of the general fat colorants and Nile blue sulfate. The peripheral cells in the fundus of the acini contain little or no lipid. TLe more centrally located cells contain lipid globules which appear to become larger and/or more numerous in the central portion of the acini. In the preductAl region, the cells are disintegrated and the lipids coalesce and fill the excretory duct and hair follicle. In sebaceous glands of acne vulgaris and chloracne, the increased secretion of fat is visualized in the exaggerated pattern of the formation of lipid globule (Figs. 1, 4a—d and 7a). 2) The presence of phospholipids is limited in many glands to cytoplasmic granules and rodlets which are also found bet\veen the lipid globules. These
appear to be increased in number in the hyperactive glands of acne vulgaris and chloracnc. They are tentatively identified as Golgi bodies. In 21 per cent of persons studied, collections of phospholipid were observed in the excretory duct and follicle (Figs. 1, 5a, b, d, e and 7b). 3) ilistochemical methods give evidence of the presence of choleteryl esters in the sebaceous cell and in the sebum within the acini. There is no evidence of the presence of free cholesterol (Figs. 1, 2, Ga, b). 4) No alkaline phosphatase activity is observed within the sebaceous gland. 5) The uninfiamed plugged follicles and milia of acne vulgaris, the milia-like cysts of chloracne and epithelial cysts have comparable histological and histochemical features. Their lipids do not differ qualitatively from each other. In acne vulgaris there may be considerable or little sudanophilic lipid present with the keratin or the keratin may predominate. The same morphologic and histochemical variation is noted in the cysts of chloraene and in epithelial cysts. Each cyst contains identifiable phospholipids and free and combined cholesterol in varying amounts (Figs. 2, 3, Sb). 6) Nile blue sulfate has no value as a means of identifying a specific class of lipids in a tissue in which several classes of lipid are present. 7) ilistochemical identification of lipids in human sebaceous glands is, with present available methods, an indirect approach and requires corroboration by direct chemical analysis of sebaeeous secretion from individual glands.
The author is most grateful to Professor Joseph E. Homan, Director, Department of Medical Art, College of Medicine, University of Cincinnati, for his indefatigable efforts and sound advice in the preparation of the photographs of this report. REFERENCES
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