Lipoxygenase activity of Pityrosporum in vitro and in vivo

Lipid peroxidation has been investigated both in cultures of Pityrosporum supplemented with different lipid classes and in skin surface lipids from patients affected with pityriasis versicolor. Thin-layer chromatography (TLC) and 2 spectrophotometric methods were used: the indirect thiobarbituric acid test and the direct N,N-diethyl-1,4-phenylene-diammonium sulfate (DEPD) test. The coupling of the DEPD test with the TLC technique performed by different eluent systems allowed the detection of the specific lipoperoxides deriving from the oxidation of the different lipid classes. In the cultures, Pityrosporum was capable of peroxidating not only unsaturated free fatty acids, but also unsaturated triglycerides, cholesterol, and squalene. A similar lipid peroxidation was observed in patients with pityriasis versicolor in skin lipids from areas positive for fungal hyphae and spores and fluorescent under the UV lamp (366 nm). The lipoperoxide values were significantly higher (p less than 0.05) than in skin lipids from normal controls. Hyphae and spore-negative areas of patients with pityriasis versicolor, whether apparently normal or achromic, showed no evidence of a significant lipid peroxidation and neither did skin areas of patients with pityriasis alba. Though further investigations are necessary, it seems reasonable to suggest, in analogy with other biologic systems, that the presence in skin lipids of a significant amount of highly reactive and cytotoxic lipoperoxides may play a role in the pathogenesis of skin alterations in pityriasis versicolor, including damage to melanocytes and resulting achromia.     

Growth requirements and lipid metabolism of Pityrosporum orbiculare.

The yeast, Pityrosporum orbiculare, isolated from lesions from lesions of tinea versicolor, grows in vitro only if fatty acids from the C12 to C24 series are added to the culture medium. Except for elaidinic and nervonic acids, all saturated and unsaturated fatty acids tested support growth. P. orbiculare can synthesize various lipid fractions containing both saturated and unsaturated fatty acids from a single fatty acid. Glucose and asparagine stimulate growth but exogenous vitamins do not.     

Relative irritancy of free fatty acids of different chain length

Free fatty acids of human skin surface lipids have previously been implicated in the pathogenesis of acne vulgaris because of their apparent irritant and comedogenic properties. Prior studies on the relative irritancy of free fatty acids revealed the saturated C8 to C14 fatty acids and a C18 dienoic unsaturated fatty acid (linoleic) to be most irritating. Saturated free fatty acids from C3 to C18, and unsaturated C18 free fatty acids were applied daily under occlusive patch tests to human skin until detectable erythema appeared. The most irritating fatty acids were C8 through C12. Of the unsaturated fatty acids tested, only linoleic acid produced irritation.     

ONYCHOMYCOSIS DUE TO PITYROSPORUM

Ten cases of onychomycosis due to Pityrosporum are reported. The epidemiology, clinical features and treatment of these cases are described in detail. These cases are reported to alert medical practitioners and mycologists that Pityrosporum can cause onychomycosis in our community.     

In vitro susceptibility testing of Pityrosporum ovale against antifungal, antiseborrheic and antipsoriatic agents

Objective The in vitro antimicrobial susceptibility of Pityrosporum ovale strains isolated from patients with seborrheic dermatitis was determined. Method Minimum inhibitory concentrations of a total of 11 agents - including true antifungal, antiseborrheic, and antipsoriatic drugs - were measured. Results Ketoconazole was the most effective of the tested antifungal agents against Pityrosporum ovale in vitro (minimum inhibitory concentration 0.1 μg/ml). The antiseborrheic agents zinc pyrithione and selenium disulfide showed a good in vitro efficacy against Pityrosporum with similar minimum inhibitory concentrations. Liquor carbonis detergens and dithranol were also able to inhibit growth of Pityrosporum ovale in vitro, but much higher concentrations were necessary. Conclusion The tested agents commonly used against seborrheic dermatitis might exert their efficacy, at least in part, due to inhibition of Pityrosporum ovale.     

Identification of tyrosinase inhibitors in cultures of Pityrosporum.

Lipid fractions capable of inhibiting the dopa-tyrosinase reaction in vitro were isolated by thin-layer chromatography from submerged cultures of Pityrosporum supplemented with oleic acid or vaccenic acid. Analysis of these fractions by gas chromatography-mass spectrometry revealed the presence mainly of C(9) and C(11) dicarboxylic acids. Standard dicarboxylic acids from C(8) to C(13) were capable of inhibiting tyrosinase in vitro to varying extents. Enzymatic kinetic studies showed that they act as competitive inhibitors of tyrosinase. These observations suggest that dicarboxylic acids could be used in the treatment of people with hyperpigmentary disorders.     

Squalene peroxides may contribute to ultraviolet light-induced immunological effects.

Ultraviolet (UV) irradiation is capable of producing a dose-dependent decomposition of skin surface lipids and particularly of squalene, with the concomitant generation of active lipoperoxides. The biological effects of UV-peroxidated squalene were tested, compared with those produced by synthetic lipoperoxides (cumene hydroperoxide), on some immunological parameters in vivo modified by UVB irradiation. Application of UV-peroxidated squalene as well as cumene hydroperoxide significantly inhibited the induction of contact hypersensitivity to dinitrofluorobenzene in mice, which was associated with a decrease in the number of ATPase positive cells. The effect was dose-dependent (over 40 micrograms for peroxidated squalene and over 20 micrograms for cumene) and relevant after 2 d of treatment. Down-regulation towards the applied hapten was demonstrated. The results indicate that UV-induced lipoperoxides of squalene are capable of inhibiting the induction of contact hypersensitivity in mice and suggest that, among the other photoproducts generated in humans, squalene peroxides may play a role as biochemical messengers of the biological effects of UV irradiation of the skin.     

Incorporation of linoleic acid by cultured human keratinocytes

Abstract Linoleic acid is required for the formation and maintenance of the epidermal barrier, but most of the current in vitro keratinocyte culture systems are linoleic acid-deficient. The aim of the present study was to examine the efficiency of linoleic acid uptake in human keratinocyte cultures grown under submerged and air-exposed conditions in serum-free medium. The water-insoluble linoleic acid was bound to carrier molecules (cyclodextrin or bovine serum albumin). Comparable results were obtained with home-made and commercially available linoleic acid complexes. In the submerged cultures, the increase of the linoleic acid medium concentration (ranging from 0 to 20 μg/ml) resulted in a gradual increase in the linoleic acid cellular content, which exceeded 1.4 times the value found in native epidermis when the highest concentration of linoleic acid was used. The addition of linoleic acid did not alter the profile of the other epidermal fatty acids, with the exception of oleic acid, which decreased in parallel with the increasing linoleic acid content. While the content of linoleic acid found in phospholipids was similar to that in native epidermis, a large excess of linoleic acid was detected in triglycerides, the synthesis of which was markedly increased in cultures grown submerged in medium containing higher concentrations of linoleic acid. Under air-exposed conditions, the dermal substrate used seemed to be the most limiting factor for efficient linoleic acid supplementation. A low linoleic acid cellular content was detected when an inert filter was used. De-epidermized dermis was found to be the most permeable substrate for linoleic acid complexes. The cellular linoleic acid content increased in a parallel with the increasing linoleic acid concentration (ranging from 4 to 30 μg/ml), but the overall amount incorporated was lower than that in submerged cultures. The content of linoleic acid in the phospholipid and ceramide fractions isolated from reconstructed epidermis grown under air-exposed conditions was close to that of native epidermis, but the triglycerides remained abnormally enriched in linoleic acid, indicating persistence of some anomalies in epidermal lipogenesis in vitro. Received: 18 June 1998 / Received after revision: 15 January 1999 / Accepted: 22 January 1999     

Role of skin surface lipids in UV-induced epidermal cell changes

Summary Ultraviolet irradiation is capable of affecting skin surface lipids, especially squalene and cholesterol, both in vitro and in vivo, with generation of active lipoperoxides. The photodecomposition of the skin lipid component was carefully evaluated by capillary gas-chromatography. The effects of UV-induced lipoperoxides on human keratinocytes in culture and on guinea pig ear slices were compared with those of synthetic lipoperoxides, i.e. cumene hydroperoxide and 13-hydroperoxylinoleate. A time- and dose-dependent effect on protein synthesis and mitotic activity was observed. In cell culture low concentrations (0.05–5 g/ml) of peroxidated squalene and synthetic lipoperoxides stimulated the incorporation of radiolabelled thymidine and phenylalanine, while higher concentrations (>10 g/ml), or longer periods of treatment, induced cellular damage. In guinea pig ear slices, the lipoperoxides (5–50 g/ml) increased aminoacid incorporation and the number of epidermal pigment cells; higher concentrations (>100 g/ml) caused a derangement of epidermal structure. The results suggest that UV irradiation of skin generates lipoperoxides from the surface lipids which, in vitro, are capable of producing a number of changes in epidermal cells.     

Induction of epidermal hyperproliferation by topical n-3 polyunsaturated fatty acids on guinea pig skin linked to decreased levels of 13-hydroxyoctadecadienoic acid (13-hode)

Reversal of essential fatty acid deficiency (EFA) induced epidermal hyperproliferation was recently suggested to require linoleic acid and an active lipoxygenase product. Because the nature of this lipoxygenase product is unknown, we employed a model of n-3 polyunsaturated fatty acid (PUFA) induced hyperproliferation in guinea pig skin to test a possible reversal of the hyperproliferation by an oxidative metabolite of linoleic acid. Topical applications of two n-3 PUFA: 0.5% of eicosapentaenoic acid (20:5n-3) and/or of docosahexaenoic acid (22:6n-3) for 5 d induced severe epidermal hyperproliferation. Development of the epidermal hyperproliferation paralleled a marked decrease in the major epidermal linoleic acid lipoxygenase product (13-hydroxyoctadecadienoic acid; 13-HODE). The application of 0.1% of 13-HODE to the n-3 PUFA-induced guinea pig hyperproliferative skin resulted in the restoration of normal epidermal histology and reversal of hyperproliferation as determined by epidermal uptake of 3H-thymidine. These data support the view that 13-HODE may represent the endogenous cutaneous mediator necessary for full restoration of cutaneous symptoms of essential fatty acid deficiency. Furthermore, the topical use of n-3 PUFA for the disruption of normal metabolism of skin n-6 EFA (linoleic acid) does serve as a useful tool for further investigations into the regulatory mechanisms of in vivo epidermal proliferation/differentiation.     

Linoleic acid and α-linolenic acid lightens ultraviolet-induced hyperpigmentation of the skin

Abstract This study was conducted to evaluate the effects of unsaturated fatty acids on ultraviolet-induced hyperpigmentation of the skin. An efficient lightening effect was observed following topical application of linoleic acid or α-linolenic acid to UV-stimulated hyperpigmented dorsal skin of brownish guinea pigs. The number of melanocytes in the treated skin was similar to the number in the skin of the pigmented control, indicating that the pigment-lightening effect was not due to depletion of melanocytes. In vitro experiments using cultured murine melanoma cells showed that melanin production was inhibited most effectively by α-linolenic acid, followed by linoleic acid and then by oleic acid. Furthermore, the turnover of the stratum corneum, which plays an important role in the removal of melanin pigment from the epidermis, was accelerated by linoleic acid and by α-linolenic acid. Taken together, the results suggest that the pigment-lightening effects of linoleic acid and α-linolenic acid are, at least in part, due to suppression of melanin production by active melanocytes, and to enhanced desquamation of melanin pigment from the epidermis. Received: 8 October 1997     

In vivo chemotaxis induced by polyunsaturated fatty acids.

Intradermal injection of as little as 500 ng of arachidonic acid or the metabolites from arachidonic acid incubated with soybean lipoxygenase produced infiltration of the upper dermis by polymorphonuclear leukocytes 18 hours after injection. In experiments comparing the chemotactic properties of four fatty acids in varying concentrations, oxidative products of arachidonic acid by soybean lipoxygenase were the most powerful followed by free arachidonic acid and free linoleic acid while stearic acid did not produce significant infiltration. These findings suggest that the elevated levels of free arachidonic acid and an arachidonic acid metabolite (12L-hydroxy-5, 8, 10, 14-eicosatetraenoic acid), recently found in psoriatic epidermis, may at least in part be attracting polymorphonuclear leukocytes into psoriatic skin.     

Changes in transepidermal water loss and the composition of epidermal lecithin after applications of pure fatty acid triglycerides to the skin of essential fatty acid-deficient rats

The importance of various unsaturated fatty acid triglycerides to the repair of faulty skin barrier function was studied in essential fatty acid-deficient rats. Following cutaneous application of the pure triglycerides for up to 5 days, the hitherto high rate of transepidermal water loss, characteristic of essential fatty acid deficiency in rats, was reduced by the triglycerides of linoleic and γ-linolenic acids. Incorporation of the applied fatty acids into the lecithin of the epidermis accompanied these changes in water loss, indicating that cutaneously applied triglycerides may be metabolized by the skin and incorporated into complex lipids. Other fatty acid triglycerides, including α-Unolenic, dihomo γ linolenic, arachidonic and ω-7-heneicosatrienoic acid, did not lower the rate of ransepidermal water loss, although all were incorporated into epidermal structural lipids. The non-essential oleic acid also had no effect upon the rate of transepidermal water loss. These data suggest that of the two main essential fatty acids that occur in skin, linoleic acid and arachidonic acid, the former specifically plays an important role in regulating barrier function whereas the latter may have a separate function, such as serving as a precursor of prostaglandins.     

Steroid acne vs. Pityrosporumfolliculitis: the incidence ofPityrosporum ovaleand the effect of antifungal drugs in steroid acne

Background Steroid acne is a folliculitis that can result from systemic or topical administration of steroid, and has been described as showing a similar clinical picture to Pityrosporum folliculitis, but there have been few reports about the incidence of Pityrosporum ovale and the effect of antimycotic drugs in steroid acne and other acneiform eruptions. Our purpose was to describe the association between steroid acne and P. ovale, and to confirm the superior efficacy of oral antifungal drugs over anti-acne drugs in the treatment of steroid acne. Methods The history, clinical features direct microscopy, histopathologic analysis, and therapeutic results of 125 cases with steroid acne or other acneiform eruptions were described and compared. Results Over 80% of patients with acneiform eruption receiving systemic steroid revealed significant numbers of P. ovale in the lesional follicle. Furthermore, oral antifungal drug (itraconazole) showed significantly better clinical and mycologic effects than any other group of medications used in this study. Conclusions Steroid acne and other acneiform eruptions showing discrete follicular papules and/or pustules localized to the upper trunk and acneiform facial skin lesions associated with multiple acneiform lesions on the body in the summer period should be suspected as Pityrosporum folliculitis. In addition, oral antifungal drugs recommended for Pityrosporum folliculitis; however, it will require a larger case-control study to confirm the superiority of antifungal therapy over anti-acne treatment.     

Murine keratinocyte cultures grown at the air/medium interface synthesize stratum corneum lipids and "recycle" linoleate during differentiation

In a recent investigation we showed that murine keratinocyte cultures grown at the air/medium interface in the presence of dermis exhibit morphologic differentiation comparable to that seen in vivo, including the formation of lamellar granules and stratum corneum intercellular lipid lamellae. In the present study, lifted cultures were found to more closely reproduce the lipid composition of the parent epidermal tissue than submerged cultures grown on plastic. In addition, the specific fatty acid profile of individual lipid classes in lifted cultures was, in general, remarkably well maintained in vitro. Acylceramides, which are highly enriched in linoleic acid in vivo, remained enriched in vitro; however, the linoleic acid content of the cultures was substantially lower than that in vivo, confirming previous reports of the relative essential fatty acid deficiency of standard culture media. As the lifted cultures differentiated over time, the lipid composition changed to reflect the formation of a stratum corneum with its different complement of lipids. Label from [U-14C]linoleic acid was specifically incorporated into linoleate-containing lipids during short pulses in both submerged and lifted cultures. Changes in label distribution over a long chase period in lifted cultures indicated that linoleate was transferred from phospholipids to ceramides, providing evidence for the "recycling" of essential fatty acids in epidermis.     

Sj?gren-Larsson syndrome: early diagnosis, dietary management and biochemical studies in two cases.

BACKGROUND: Sj?gren-Larsson syndrome (SLS) is a rare autosomal recessive disorder with worldwide distribution. It consists of ichthyosis, spastic diplegia and mental retardation caused by an enzymatic defect in fatty alcohol oxidation. OBJECTIVE: To study the effects of dietary management on clinical outcome and plasma/red blood cell fatty alcohol and plasmalogen concentrations. METHODS: To reduce fatty alcohol production, we reduced total fat intake to 30% of total intake of calories. To correct d 6 desaturase deficiency, we supplemented the diet with both n-3 and n-6 fatty acids to obtain a linoleic/linolenic acid ratio of 6 with low erucic acid rapeseed oil, plus high unsaturated fatty acids. We used gas liquid chromatography to assay blood cell membranes and plasma fatty alcohols/plasmalogens. RESULTS: Two SLS infants with proven fatty alcohol/NAD+ oxidoreductase deficiency were studied. Good clinical results were obtained in one of the patients when dietary intervention was started in early infancy and correlated well with plasma fatty alcohol decrease. However, no clinical improvement was seen in the other patient who started later with low compliance. Acitretin therapy was necessary to control skin symptoms in this second patient. CONCLUSION: Dietary intervention using the combined approach described here may improve fatty alcohol metabolism in SLS. However, only very early intervention seems clinically beneficial.     

Analysis of the antifungal activity of ketoconazole, zinc pyrithione, and ciclopirox olamine against Pityrosporum ouale. A diffusion assay for cultures in solid media

Background Recently dandruff has been associated with a local increment in the number of Pityrosporum yeasts. Due to this fact, several anti-dandruff shampoos containing antifungals have been marketed. Objective We studied the sensitivity of Pityrosporum ovale (PO) to three different groups of antimycotics (ketoconazole, Zn pyrithione, and ciclopirox olamine). Methods /Results The drugs were tested by an inhibition assay in solid medium. Ketoconazole proved to be the most effective drug in inhibiting PO growth in a dose-dependent fashion. The results of our evaluation revealed that the inhibitory effects of the drugs were ketoconazole > Zn pyrithione > ciclopirox olamine. Conclusion We postulate that the diffusion assay is a reliable medium for evaluating the effectiveness of antifungals contained in anti-dandruff shampoos.     

Fatty acid uptake by cultured human keratinocytes grown in medium deficient in or supplemented with essential fatty acids

Abstract Epidermal linoleic acid, i.e. essential fatty acid (EFA), is essential for cutaneous barrier function. Cultured human keratinocytes, routinely used for studies of lipid metabolism, are grown in a keratinocyte serum-free medium (KSFM), under conditions that reveal EFA-deficient cells. Here, fatty acid (FA) uptake was analysed in human adult keratinocytes grown either under EFA-deficient conditions [KSFM supplemented with 10% FCS (A) or 1% UltroserG (B)] or EFA-supplemented conditions [KSFM supplemented with a devised FA cocktail (C) or evening primrose oil (D)]. The FA composition of the total cellular lipid and major lipid fractions was analysed by gas chromatography. Cells grown with supplements A or B balanced their EFA-deficient state primarily with oleic acid. Cells grown with supplements C or D normalized to the epidermal FA composition in vivo with raised linoleic and lower oleic acid contents. When cells were grown longer than 48 h with supplements C or D decreased cell growth was observed. FA uptake was curvilinear with preference for linoleic over oleic acid under all culture conditions. The uptake of linoleic acid by cells cultured with supplement B was twice the uptake of those cultured with supplement A, while the uptake of oleic acid was similar under both culture conditions. Oleic acid uptake of cells cultured with supplement C or D was lower. These results show that the uptake of linoleic, but not that of oleic acid, is influenced by the extracellular FA composition, and that EFA-supplemented keratinocytes compared to EFA-deficient cells might serve as an in vitro model for the study of EFA metabolism. Received: 10 February 1997 / Received in revised form: 10 September 1998 / Accepted: 17 September 1998     

Fatty acid metabolism in human keratinocytes cultivated at an air-medium interface

Stratum corneum lipids, which provide the mammalian permeability barrier, display a distinctive fatty acid profile with a predominance of long chain, saturated fatty acids. In addition, linoleic acid (18:2) is present in substantial quantities, implying that it is an important structural component. To investigate selectivity of fatty acid incorporation into epidermal lipids, we examined the metabolism of exogenous fatty acids in cultured human keratinocytes, grown at the air-medium interface to enhance differentiation. Keratinocytes were pulsed with [3H] oleic, [14C] stearic, [14C] palmitic, or [14C] linoleic acids; lipids were extracted and fractionated by thin layer chromatography. All fatty acids were taken up and incorporated into complex lipids in a dose-dependent manner that was linear over the first 60 min. These fatty acids were incorporated predominantly into phospholipids and triacylglycerols; their incorporation could be rank ordered: linoleic greater than oleic greater than or equal to palmitic greater than stearic acid. Less than 2% of each fatty acid taken up by keratinocytes was oxidized to CO2; therefore, these differences in utilization cannot be ascribed to differences in rates of beta-oxidation. In pulse-chase studies fatty acids incorporated initially into triacylglycerols, subsequently chased into phospholipids. [14C]Palmitic acid and [14C] acetate were incorporated into sphingolipids more efficiently than the other fatty acids studied. These studies demonstrate that 1) keratinocytes have the ability to incorporate exogenous fatty acids preferentially into complex lipids; 2) triacylglycerols provide a pool of fatty acids for phospholipid synthesis; and 3) palmitate and de novo synthesized fatty acid are preferably utilized for sphingolipid synthesis.     

Modulation of stratum corneum lipid composition and organization of human skin equivalents by specific medium supplements

Our in‐house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono‐unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono‐unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements.