The suitability of saliva for detection of glucose-6-phosphate dehydrogenase deficiency.

Saliva was investigated for its suitability as a biopsy tissue for the determination of glucose-6-phosphate dehydrogenase deficiency. It appears that there is a significant difference between the activity of the enzyme in patients and controls. However, some controls have very low values making discrimination between patients and controls using a qualitative method impossible. Glucose-6-phosphate dehydrogenase deficiency is a relevant clinical problem in many rural areas in developing countries. Existing methods for determination of the deficiency in blood and hair follicles do not meet the criteria necessary for their large scale introduction in the areas of the world that are concerned by the problem. The present study shows that saliva is not a suitable alternative. Between the three biopsy tissues compared: blood, hair follicles and saliva, hair follicles remain most attractive since their isolation hardly involves the risk of infection. A simplified method for the detection of glucose-6-phosphate dehydrogenase activity in hair follicles that would allow health service workers in the field to determine the carrier status of pregnant women might form the basis for a future kernicterus prevention programme.

Is increased 5 alpha-reductase activity a primary phenomenon in androgen-dependent skin disorders?

Testosterone metabolism was investigated in fractions of human skin, enriched in epidermis, dermis, sebaceous glands, and sweat glands, by histologic sectioning of skin punch biopsies, and the results were compared with two culturable skin cells, i.e., keratinocytes and fibroblasts. Since sebocytes could not be brought in culture, metabolism was also investigated in the hamster flank model. In the epidermal tissue of the skin biopsies the predominant metabolite was androstenedione, formed by the enzyme 17 beta-hydroxysteroid dehydrogenase. The same was true for cultured hair follicle keratinocytes. In the deeper skin layers the formation of androstenedione was markedly reduced, whereas the formation of 5 alpha-reduced metabolites was highly increased, with a maximum in the skin fractions containing large sebaceous glands. Cultured shoulder skin fibroblasts showed a markedly different testosterone metabolism compared with the sectioned skin biopsies, suggesting that dermal fibroblasts play a less important role in the overall skin testosterone metabolism. The present approach, allowing the comparison of testosterone metabolism in different substructures of the same skin biopsy provides new evidence that the high 5 alpha-reductase activity in the specific skin fractions must be mainly ascribed to the sebaceous glands. These results render a previous hypothesis, stating that the elevated level of 5 alpha-reductase and subsequent formation of dihydrotestosterone in androgenetic alopecia and acne (usually accompanied by seborrhea) could therefore simply be the consequence of sebaceous gland enlargement, much stronger. This hypothesis is further evaluated by quantitative correlation of sebaceous gland size with enzyme activity in the hamster flank model.

Dydrogesterone has no peripheral (anti)-androgenic properties.

Dydrogesterone, an orally effective progestational compound, frequently used in a large spectrum of endocrine and gynecologic problems was evaluated for possible peripheric androgenic or anti-androgenic activity. The drug was tested in vivo in the hamster flank organ test and in vitro in tests based on molecular mechanisms of androgen action. Dydrogesterone appeared to have neither androgenic nor anti-androgenic properties. This is important in view of the fact that dydrogesterone can also be indicated during pregnancy.

Psoriatic hair follicle cells. III. Characterization of aberrant morphology in differentiating cultures.

Psoriatic and control human hair follicle keratinocytes were cultured on bovine eye lens capsules in Epicult dishes for a period of 5-6 weeks and examined using light microscopy. The following morphological differences between cultures were observed: 1. The lower cell layers contained predominantly flattened cells in psoriatic cultures instead of roundish in control cultures. 2. The differentiation pattern was irregular in psoriatic cultures instead of regular in control cultures. 3. The differentiated zone of psoriatic cultures was more compact and thicker in comparison to normal cultures. These differences might allow discrimination between normal and psoriatic cultures.

Determination of glycogen and enzymes of glycogen metabolism in human hair follicles.

The skin epithelium and its organelles use glycogen as well as glucose as source of energy. Therefore the characterisation of glycogen metabolism and the enzymes involved is important in the study of mechanisms regulating the normal or abnormal differentiation of skin organelles such as sebaceous glands and hair follicles. The present paper describes fluorimetric methods for the determination of glycogen and for the measurements of phosphorylase and phosphorylase kinase activity in one and the same lysate of minute tissue samples. The methods were tested for their suitability on freshly isolated human hair follicles and cultured hair follicle cells. The possible use of these techniques for studies on the pathophysiology of acne and hirsutism is discussed.

Phosphoglycerate kinase deficiency: biochemical studies on hair follicles.

A fluorimetric procedure for the determination of phosphoglycerate kinase in single human hair follicles is described. Enzyme studies on different parts of hair follicles after dissection show that the distribution of glucose-6-phosphate dehydrogenase matches that of phosphoglycerate kinase. Glucose-6-phosphate dehydrogenase can therefore be used as a reference enzyme to compensate for differences in hair follicle sizes. It was shown that the variation in the values found in individual hair follicles is improved by relating phosphoglycerate kinase to glucose-6-phosphate dehydrogenase activity. In areas of the world where glucose-6-phosphate dehydrogenase deficiency occurs frequently, an autosomally inherited reference enzyme may be preferred. It is shown that 6-phosphogluconate dehydrogenase is useful in this respect. Upon storage a gradual drop in the activity of all three enzymes was observed, but the rate of decrease was about equal: the enzyme activity ratio was, therefore, almost unaffected for a period of one week. This allows the determination of phosphoglycerate kinase even in mailed hair follicles.

Glutathione-S-epoxide transferase in human hair follicles.

A fluorometric assay for determination of glutathione-S-epoxide transferase (GSH-T) activity in freshly isolated human hair follicles or cultured cells is described. With this assay, basal levels of the enzyme in hair follicles have been compared between smokers and non-smokers. No significant difference between both groups could be detected. GSH-T was not elevated after treatment of cultured hair follicle keratinocytes with benz[a]anthracene or clotrimazole. The same result was obtained with cultured skin fibroblasts from Ah-responsive and Ah-non-responsive mice. The possible significance of basal GSH-T levels for assessment of the risk of individuals for the carcinogenic action of polycyclic aromatic hydrocarbons is discussed.

Actin distribution patterns in patients with adenomatosis of colon and rectum.

The actin cytoskeleton was examined in cultured skin fibroblasts of control individuals and patients with adenomatosis of colon and rectum using a fluorescent dye specific for F-actin, NBD-phallacidin. We confirmed that the actin distribution pattern differs significantly between the group of controls and the group of patients. However, the method is not practicable for the diagnosis of individual patients due to the number of false positives and negatives to be expected.

Studies on the local activity of antiandrogens at the molecular and histological level.

Androgen dependent skin disorders are important in clinical practice. Effective topical antiandrogens would lead to a breakthrough in their treatment. Although many attempts have been performed to develop such compounds, major successes have not been forthcoming. In the present study three existing antiandrogenic molecules have been compared with regard to their effect on androgen metabolism, receptor competition and on histological parameters in the hamster flank organ test. It appears that the effect on the hamster pigmented spot can be predicted on the basis of molecular mechanism. However, the effects on histological parameters are apparently dependent on additional factors such as metabolism of the active substance before reaching the sebaceous structure or limited penetration through the skin surface. The results indicate that in the development of new antiandrogens pre-screening can be performed with the aid of metabolic and receptor studies, while the histological parameters in the hamster flank organ test provide an animal model with a good predictive value.

Psoriatic hair-follicle cells. II. Morphological differentiation of outer root-sheath cells in culture.

Psoriatic human hair-follicle keratinocytes were cultured and then examined using light and electron microscopy. In comparison to control cultures derived from non-psoriatics, there were significant differences: stratification in general was more extensive; suprabasal cells were flat instead of round; there were almost no depositions of basal lamina or of cellular debris on the growth substrate; numerous membrane coating granules and a few keratohyalin granules were present earlier in psoriatic cultures than in control cultures; and the differentiation pattern resulted in an earlier appearance of corneocyte-like cells, and clusters of these corneocyte-like cells appeared to have been shed into the culture medium. As in control cultures, no distinct stratum corneum was found. Whether these differences between psoriatic cultures and control cultures reveal an aberrant differentiation pattern for psoriatic cells in vitro is as yet unknown: due to the faster outgrowth in psoriatic cultures, a multilayered and therefore further-differentiated structure near the hair follicle could be obtained more rapidly in psoriatic than in normal skin.

The use of liposomes in the topical application of steroids.

The effect of topical application of the androgen 5 alpha-dihydrotestosterone (DHT), both encapsulated in liposomes and solved in acetone, has been evaluated using the female hamster flank organ as a model system. Systemic absorption of DHT was significant from the acetone solution, but negligible from the liposome system. The topical biological effect is, however, proportionally diminished when the liposome system is used. Under the experimental conditions used, the lited using the female hamster flank organ as a model system. Systemic absorption of DHT was significant from the acetone solution, but negligible from the liposome system. The topical biological effect is, however, proportionally diminished when the liposome system is used. Under the experimental conditions used, the liposome system had no advantages over application in acetone in this model.

Population screening for glucose-6-phosphate dehydrogenase deficiency on the Baleares.

Two thousand people on the Isles of the Baleares were screened for glucose-6-phosphate dehydrogenase deficiency using a commercially available kit. Among the thousand males tested, five were found deficient; of the thousand women, one had low enzyme activity according to this test. Diagnosis of glucose-6-phosphate dehydrogenase deficiency could be verified using hair follicle analysis on mailed hair samples. The same technique also allowed heterozygotes to be identified unequivocally.

Human hair follicles may be used for population screening of heterozygotes of glucose-6-phosphate dehydrogenase deficiency.

The carrier status for glucose-6-phosphate dehydrogenase deficiency can still be detected with the use of hair follicles after a period up to 10-14 days after plucking, even under conditions of temperature and humidity that are encountered in tropical countries, where the disease is most common. Furthermore, it is shown that the hair colour or age of the donor has no influence on the enzyme activity. As a consequence, the hair follicle is indeed suitable for screening in rural areas since mailing to existing medical centres is possible.

Absence of induction of aryl hydrocarbon hydroxylase in mice after topical application of beclomethasone dipropionate.

The effects of the topical application of beclomethasone dipropionate (a corticosteroid) on the aryl hydrocarbon hydroxylase (AHH) activity in epithelial tissue (lung and skin) were studied. To evaluate the results of the pre-treatment with beclomethasone dipropionate a comparison was made with the effects of the topical application of benzanthracene (a polycyclic aromatic hydrocarbon and a known inducer of AHH activity). AHH activities were increased 2.5 to 5-fold in lung and about 20-fold in skin of mice after topical application of benzanthracene. However, after pre-treatment with beclomethasone dipropionate no induction of AHH activity could be observed, either in lung or in skin. The implications of these findings are discussed.

An improved method for evaluating antiandrogens.

In recent years, the hamster flank organ test has been frequently used to screen compounds for antiandrogenic activity. When used routinely, the diameter of the pigmented spot is used to evaluate the androgenic or antiandrogenic effect of the drug tested. In this article, it is shown that the pigmented spot and the sebaceous glands may respond to a quite different degree to hormonal stimuli, indicating that visual observation alone is insufficient for screening purposes. Determination of sebaceous gland sizes by histochemical techniques allows a better evaluation of androgenic or anti-androgenic effects.

A method for the evaluation of the local antiandrogenic action of 5 alpha-reductase inhibitors on human skin.

A method has been developed for the first time that allows the evaluation of the effect of therapeutic concentrations of 5 alpha-reductase inhibitors in human skin, without applying radioactivity to the skin. Moreover, the method makes it possible to determine whether inhibition is found only at the application site or also in other parts of the skin.

The antiandrogenic effect of progesterone on the hamster flank organ.

Progesterone, topically applied, prevents the stimulation of the hamster flank organ by testosterone, but not by dihydrotestosterone. 5 alpha-Dihydroprogesterone does not prevent stimulation by either of the two androgenic hormones. Neither progesterone nor 5 alpha-dihydroprogesterone stimulate the flank organ to any extent. Implications on the use of progesterone in acne therapy are discussed.