The fatty acids of human sebaceous gland phosphatidylcholine.

The fatty acids of human sebaceous gland phosphatidylcholine were examined by gas chromatography and by analysis of the double bond positions in the C-16 and C-18 monoenes. Compared to phosphatidylcholine from other organs, sebaceous gland phosphatidylcholine was found to be deficient in essential fatty acids and poly-unsaturated fatty acids. The relatively large mono-unsaturated fatty acid fraction consisted predominantly of fatty acids with delta6- and delta8-unsaturation. Analysis indicates that the fatty acids of sebaceous gland phospholipids are predominantly of types synthesized in sebaceous glands.

A reevaluation of fatty acids as inflammatory agents in acne.

The currently widely held theory that intrafollicular free fatty acids (FA) are the primary agents instigating inflammatory changes in acne is based on circumstantial evidence. There is no direct evidence that FA in physiologic concentrations are inflammatory. In the present study the quantities of FA present in isolated pilosebaceous ducts and in isolated comedones were analyzed. Using these values, the effect of FA on intracutaneous injection into human skin was investigated. The range of FA in 257 isolated pilosebaceous ducts from skin of the upper back of 10 male subjects was 0.19 to 2.43 mug, with an average of 0.89 +/- 0.75 mug of FA per duct. The mean FA content in 45 open comedones was 63.6 +/- 24.8 mug per comedone. Fatty acids for intracutaneous testing were isolated from human skin surface lipids and from hydrolyzed triglycerides purified from pooled isolated sebaceous glands. Twenty-six subjects received 100 mug of FA intracutaneously in the upper back. The response to FA injections could not be distinguished from the response to saline control injections. By 24 hr no erythema, induration, or any visible marks of inflammation were present in the skin of any of the subjects tested. At the histologic level a mild inflammatory infiltrate consisting perdominantly of lymphocytes was slightly more marked in the FA injection site than in the saline control injection site. Increasing the amount of FA injected to 500 mug still produced no visible inflammatory response in human skin. We conclude that intracutaneous injections of FA in physiologic concentrations do not produce more than a very mild inflammatory reaction in human skin and suggest that the role of Propionibacterium acnes in the pathogeneisis of acne may be more complex than merely as a source of intrafollicular lipases.

Characterization of wax esters, triglycerides, and free fatty acids of follicular casts.

The abnormal impactation of a sebaceous follicle (the follicular cast) has been implicated as the preclinical lesion of acne vulgaris. We have characterized the lipid composition of these structures in the first of a series of studies aimed at the identification of sebaceous lipids that may be associated and/or responsible for the initiation of clinical lesions. The lipid composition of follicular casts was analyzed using thin-layer chromatography, gas chromatography, and mass spectrometry. The mean wet weight of the casts was 24.7 +/- 8.6 micrograms and 7.2 +/- 5.6 micrograms (29.4 +/- 13.5%) was lipid. Cholesterol (3.8 +/- 1.8%) and cholesterol esters (2.0 +/- 2.7%), wax esters (25.3 +/- 6.0%), squalene (19.9 +/- 6.6%), triglycerides (16.1 +/- 7.8%), and free fatty acids (33.0 +/- 10.0%) were all present in cast lipid. Fatty acids of the free fatty acid and triglyceride fraction ranged from C12 to C22. The major components of the free fatty acids were C14:0, C15:0, C16:1, C16:0, 2-me-C17:0, and C18:1. In the triglyceride fraction C14:0, C15:0, C16:0, C18:1, and C18:0 dominated. The free fatty acids were composed of normal saturated (50.6%), normal unsaturated (32.8%), and monomethyl branched (16.6%) acids; the triglyceride fraction contained (86.3%) normal saturated (10.8%), normal unsaturated, and (3.0%) monomethyl branched fatty acids. Wax esters of follicular casts included esters ranging from C26:1 to C38:0. Saturated esters predominated and both odd- and even-numbered esters were present. The most abundant fatty acid moieties of these esters were C16:0 and C15:0, whereas C14:0, C17:0, and C20:0 were the most frequently detected alcohol moieties.

Autoradiographic localization of tritiated dihydrotestosterone in the flank organ of the albino hamster.

In the hamster flank organ, the growth of hair and growth of sebaceous glands are androgen-dependent functions. Although dihydrotestosterone (DHT) is known to be a potent stimulator of flank organ growth, there is no information about localization of DHT receptor sites in this organ. The purpose of this study was to use steroid autoradiography to localize DHT receptors in the hamster flank organ. Because steroid hormones are functional when translocated to nuclear receptors, nuclear localization by autoradiography defines receptor sites. In order to be able to visualize autoradiographic grains from radiolabeled androgens around hair follicles, albino hamsters were studied to avoid confusion between the grains and pigment granules which are abundant in the more common Golden Syrian hamster. Mature male hamsters castrated 24 hours earlier were given tritium-labeled dihydrotestosterone ( [3H]DHT). Using the technique of thaw-mount steroid autoradiography, 4-micron unfixed frozen sections were mounted in the dark onto emulsion-coated glass slides and allowed to develop for 4-6 months. [3H]DHT was found to be concentrated over sebocyte nuclei. The label was present peripherally as well as in differentiating sebocytes. There was no nuclear localization of [3H]DHT in animals pretreated with excessive quantities of unlabeled DHT. Steroid metabolites of [3H] DHT were assessed by thin-layer chromatography in paired tissue samples. Most of the label remained with DHT. Uptake was inhibited in the flank organ of hamsters pretreated with unlabeled DHT. Specific DHT receptors in the albino hamster flank organ are located in peripheral and differentiating sebocytes. Steroid autoradiography is a useful tool to study androgen interaction in the skin.

Analysis of lipid composition of isolated human sebaceous gland homogenates after incubation with cutaneous bacteria. Thin-layer chromatography.

The effects of specific species of skin bacteria on human sebaceous gland lipids in vitro were analyzed. Isolated dissected sebaceous glands were pooled, homogenized, and sterilized, then incorporated into peptone-yeast extract medium and used as substrate for growth of Propionibacterium acnes, P. granulosum, and Staphylococcus epidermidis subgroup II. The sebaceous lipids were analyzed by thin-layer chromatography before and after bacterial growth. The most striking effect of bacteria on sebaceous gland lipid composition was the hydrolysis of sebaceous triglycerides. The degree of hydrolysis varied with bacterial strain but was most complete with P. acnes and P. granulosum. Staphylococci were not effective in hydrolyzing sebaceous triglycerides at pH 4.5 although, when the pH of the medium was raised to pH 6.4, some strains of staphylococci were as effective as the propionibacteria in hydrolyzing sebaceous triglycerides to free fatty acids. Thus minor changes in acidity may play asignificant role in controlling the lipolytic activity of staphylococci on skin. Another effect of bacterial action on sebaceous gland lipids was the esterification of sebaceous cholesterol to cholesteryl esters. Thus, bacterial action must be taken into account in evaluating studies of alterations in cutaneous cholesterol and cholesteryl esters in skin surface lipids in normal and disease states.

Isotretinoin treatment of severe acne affects the endogenous concentration of vitamin A in sebaceous glands.

An investigation of pooled skin samples from 22 acne patients has shown that isotretinoin and its major metabolite, 4-oxo-isotretinoin, can be detected in sebaceous glands during treatment with isotretinoin (1 mg/kg/d for 4 months). The levels are less than those in the epidermis, thus excluding selective drug distribution as a prime explanation for drug function. Oral isotretinoin markedly increases retinol levels and decreases dehydroretinol levels in the skin while on therapy. The effect is more pronounced in sebaceous glands than in epidermis and dermis. The increased retinol levels probably reflect a metabolic interference with endogenous vitamin A, since isotretinoin cannot be converted into retinol in vivo. Previous studies have shown that dehydroretinol accumulates in hyperproliferative, keratinizing skin lesions and so its reduction with isotretinoin therapy may relate to a reduction in cell proliferation or to dedifferentiation. However, the precise interrelationships of these observations need further elucidation.

Fatty acids of acylceramides from comedones and from the skin surface of acne patients and control subjects.

Comedonal lipids and skin surface lipids were collected from six acne patients and surface lipids were collected from sex- and age-matched controls without acne. Six series of ceramides were found in each sample, the relative amounts of which were determined by thin-layer chromatography/photodensitometry. Acylceramides (ceramide 1) were isolated by preparative thin-layer chromatography and their ester-linked fatty acids were analyzed by gas-liquid chromatography. The comedonal acylceramides contained higher proportions of 16:0, 16:1 delta 6, and 18:1 delta 6 + delta 8 and much less linoleate (18:2 delta 9,12) than the acylceramides from the skin surface. In the surface lipids from legs, acylceramides from the acne patients contained less linoleate than the acylceramides from control subjects. Free fatty acids from the comedones were also isolated and analyzed, and had a composition very similar to the esterified fatty acids of comedonal acylceramides. The results confirm that fatty acids derived from sebum become incorporated into comedonal acylceramides, displacing linoleate, and show that this process even affects the acylceramides of surface epidermis, more so in acne patients than in normal subjects.

Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages.

To localize epidermal growth factor (EGF) receptors in normal human epidermis and other skin structures, two different light microscopic methods were used. EGF binding [( 125I]EGF/R) to the extracellular portion of the EGF receptor was studied by incubating intact skin samples with [125I]EGF, sectioning the tissues, and performing autoradiography. Immunoreactive EGF receptor molecules (IR-EGF/R) were localized with a mono-specific anti-EGF receptor antibody using a 2-step indirect immunocytochemical method (horseradish peroxidase) and detergent permeabilized tissues. This latter method measured the total pool of EGF receptors: occupied and/or internalized forms, precursor forms, and partially degraded forms of the EGF receptor that retain immunoreactivity. Both the [125I]EGF/R and IR-EGF/R localization studies indicated that EGF receptors were present in basal epidermal keratinocytes, sebocytes, outer root sheath cells in hair follicles, smooth muscle cells of arrector pili muscles, and dermal arteries. The highest levels of [125I]EGF/R and IR-EGF/R were found in the dermal ducts of eccrine sweat glands. The distribution of both [125I]EGF/R and IR-EGF/R was not consistent with the concept that EGF exclusively is involved in cellular division and proliferation in normal human epidermis and its appendages, i.e., EGF receptors were also found in tissues that do not undergo rapid proliferation. The present study indicates that EGF may have a more complex regulatory role in the skin than was previously thought.

Presence of sex pheromone in preputial glands of male rats.

Female rats consistently preferred the odour of male rat preputial gland compared with that of foot pads, submaxillary-sublingual glands, coagulating glands, liver, fat or muscle. Both saline homogenates and ether extracts were effective. Female rats did not respond to the odour of female preputial extract and they preferred the odour of normal male preputial extract to that from castrated rats. The pheromone was not associated with the fatty acids of the preputial extract. The fractionation of the volatile components of preputial extracts by gas chromatography revealed that most of the biological activity resided in a specific fraction.

Diesters of alkane diols and 2-hydroxy fatty acids: identification and discrimination of isomers with the aid of NMR spectroscopy.

High resolution nuclear magnetic resonance spectroscopy has been shown to be extremely useful for the identification and discrimination of naturally occurring diesters of 1,2- and 2,3-alkanediols as well as for fatty alkyl esters of acylated 2-hydroxy fatty acids. A comparison of 220 MHz spectra of 1,2 and erythro- 2,3-alkanediol diesters exhibits the following distinguishing features: (1) two non-equivalent methylene protons from the glycol group of 1,2-alkanediol diesters resonate at 3.87 ppm and 4.17 ppm respectively while these resonances are completely absent in the spectrum of 2,3-isomer; (2) methylene protons adjacent to esther carbonyl groups appear as two overlapping triplets at 2.22 ppm in 1,2-alkanediol diesters while the corresponding protons in the 2,3-isomer are displayed as two partially overlapping triplets centered at 2.15 ppm and 2.2 ppm respectively; and (3) methyl protons adjacent to glycol group in 2,3-isomer appear as downfield doublet at 1.13 ppm; this downfield doublet is not shown by 1,2-alkanediol diesters. Erythro- and threo-2,3-alkanediol diesters have also been distinguished from each other; two alpha-methylenes in erythro isomers appear as partially overlapping triplets while these protons in threo isomer display an apparent quartet centered at 2.22 ppm. Fatty alkyl esters of acylated 2-hydroxy fatty acids display a triplet at 4.79 for 2-position methylene proton, a distinguishing feature not shown by diacyl alkanediols. A distinction between diester lipids and other classes of neutral lipids has also been achieved by the study of nuclear magnetic resonance spectra, particularly in the region of 3-6 ppm.

Evidence of androgen and estrogen receptors in the preen gland of male ducks.

Androgen receptors have been found in duck preen glands by using dextran-coated charcoal adsorption. They bound DHT with high affinity (KD = 0.2 nM), limited capacity (45 fmoles/mgP) and good specificity. They sedimented at 8 S in a sucrose gradient and were destroyed by pronase digestion and heating. An estrogen receptor having different binding specificity was also demonstrated. On the basis of a marked annual cycle of gonadal activity in ducks, this system appears appropriate for studying the regulation of sex steroid hormone receptors.

Side gland of Suncus murinus as a new model of sebaceous gland: 5 alpha-reductase, androgen receptor, and nuclear androgen content in male and female animals.

The side gland of Suncus murinus is composed of well-developed sebaceous glands in both males and females. We measured the 5 alpha-reductase activity, androgen receptor content, and the intranuclear concentrations of testosterone and dihydrotestosterone in this side gland taking it as a new experimental model of human sebaceous glands. Lineweaver-Burk plot analysis suggested the presence of two classes of 5 alpha-reductase with different Km for testosterone in the homogenate. The enzyme activity was slightly higher in males than in females in the presence of a high concentration of testosterone. The levels of androgen receptors were approximately 40 and 30 fmol/mg protein in the cytosol and nuclei, respectively. The values did not differ significantly between males and females. The intranuclear concentration of dihydrotestosterone in the side gland was higher than that of testosterone in each sex. The intranuclear level of each of these two androgens in the female side gland was comparable to that in the male side gland despite the fact that the serum level of testosterone was much lower in the female. These data clearly indicate that the side gland is a typical target tissue for androgens in the female as well as in the male. Androgens other than testosterone may serve as precursors of dihydrotestosterone in the female side gland.

Steroid hormone receptors and their relevance for sebum production in the sebaceous gland ear model of the Syrian hamster.

We determined the capacity of steroid hormone receptors in the sebaceous glands of intact nontreated, castrated, with testosterone substituted castrated male, intact female, and intact with testosterone substituted female animals using the animal ear model of the Syrian hamster. The steroid hormone binding capacity was compared with the sebaceous gland areas and sebogenesis. Intact male animals showed large sebaceous follicles, a high sebogenesis rate, and high capacity for sexual hormone binding proteins. In castrated males, the sebaceous gland areas and sebogenesis were both diminished, and androgen and estrogen receptors were decreased. When the castrated males were substituted with testosterone propionate, the sebaceous glands showed large volumes, high sebum production, and androgen binding activity again. In female animals having small sebaceous follicles and a low rate of sebogenesis, testosterone propionate enlarged the sebaceous glands and increased sebogenesis and the capacity of androgen binding. One can conclude from these data that testosterone is not only the main hormone for sebum production but also induces the synthesis of its own receptor.

Immunohistochemical identification of epithelial membrane antigen in sweat gland tumors by the use of a monoclonal antibody.

A total of 78 cases of adnexal tumors of the skin were examined with the use of monoclonal antibody against epithelial membrane antigen (EMA). The EMA reaction was confined to luminal surfaces and lateral borders of sweat glands, both eccrine and apocrine, being usually absent in ductal segments. Neoplastic lesions of all the adenomatous tumours and mixed tumours of sweat glands showed specifically positive EMA staining of luminal surfaces and lateral borders of tubular, duct-like, and cystic structures. However, solid foci of those tumours were negative for EMA. Tumours of ductal origin, e.g. syringomas and poromas, exhibited positive EMA staining in their plasma membrane, although normal intact keratinocytes did not stain for EMA. Immunohistochemical distribution of EMA in skin adnexal tumours was classified into two types: one in which the luminal surfaces and lateral outer borders were positive, similar to that of the normal secretory coil, and the other in which the plasma membrane of neoplastic cells of ductal origin was positive.

Uropygial gland alkane diol diesters in the Kn mutation of the domestic chicken.

Uropygial glands from chickens which are known to have the Kn gene secrete qualitatively the same types of alkane diol diesters as the normal domestic chicken. However, there are many significant differences in the mole percent of individual fatty acids and diols within the diester between the two genetic types of chickens. In general, the mutant chickens have lesser amounts of the shorter chain fatty acids and diols and greater amounts of the longer chain fatty acids and diols. There is also a gene dosage effect with two mutant genes having a greater effect in many cases than a single mutant gene.

The occurrence of long chain alpha, omega-diols in the lipids of steer and human meibomian glands.

A group of long chain alpha, omega-diols (C29 to C34) has been identified in the lipids of steer and human meibomian gland excreta (meibum). These new lipids were isolated from the steer meibum unsaponifiables. Proof of structure was provided by 1) the column chromatographic behavior and TLC of the diols and their diacetates; 2) GLC on glass capillary columns; 3) fragmentation patterns in GC-MS; 4) NMR data, and 5) ozonolysis studies of the unsaturates. Chain types for the steer sample were 51% straight monoenes, 8.5% straight saturates, 39% iso and anteiso saturates and 1.5% iso and anteiso unsaturates. GC for the human sample gave straight monoenes 83%, straight saturates 8%, and iso plus anteiso saturates 9%. Close correspondence of the alpha, omega-diol chain lengths and types with meibum omega-hydroxy fatty acids suggests a biochemical precursor relationship.

Diester waxes containing 2-hydroxy fatty acids from the uropygial gland secretion of the white stork (Ciconia ciconia).

The uropygial gland of the white stork secrets mono- and diester waxes as well as triglycerides, all of which contain unbranched medium chain fatty acids. n-Decanol and n-dodecanol have been the only alcohols detected in both types of waxes. The diester waxes contain 2-hydroxy fatty acids.

Composition of uropygial gland secretions of birds of prey.

The chemical composition of the uropygial gland secretion of five species of birds of prey was investigated by gas liquid chromatography-mass spectroscopy technique, and the results are discussed from the chemotaxonomical point of view. The secretion is a complex mixture of monoester waxes, the fatty acids of which are mainly dimethyl-branched, with the first substituent in 2 position and the other near the methyl end of the molecule. Mono-, trimethyl-, and unbrached fatty acids also are observed. The wax alcohols are mainly mono- and dimethyl-substituted. Unbranched alcohols and traces of trimethyl-substituted alcohols also were detected. Chemotaxonomically, the birds of prey differ from all orders hitherto investigated. The degree of substitution increases from the Falconidae to the Accipitridae.

A chemist's view of the analysis of human hair for trace elements.

With the comparatively recent development of analytical techniques of great power and sensitivity, the significance of the levels of trace elements in human hair has attracted the attention of many disciplines including the environmental sciences. This paper presents the view that an agreed basis for the chemical analysis of trace elements in hair has not been established by the many workers in the field; a chemical basis is proposed here. Levels of 37 trace elements found in human hair are tabulated. Endogenous and exogenous sources of such trace elements are described and discussed. An extended review of the many pre-analysis treatments of hair (for the removal of exogenous elements) is presented. Twenty-four representative treatments are tabulated. Some of these treatments clearly removed significant fractions of endogenous elements along with exogenous elements. It is clear that method of cleaning have frequently been chosen without knowing enough about the basic chemistry and behaviour of the hair shaft. The significance of the results obtained cannot therefore be reliably assessed. A collation of recent literature reports leads to the tentative conclusion that disulphide bonds in the cuticular proteins of hair are major sites both for the deposition of metals during formation of hair and for interaction with exogenous elements. The feasibility of a holistic, no-wash policy for hair analysis is outlined and supported.