J. A. Milne

HISTOCHEMICAL DISTRIBUTION OF HYDROXYSTEROID DEHYDROGENASES IN HUMAN SKIN.

Twelve pieces of skin were obtained from luiman subjects ; six were from the upper part of the back and six from the forearm or lower limb. The subjects ranged in age from 20-35 years and the specimens were full thickness biopsies about 5 mm.in size. In addition, biopsy specimens from the backs of four subjects with severe acne were studied. Each specimen was frozen on solid carbon dioxide within two minutes of interruption of its blood supply and sectioned at 12^ in a cryostat maintained at — 20^ C. The sections were attached to clean, dry glass slides by momentary thawing and incubated individually to demonstrate NAD and NADP dependent hydroxysteroid dehydrogenase activity using techniques described previously. (Baillie, Caiman, Ferguson and Hart, 1965a and b ; Baillie, Ferguson, Caiman and Hart, 1965). Propylene glycol was found to be an unsatisfactory steroid solvent for dermatological work on account, apparently, of the presence in epidermal and sebaceous cells of an alcohol dcliydrugenasc capable of acting on propylene glycol, and a similar situation obtains in rodent skin (Ferguson. 1965). Dimethyl formamide was accordingly chowen as the steroid vehicle and the fallowing steroids were used :

THE DISTRIBUTION OF HYDROXYSTEROID DEHYDROGENASE IN HUMAN SEBACEOUS GLANDS.

IN a preliminary communication (Baillie, Caiman and Milne, 1965) the presence of histocbemically demonstrable 3a-, 3yff-, Ily5-, 16/?and 17^-hydroxysteroid dehydrogenases in human sebaceous glands from the upper part of the back was recorded. Moreover, no hydroxysteroid dehydrogenase activity was observed in sebaceous glands from the forearm or lower limb. These results represent the first significant morphological differences to be noted between the sebaceous glands in areas prone to acne vulgaris and those in other parts of the body. The present paper is an account of the age, sex and anatomical distribution of sebaceous glands with hydroxysteroid dehydrogenase activity in man.

THE METABOLISM OF ANBHOGENS BY SEBACEOUS GLANDS

THE first indication tlmt Iniinan sebaceous glands might be involved in steroid metabolism was \\\nm Baillie et al. (1965) reported their results on the histochemical utilization of a variety of hydroxysteroids in normal human skin. Using established techniques (Baillitet al., l!Ki5. 1966} based on the following reaction they showed that sebaeeous glands of normal subjects from areas of tlie body prone to develop acne vulgaris exhibited a characteristic jmttern of NAD-dependent hydroxysteroid dehydrogenase (HSD) activity whieh is summarized in Table I. A weak ])ositive reaction results in a deposition of j>inl\ monoformazan (M) in the cells \\ hile a moderate reaction results in the deposition of pink monoand blue diformazan (MD) (Fig. 1). A strong reaction is indicated by abundant blue diformazan granules in the sebaceous gland cells. In discussing the wignificance of these results Baillie et al. (U165) remarked that their possii)lo biochemical sigiiificauee was difficult to evaluate. While there was a possibility

SURVEY OF THE DISTRIBUTION OF STEROID DEHYDROGENASES IN SEBACEOUS GLANDS OF HUMAN SKIN

SUMMARY.— This paper reports the results of a survey carried out using human skin to investigate the distribution of 3β‐hydroxysteroid dehydrogenase, 16β‐hydroxysteroid dehydrogenase and 17β‐hydroxysteroid dehydrogenase over the skin surface. In common with other work, sebaceous glands were found in greater numbers in the face, scalp and thoracic region. The enzymes occur at all ages, although there was an apparent increase in activity in the 20–30 age group. The enzymes occur in both sexes, although there is a difference with the 17β‐HSD, the enzyme occurring more frequently in male skin biopsies. The enzymes occur in all surface areas of the human body and differences from a previous survey are noted. In particular the enzymes are found in the scalp, the perineal region, the arms, legs and abdomen, as well as the face and chest. The difference between the localization of the 3β‐HSD and 17β‐HSD within sebaceous glands is described.

The use of retinoic acid in congenital ichthyosiform erythroderma.

SUMMARY. .

HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN HUMAN SKIN

SUMMARY. A method has been established to estimate semiquantitatively the activity of HSD enzymes and is described in detail. In the main, the method depends on demonstrating NAD‐dependent hydroxysteroid dehydrogenase by using nitro‐BT as a hydrogen acceptor leading to the deposition of blue diformazan particles. The latter are then counted in a sebaceous gland by means of a Wild M20 microscope with a drawing tube attachment. The method is limited in use as a semi‐quantitative comparison of enzyme activity in a few adjacent sections of a sebaceous gland.

THE IN VITRO METABOLISM OF 7α-3H DEHYDROEPIANDRO-STERONE BY HUMAN SKIN

SUMMARY.— Slices of skin from human subjects have been shown to transform 3H‐dehydroepiandrosterone predominantly to 4‐androstene‐3,17‐dione and 5‐androstene‐3β,17β‐diol. Testosterone, testosterone sulphate and dehydro‐epiandrosterone sulphate were not formed in detectable amounts under the conditions of the investigation. Skin from the shoulder appeared significantly more active than that from the thigh or axilla in metabolizing the dehydro‐epiandrosterone.

HYDROXYSTEROID DEHYDROGENASE ACTIVTTY IN HUMAN SKIN

SUMMARY.— The effects of various factors on the histochemical demonstration of 3β‐hydroxysteroid dehydrogenase activity in human skin were examined by a semiquantitative technique of granule counting. The optimum concentration of DHA was found to be 0·1 mg./ml. and of NAD 0·3 mg./ml. Vitamin K and nicotinamide enhanced the histochemical demonstration of the enzyme whereas cyproterone and cyproterone acetate inhibited the reaction.

THE PRESENCE OF HYDROXYSTEROID DEHYDROGENASES IN MAMMALIAN SEBACEOUS STRUCTURES

Histochemical localization of HSD activity was investigated in sebaceous glands in skin of rat, guinea‐pig, hamster and gerbil. Rat preputial gland, hamster costovertebral organ and gerbil ventral gland were also used. Rat skin and preputial gland showed HSD activity with DHA. oestradiol and 5‐androstene‐3β‐16β‐diol‐3 methyl ether substrates while testosterone was poorly utilized in the 17β‐HSD reaction.

THE CHOICE AND USE OF EQUIPMENT FOR PHOTOMICROGRAPHS

Sharp black and white photomicrographs of good contrast and gradation can considerably enhance the value of a publication, and can often make a point more clearly than many lines of text. The production of such micrographs is not difficult if suitably matched microscope eyepieces, objectives and condenser and illumination system are chosen and used at their optimum settings. It should be emphasized that a good photomicrograph starts with an atraumatic biopsy of a suitable lesion, proper fixation, dehydration, embedding, and the eventual production of a well-stained section of around 5 /(m in thickness. It is advisable to use the same thickness of good quahty glass slides for sections intended for photography as thick, poor quality shdes may distort the final image. Broadly speaking, most microscope objectives are computed for use with a cover glass of 0-17 mm, and the extra cost of using cover glasses of this guaranteed thickness is more than justified. While there is no doubt that photomicrographs can be produced by a compound microscope with a camera and lens applied to the eyepiece, these are rarely of sufficient quality for reproduction. The use of a camera body without lens produces better results and many manufacturers of cameras and microscopes produce adaptors for this purpose. One of the main drawbacks of this type of system is the difficulty in focusing and maintaining sufficient rigidity in the system to obviate camera shake. The majority of good microscope manufacturers supply a special tube which fits on to a monocular body and contains an angled observation tube and eyepiece through which the specimen may be focused, the point of focus in the observation tube corresponding with the focal plane of the film. If such a system is used it is advisable to have the entire microscope-camera combination clamped to a rigid metal rod screwed into a firm base. It is more common now to purchase one of the photomicrographic systems and these are offered by leading British, German, Swiss and Japanese manufacturers. Perusal of their most attractive coloured brochures indicates that purchase of the system is a simple matter, and it is only when the purchaser is confronted with the long lists of various objectives, eyepieces and condensers available that he realizes that it is far from simple. The first thing which has to be decided is the negative size. This varies from the standard 35 mm (i X i\ in) roll film, 2^ x 2^ in roll film, 3^ x 4^ in sheet film to 5 x 4 in sheet film. There is no doubt that the larger the negative the less the degree of enlargement required for the final print, and this gives the highest quality. However, for convenience 35 mm film is the most commonly used. With modern fine grain emulsions and developers very high quality results can be obtained with this film size. There is a wide variety of equipment available today for photomicrography, from a relatively simple compound microscope with a 35 mm camera body mounted on top of it, through more versatile semi-automatic systems to the very expensive, sophisticated, fully automatic systems incorporating a research type microscope capable of phase and fluorescence microscopy. While the type of equipment purchased depends primarily on the kind of work to be undertaken and on the budget available, it is always advisable to buy the best available from the chosen manufacturer. The following remarks apply to any system, from the simplest to the most complex.