Biomedical subjects

D T Downing

Publications and source records attributed to D T Downing.

At least 73 records · Page 4Linked to original sources

PubMed1988

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.

Acne Vulgaris↗

PubMed1988

Lamellar granule extrusion and stratum corneum intercellular lamellae in murine keratinocyte cultures.

Lamellar granules are specialized epidermal organelles containing stacks of membranous disks that are extruded into the intercellular spaces in the upper portion of the granular layer. The extruded disks are believed to undergo biochemical and biophysical changes to form the stratum corneum intercellular lipid sheets that constitute the epidermal permeability barrier. Little is known about this important component of epidermal differentiation, in part due to lack of a suitable in vitro model. We have demonstrated microscopically the presence of characteristic lipid membrane structures in a primary keratinocyte culture system which shows morphologic differentiation comparable to that seen in vivo. A basal cell-enriched fraction of isolated neonatal mouse keratinocytes was plated into Vitrogen-coated 30 mm Millicell (Millipore, Bedford, Massachusetts) wells, fed daily with Medium 199 containing 10% fetal bovine serum, 10 micrograms/ml each of insulin and hydrocortisone, and kept at 32 degrees C in a 5% CO2/95% air atmosphere in a humidified incubator. Three days after plating, cultures were placed on living, epidermis-free mouse dermis at the air/liquid interface. At 2 wk, histologic examination showed multiple well-organized cell layers, including a distinct granular layer and a well-developed stratum corneum. Transmission electron microscopy demonstrated numerous lamellar granules and extrusion of their contents into the intercellular space. After fixation with ruthenium tetroxide, stacked intercellular lamellae in the stratum corneum were seen. Both the presence of dermis and growth at the air/liquid interface were necessary to achieve complete differentiation. This system conclusively demonstrates the formation of complex epidermal lipid structures in vitro and should allow the mechanisms and regulation of their synthesis to be elucidated.

Animals↗

PubMed1988

The nonpolar lipids of pig epidermis.

Epidermis was removed from several sites on the intact bodies of each of seven pigs and the lipids were extracted and analyzed by thin-layer chromatography. The principal nonpolar components were cholesterol, free fatty acids and cholesteryl esters. Small and variable amounts of paraffin hydrocarbons, triglycerides, and an unidentified lipid were noted. In a series of time-course experiments, epidermal lipids were labeled by intradermal injection of [1-14C] acetate at varying times up to 21 days before removal of the epidermis. The hydrocarbons never acquired radioactivity, while the triglycerides retained label only for about 1 day after injection. Squalene and lanosterol retained considerable radioactivity for up to 12 h after labeling. Radioactivity appeared in free fatty acids and cholesteryl esters 2-3 days after labeling and began to decline after 21 days. Epidermis from uninjected sites did not acquire significant radioactivity. From these results it was concluded that cholesterol, cholesteryl esters and free fatty acids are synthesized locally in the epidermis and remain until lost by desquamation. Triglycerides are also synthesized but do not persist into the stratum corneum, so the small amounts found there may be environmental contaminants. Alkanes are not synthesized, and are most probably derived from petroleum waxes.

Animals↗

PubMed1988

The time course of lipid biosynthesis in pig epidermis.

In order to examine the time course of lipid metabolism in the epidermis, pairs of sites on each of two pigs were given seven intradermal injections of 10 microCi of [1-14C]-acetate at 1, 3, 6, 12, 24, 36, and 48 h before the animals were killed. A second time course was obtained with two other pigs at 1 h and 3.5, 7, 10.5, 14, 17.5, and 21 d after labeling. The epidermis over each injection site was recovered, and the lipids were extracted and analyzed by thin-layer chromatography and by autoradiography. Virtually all of the radioactivity in aliphatic chains was found first in phospholipids and was then transferred to glucosylceramides, and then into ceramides, between 1 and 7 d after labeling. Labeling of free fatty acids showed a similar time course. Most of the radioactivity acquired by acylglucosylceramide was transferred to hydroxyceramide, bound to the corneocyte envelope, between 2 and 10 d after labeling. The hydroxyacid and sphingosine moieties of the hydroxy-ceramide had equal specific activity. The results indicate that the aliphatic lipids of the stratum corneum are synthesized de novo in the epidermis via phospholipids. Cholesterol, however, was synthesized directly from acetate within hours of labeling, and its esters acquired label 3 to 7 d later.

Acetates↗

PubMed1987

Effects of essential fatty acid deficiency on epidermal O-acylsphingolipids and transepidermal water loss in young pigs.

Linoleate-rich O-acylglucosylceramides and acylceramides are thought to be of major significance for the physical structure and function of the epidermal permeability barrier. In the present investigation, the effects of a linoleate-free diet on O-acylsphingolipids and their associated functions were investigated. Starting at 5 days of age, male pigs were fed diets containing 12% of either lard or hydrogenated coconut oil. Transepidermal water loss was measured with an electrolytic water analyzer at weekly intervals. Pigs were killed at intervals, and epidermal lipids were isolated and analyzed. Fatty acid compositions were determined by gas-liquid chromatography (GLC). Within 2-3 weeks, pigs on the diet containing coconut oil began to display biochemical and physiological symptoms of essential fatty acid deficiency. Within 2 months, this group had extremely scaly skin and transepidermal water loss was elevated to five times that of controls. The progressive increase in transepidermal water loss correlated with replacement of linoleate by oleate in both acylceramide and acylglucosylceramide. The formation of lamellar granules and intercellular lipid sheets in the stratum corneum was not impaired in essential fatty acid deficiency as judged by electron microscopy. These results suggest that the linoleic acid normally found in the O-acylsphingolipids is not essential for formation of the epidermal membrane system. Rather, it appears that the nature of the ester-linked fatty acid in the O-acylsphingolipids regulates the permeability of this membrane system.

Acylation↗

PubMed1987

Covalently bound omega-hydroxyacylsphingosine in the stratum corneum.

Pig epidermis was heat separated, and the stratum corneum was isolated after trypsinization. Exhaustive extraction of the stratum corneum fraction with chloroform/methanol mixtures yielded 14.7% lipid on a dry weight basis. After mild saponification of the extracted residue, additional lipid could be extracted which accounted for 2.1% of the stratum corneum weight. This bound lipid proved to consist mainly (91.9%) of N-(omega-hydroxyacyl)sphingosines in which the amide-linked omega-hydroxyacids were 28 to 34 carbon atoms in length. The release of this lipid by mild alkaline hydrolysis indicates that it is bound through an ester linkage. Half of the hydroxyceramide molecules reacted in situ with acidic acetone, suggesting that half of these molecules are attached to the stratum corneum through the omega-hydroxyl function, while the other half may be linked through one of the hydroxyl groups of the sphingosine.

Animals↗

PubMed1987

Evaluation of a method for measuring the sustainable rate of sebaceous wax ester secretion.

Sebaceous wax ester secretion rates were measured in six subjects on six occasions each, using absorption into bentonite clay and analysis of the collected lipid by quantitative thin-layer chromatography. On each occasion eight samples were collected, four from the left and four from the right side of the forehead in four successive intervals. The first two intervals, which totaled 14 h, were intended to deplete the follicular reservoir of sebum so that a constant rate of absorption could be obtained during the third and fourth intervals, which were 3 h each. Thin-layer analysis of each sample was done in triplicate. The data were examined using analysis of variance techniques to determine the reproducibility of the measurement method and to identify possible sources of variability. The intraclass correlation coefficient (rI) for all 432 post-depletion determinations was 0.80. The reproducibility was considerably better for three of the subjects (rI = 0.93) than for the other three (rI = 0.75). Variability within the latter three subjects did not seem to be attributable to lack of reproducibility in the thin-layer analysis. Real biological variability also seems unlikely considering the holocrine mechanism of sebum secretion. Therefore, the variability probably arises from non-representative collection of sebum into the bentonite absorbent.

Adult↗

PubMed1987

Skin surface lipids of the mole Scalopus aquaticus.

Skin surface lipids of the mole Scalopus aquaticus were found to consist principally of squalene (70%), wax esters (15%), and sterol esters (5%), together with small amounts of triglycerides, free fatty acids, free fatty alcohols, and free sterols. Analysis of the fatty acids occurring free and as wax esters and sterol esters showed these to consist of approximately equal amounts of saturated and monounsaturated compounds. The saturated fatty acids consisted predominantly of odd-carbon anteiso and even-carbon straight-chain compounds, with minor amounts of even-carbon iso-branched chains. The unsaturated fatty acids had double bond positions that would have been produced by delta 9-desaturation of C14, C16 and C18 straight chain saturated precursors. Both the free and the esterified fatty alcohols had chain structures corresponding with those of the fatty acids but of somewhat greater average chain length. Discovery of a major proportion of squalene in the sebum of this animal extends the number of non-human species that have this characteristic to four, all of which inhabit a damp environment, suggesting that squalene conveys some biological advantage under these conditions.

Animals↗

PubMed1987

Polar lipid composition of mammalian hair.

The types and amounts of polar lipids from the hair of monkey (Macacca fascicularis), dog (Canis familiaris), pig (Sus scrofa) and porcupine (Erethizon dorsatum) have been determined by quantitative thin-layer chromatography. The polar lipid content of the hair samples ranged from 0.6 to 1.6 wt%. Lipid compositions included ceramides (57-63% of the polar lipid by weight), glycosphingolipids (7-9%) and cholesteryl sulfate (22-29%). Several minor components (4-7%) remain unidentified. The results suggest that cholesteryl sulfate may be an important determinant of the cohesiveness of hair.

Animals↗

PubMed1987

Skin lipids: an update.

The stratum corneum lipids, responsible for the epidermal water barrier, consist principally of ceramides, cholesterol, cholesteryl sulfate, and free fatty acids. These lipids are arranged in multiple intercellular lamellae that provide an efficient water barrier because of the crystalline array of the straight and predominantly saturated lipid chains. Interlamellar linkages provided by lipids based on 30-carbon omega-hydroxyacids may be responsible for holding together the intercellular lamellae as well as for assembly of the lamellar granules of the granular cells. The normally ordered exfoliation of corneocytes as they arrive at the surface seems to require hydrolysis of the cholesteryl sulfate to free cholesterol. The sebaceous glands secrete continuously, producing sebum that consists predominantly of triglycerides, wax esters, and squalene. High rates of sebum production per sebocyte result in low levels of linoleate in the sebaceous esters, subjecting the follicular epithelium to essential fatty acid deficiency and the characteristic hyperkeratosis that results in comedo formation. Suppression of sebum production by drugs elevates sebum linoleate concentration and relieves follicular hyperkeratosis. Thus, sebum continues to be a prime suspect in the crime of acne. Low levels of sebaceous gland activity are not correlated with the occurrence of dry skin.

Acne Vulgaris↗

PubMed1987

Evidence that the corneocyte has a chemically bound lipid envelope.

The stratum corneum of mammalian epidermis contains a mixture of ceramides, free fatty acids, cholesterol, and cholesteryl sulfate, amounting to 14% of the dry weight of the tissue, that can be removed by exhaustive extraction with chloroform/methanol. Subsequent mild alkaline hydrolysis liberates additional lipid, consisting almost exclusively of C30-C34 omega-hydroxyacids in amide linkage with sphingosine, equal to 2% of the tissue mass. In the present study, transmission electron microscopy was used to demonstrate that the initial extraction removes the intercellular lamellae that constitute the epidermal water barrier but leaves the lucent band that has been termed the corneocyte plasma membrane. The subsequent alkaline hydrolysis and lipid extraction remove the lucent band, which must therefore contain the omega-hydroxyacylsphingosines. From the results of in situ derivatization of these lipids and the construction of molecular models, it is inferred that the bound lipids exist in ester linkage with protein on the surface of the corneocyte envelope. The tightly packed hydroxyacylsphingosine molecules thus form a lipid envelope for each corneocyte.

Animals↗

PubMed1987

Presence of intact intercellular lipid lamellae in the upper layers of the stratum corneum.

The epidermal permeability barrier necessary for terrestrial life resides in the intercellular spaces of the stratum corneum and is composed of lipids. Membrane coating granules (MCGs), small intracellular organelles found in the uppermost layers of the living epidermis, contain stacks of membranous disks which are extruded into the intercellular space and undergo both biochemical and physical changes to form the lipid sheets which constitute this barrier. Using ruthenium tetroxide as a secondary fixative, we are able to demonstrate stacks of lamellae filling the intercellular spaces in the uppermost layers of the stratum corneum. The structure of these lipid lamellae is consistent with the proposed derivation of MCG lipid disks and also suggests that the lipid bilayer adjacent to the corneocyte cell envelope may be assembled from lipids not derived from MCGs.

Animals↗

PubMed1987

Composition and morphology of epidermal cyst lipids.

The contents of epidermal cysts were used as a source of desquamated human keratinocytes uncontaminated by sebaceous, subcutaneous, or bacterial lipids. Lipids extracted with chloroform:methanol mixtures included six series of ceramides (41% of the total extractable lipid), cholesterol (27%), cholesteryl esters (10%), fatty acids (9%), cholesteryl sulfate (1.9%), a novel class of ceramide esters (3.8%), and a sterol diester (0.9%). Electron microscopy revealed that the lipids in the cyst contents existed as multiple intercellular lamellae, as in stratum corneum. One lamella, adjacent to the horny cell protein envelope, was resistant to lipid extraction and is thought to represent covalently bound lipid on the outer surface of the keratinocyte. The results indicate that the degradation of intercellular lipid lamellae is not required for desquamation.

Ceramides↗

PubMed1987

Stratum corneum lipid liposomes: calcium-induced transformation into lamellar sheets.

The epidermal water barrier in mammalian stratum corneum is formed of broad lamellar sheets of lipids consisting principally of ceramides (40%), cholesterol (25%), cholesteryl sulfate (10%), and free fatty acids (25%). Such lipid mixtures have been shown to form lipid bilayers in the form of small, unilamellar liposomes when sonicated at 80 degrees C in water containing Tris buffer and 100 mM NaCl. In the present study it is shown that such liposomes are slowly transformed into large unilamellar liposomes and then into broad lamellar sheets after the addition of stoichiometric amounts of calcium chloride. The presence of free fatty acids was a necessary condition for this calcium-induced fusion. These observations may provide a useful analogy for the transformation of flattened liposomes into broad lamellar sheets that occurs during transition of epidermal granular cells into corneocytes.

Animals↗

PubMed1987

The effect of 13-cis-retinoic acid on sebaceous glands.

The drug, 13-cis-retinoic acid, which has been demonstrated to have a marked effect on nodulocystic acne, probably has several mechanisms of action. This article summarizes the effects on the sebaceous glands, and the accompanying changes in cutaneous lipids that result from 13-cis-retinoic acid therapy. These changes in lipid composition support the concept that linoleate may be of importance in the pathogenesis of acne.

Acne Vulgaris↗

PubMed1987

Essential fatty acids and epidermal integrity.

The intercellular spaces of the stratum corneum contain multilamellar lipid sheets derived from the extruded contents of lamellar granules. In the absence of linoleic acid, lamellar granules appear empty, and only fragmentary extracellular sheets are found. This defective differentiation is attributable to substitution of oleate for linoleate in O-acylsphingolipids. Normally, linoleate is ester-linked to 30- to 34-carbon omega-hydroxyacids, which, in turn, are amide-linked to sphingosine. Acylglucosylceramides, bearing a beta-D-glucosyl moiety on the sphingosine, may provide the driving force for lamellar granule assembly. The omega-hydroxyacyl chains are long enough to span a lipid bilayer, while the linoleate inserts into an adjacent bilayer. This interaction could promote assembly of lamellar granules. It has also been proposed that acylceramides may stabilize the extracellular sheets by a similar mechanism. In addition, the horny cell has been found to possess a covalently bound lipid envelope consisting principally of omega-hydroxyacylsphingosines derived from O-acylsphingolipids.

Animals↗

PubMed1987

Glucosylsterol and acylglucosylsterol of snake epidermis: structure determination.

The structures of two classes of glycolipids, acylglucosylsterol and glucosylsterol, from snake epidermis have been determined by chemical, spectroscopic, and gas-liquid chromatographic methods. The acylglucosylsterol consists of a glucose molecule attached to cholesterol and an ester-linked fatty acid on carbon 6 of the sugar. The major ester-linked fatty acids are palmitic, stearic, and oleic. The glucosylsterol consists of glucose attached to cholesterol. These unusual glycolipids may play a role in epidermal barrier function in snakes and it is concluded that these sterol glycosides may remain in the reptiles as a vestige of a more primitive barrier-forming mechanism.

Animals↗

PubMed1986

Covalent attachment of omega-hydroxyacid derivatives to epidermal macromolecules: a preliminary characterization.

The present report establishes that fatty acids, omega-hydroxyacids and omega-hydroxyacylsphingosines are covalently attached to macromolecules in mammalian epidermis. Extensive extraction of pig epidermis yielded 11.2% lipid on a dry weight basis. Reextraction of the epidermal residue did not yield additional lipid; however, a mild saponification of the delipidized residue followed by acidification and extraction yielded an additional 0.5% by weight of lipid-soluble material. The major components of this residual lipid fraction are omega-hydroxyacylsphingosine (60% of the total), free fatty acids (24%) and free omega-hydroxyacids (7%).

Animals↗