Search PubMed⌕ Search

Biomedical subjects

D T Downing

Publications and source records attributed to D T Downing.

At least 37 records · Page 2Linked to original sources
PubMed1994

6-Hydroxy-4-sphingenine in human epidermal ceramides.

The solvent-extractable lipids of human epidermal stratum corneum consist predominantly of ceramides. In addition two non-extractable ceramides are chemically bound to the stratum corneum protein. One of the bound ceramides, constituting 50% of the bound lipids, was previously shown to consist of very long chan omega-hydroxyacids in amide linkage with sphingosine. The second bound caramide, which forms 25% of the bound lipids, was shown to contain the same hydroxyacids, but the sphingoid base was neither sphingosine nor phytosphingosine. In the present study, the undefined bound ceramide was shown by NMR and chemical procedures to be the omega-hydroxyacid derivative of a new base, 6-hydroxy-4-sphingenine. In addition, a ceramide previously known to constitute 25% of the extractable human stratum corneum ceramides has been found to contain the same novel sphingoid base, amide-linked to long-chain alpha-hydroxyacids. Finally, a new acylceramide has been isolated and identified that consists of very long chain omega-hydroxyacids in amide linkage with the novel sphingolipid, with fatty acids esterified wit the terminal hydroxyl group of the hydroxyacid.

Adult↗
PubMed1993

Partition of sodium dodecyl sulfate into stratum corneum lipid liposomes.

Synthetic detergents produce deleterious effects on human skin as the result of being taken up by the stratum corneum (SC). The present study aimed to determine to what extent a typical detergent enters the SC lipid lamellae, and what effect this might have on the physical properties of the lipids. These effects were studied in large unilamellar liposomes prepared from SC lipids (50% by weight of epidermal ceramides, 28% cholesterol, 17% free fatty acids, and 5% cholesteryl sulfate) by extrusion through successive polycarbonate filters of decreasing pore size, finally 400 nm. Freeze-fracture electron microscopy and light-scattering particle size analysis indicated a uniform liposome diameter averaging 230 nm. Partitioning of sodium dodecyl sulfate (SDS) into the lipid phase from aqueous buffer solutions was measured using the SC lipid liposomes and [U-14C]SDS. The partition coefficient was 416, 450, and 588 at pH 8.5 and 524, 507, and 807 at pH 7 for three different concentrations (0.1%, 0.02%, and 0.004%) of SDS. This high degree of partitioning into the liposomes is consistent with the high level of SDS partitioning seen in full SC. At the maximum, the SDS represented 18% of the liposomal lipids. Preparation of stable liposomes from SC lipids to which 10% or 20% of SDS had been added confirmed the ability of the liposomes to survive these high concentrations of surfactant. The permeability of the liposomes was enhanced as a result of SDS partitioning into the bilayers, as measured by the increased release of trapped [U-14C]glucose from these vesicles, and by their increased permeability to water in osmotic shock experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Freeze-fracture electron microscopic and osmotic water permeability studies of epidermal lipid liposomes derived from stratum corneum lipids of porcine epidermis.

Freeze-fracture electron microscopic studies revealed that the liposomal membrane morphology was intact before and after osmotic treatment. This finding suggested that water leakage from the liposomes was not due to fusion of two or more lipid vesicles, but rather to the osmotic salt effect. A stop-flow spectrophotometric study revealed that epidermal lipid liposomes derived from stratum corneum lipids of porcine skin underwent increases of the absorbances with decreases of volume of the vesicles. The initial rate at which the changes in optical density occurs is a measure of the water permeability through the liposomes. The reciprocal of the changes in the absorbance at the equilibrium at different salt osmotic shocks showed a linear dependence on the reciprocal of the osmotic pressure gradient, indicating that epidermal lipid liposomes are an ideal osmometer. The present investigation reports that lignoceric acid is a potent water barrier. Present findings suggest that the initial rate of water penetration decreased in the liposomes made from 30-45% (wt% ratio) of cholesterol and ceramides. Oleic acid as drug penetration enhancer facilitated the water diffusion of the stratum corneum lipid liposomes by a fluidizing effect on the liposomal membranes. Furthermore, ceramides are important in the water barrier properties of the skin. The permeability of water depends upon the amount (wt%) and the type of lipid of the membrane.

Animals↗
PubMed1993

Interaction between sphingosine and cholesteryl sulfate in epidermal lipids.

Free sphingosine, a material with multiple and potent biological activities, is known to occur in high concentration in mammalian epidermis. In the present study, thin-layer chromatography showed that in lipid extracts of human and pig stratum corneum, sphingosine forms a relatively stable compound with endogenous cholesteryl sulfate. NMR spectrometry of sphingosine and its hydrochloride, sulfate, and mixtures with cholesteryl or dodecyl sulfate showed that interaction with the organic sulfates constituted simple salt formation. Under neutral or weakly acidic conditions, such salts were only slightly dissociated and migrated on thin-layer chromatograms as discrete compounds. Thin-layer chromatography revealed undissociated salt formation between several long-chain bases and organic sulfates, and showed that their interaction is stoichiometric. However, undissociated salts were not formed between long-chain bases and fatty acids or phosphatidic acid. Undissociated salt formation may therefore be specific for organic bases and sulfates. It was concluded that the free sphingosine in the stratum corneum may be present as its cholesteryl sulfate salt and in this form be unavailable for permeation into the viable epidermal cells.

Animals↗
PubMed1992

Lamellar structures formed by stratum corneum lipids in vitro: a deuterium nuclear magnetic resonance (NMR) study.

Hydrated lipid mixtures consisting of stratum corneum ceramides, cholesterol, specifically deuterated palmitic acid, and cholesteryl sulfate were investigated by solid-state 2H NMR spectroscopy at different temperatures. The mole ratio of cholesterol to ceramides was varied from 1 to 0. 2H NMR spectra from these mixtures showed powder patterns with quadrupolar splittings smaller than those obtained from control mixtures containing dipalmitoylphosphatidylcholine (DPPC) instead of the ceramides. This result is attributed to the rigid amide group of the ceramides, with a planar configuration, which could prevent close packing of the alpha-methylenes of the acyl chains. There was a gradual loss of symmetry in the powder pattern as the amount of cholesterol was decreased and the amount of ceramides (or DPPC) was increased concomitantly. The loss was more pronounced in the ceramide-containing samples. This phenomenon is interpreted as a decrease in the axial reorientation rate of the alpha-deuterated palmitic acid in the bilayers, presumably caused by the increased hydrogen bonding resulting from the high amount of hydroxyl-bearing ceramides. Spectra obtained at temperatures above 60 degrees C indicated the formation of a hexagonal phase (HII) by the ceramide-containing mixtures. Spectra of the omega-deuterated palmitic acid in the mixture containing 76 mol% ceramides and no cholesterol indicated phase separation into a more rigid phase and a more mobile phase in the temperature range of 25 to 60 degrees C. The bilayer configuration of lipids at 25 degrees C was confirmed by thin-section electron microscopy.

Animals↗
PubMed1992

Sebaceous gland activity and serum dehydroepiandrosterone sulfate levels in boys and girls.

BACKGROUND AND DESIGN: Increases in sebaceous gland activity are often the earliest sign of the approach of puberty in children. These increases have been attributed to increases in the secretion of adrenal androgens, but the supporting data are sparse and are based on measurements of urinary, rather than serum, androgen concentrations. In this study, we examined sebum composition, serum levels of dehydroepiandrosterone sulfate, and pubertal stage in 111 boys and girls, aged 2 to 15 years. Sebum composition was evaluated by measuring the ratio of wax esters/(cholesterol + cholesterol esters), a ratio known to increase with increasing sebaceous gland activity. RESULTS: Both wax esters/(cholesterol + cholesterol esters) ratios and serum dehydroepiandrosterone sulfate levels began to increase in children 7 to 10 years old. These changes occurred in many children before the appearance of any physical signs of puberty. Wax esters/(cholesterol + cholesterol esters) ratios were correlated with dehydroepiandrosterone sulfate levels in both boys and girls. In prepubertal children, the regression lines passed through the origin. In subjects who were in early or late puberty, the y intercepts of the regression lines had positive values. CONCLUSION: Adrenal androgens appear to be the major determinants of sebaceous gland activity during the prepubertal period and to be additive to another hormone or hormones during puberty.

Adolescent↗
PubMed1992

In vivo studies of cutaneous lipid biosynthesis.

The epidermis and the sebaceous glands are both characterized as terminally differentiating tissues in which cells produced by replication in the germinative layer progress during several weeks through a programmed series of biochemical and morphological changes that end in cell death and eventual sloughing of the final metabolic products. Because of these slow changes and the presence of successive anabolic and catabolic regimes in the same tissue, classic methods of biochemistry using cell homogenates have not been widely successful. Much of what is known of the lipid metabolic pathways has therefore been obtained from experiments with biopsy materials or in intact animals. Radiotracer experiments in intact animals have been successful largely because the lipid products into which the radiolabel is incorporated are not lost to the general circulation during the several weeks in which they are retained in the tissue. Nor are they subject to continual degradation and resynthesis that would be the case in most other tissues, but progress steadily through the series of biochemical transformations that occur along with the morphologic changes characteristic of the epidermal and sebaceous tissues. Furthermore, of the radiolabel that enters the general circulation, only an insignificant proportion returns to the cutaneous tissues to obscure the local metabolic activities.

Animals↗
PubMed1991

Deuterium NMR investigation of polymorphism in stratum corneum lipids.

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.

Animals↗
PubMed1990

Ceramidase activity in porcine epidermis.

This report presents the first demonstration of a ceramide-hydrolyzing activity in mammalian epidermis. An assay using fractions derived from porcine epidermis and synthetic [3H]ceramide is described, and it is shown that under the conditions used, the Km for ceramide is 110 microM and hydrolysis is linear for up to 2 h. The enzyme activity is maximal at pH 8-9. The specific activity of ceramide hydrolase decreases as the protein concentration in the assay mixture increases, suggesting the possibility of a dissociable inhibitor. Also, the activity can be inhibited by added palmitic acid. Ceramide hydrolysis may be an important regulatory mechanism in the epidermis due to the ability of the liberated free sphingosine to modulate the activity of protein kinase C.

Amidohydrolases↗
PubMed1990

Interaction between corneocytes and stratum corneum lipid liposomes in vitro.

Small unilamellar vesicles were made from a mixture of epidermal ceramides (45%), cholesterol (35%), free fatty acids (15%) and cholesteryl sulfate (5%). Isolated corneocytes prepared from pig epidermis were added to the liposomes and the interaction between corneocytes and liposomes was studied by (1) thin-section electron microscopy and (2) monitoring the release of aqueous contents of the vesicles by following the fluorescence intensity of carboxyfluorescein entrapped in the vesicles. The vesicles adsorbed readily onto the corneocytes and slowly transformed into lamellar sheets. Enhanced fluorescence intensity indicated a corneocyte-induced membrane fusion process that resulted in the release of aqueous contents of the vesicles. The results suggest a cohesive role for the corneocyte cell envelope, which consists of a monomolecular layer of lipids covalently bound to the outside of a cross-linked protein envelope. This may be one of the major factors in the reassembly of extruded membranous disks into lamellar sheets which occurs during the final stages of epidermal differentiation.

Animals↗
PubMed1990

Composition of cerumen lipids.

Lipids were extracted from "wet" cerumen and analyzed by quantitative thin-layer chromatography to determine their composition. The lipid fraction comprised 52% of the dry weight of cerumen and consisted of squalene (6.4%), cholesterol esters (9.6%), wax esters (9.3%), triacylglycerols (3.0%), fatty acids (22.7%), cholesterol (20.9%), ceramides (18.6%), cholesterol sulfate (2.0%), and several unidentified polar components (7.5%). In addition to the extractable lipids, the residue contained an additional 0.9% lipid that could be released only after saponification. This covalently bound lipid consisted of two unusual ceramides (63.4%), omega-hydroxyacids (27.7%) and nonhydroxy fatty acids (8.8%). The composition of this bound lipid resembled that recently found in human stratum corneum, which is thought to comprise a lipid envelope on the outer surface of the corneocytes. The free and covalently bound lipids may be significant determinants of the properties of cerumen. Desquamation of corneocytes and their associated lipids from the epidermal lining of the ear canal may make a major contribution to cerumen.

Cerumen↗
PubMed1990

Metabolism of topically applied fatty acid methyl esters in BALB/C mouse epidermis.

The goal of the present study was to determine whether topically applied fatty acid esters enter into epidermal lipid metabolism. Of special interest with respect to epidermal function were the linoleate-rich O-acylsphingolipids thought to be essential in maintenance of the permeability barrier. Neonatal BALB/C mice were coated at 24 h intervals with 50 mg portions of methyl oleate or methyl palmitoleate. One day after the second application, the animals were sacrificed, and individual epidermal lipid classes were isolated. Fatty acid methyl esters were prepared from the isolated lipids and analyzed by gas-liquid chromatography. These analyses revealed that acylceramide was affected only slightly by application of either methyl ester. However, treatment with methyl oleate resulted in a significant replacement of linoleate by oleate in both acylglucosylceramide and phosphatidylethanolamine. This treatment also led to small increases in palmitoleate and possibly unresolved isomers of palmitoleate (16:1) in both lipids. Topical methyl palmitoleate resulted in a more complex pattern of substitution in both of these lipids. After methyl palmitoleate treatment, palmitate 16:0, 16:1 and vaccenate (18:1 delta 11) were elevated at the expense of linoleate in acylglucosylceramide. In the phosphatidylethanolamine, 16:1 and 18:1 delta 11 both increased while oleate (18:1 delta 9) and linoleate (18:2) decreased. These results demonstrate that topically applied fatty acid methyl esters can penetrate to the living cells of normal epidermis, enter into metabolism and significantly modify endogenous epidermal lipids.

Administration, Topical↗
PubMed1990

Unusual cholesterol esters in the sebum of young children.

Cholesterol esters (CE) having fatty acids of more than 18 carbons are a prominent feature of fetal skin surface lipid (vernix caseosa), but are a minor component of adult lipid. The difference may be related to the fact that fetal sebaceous glands generally synthesize little lipid. If so, it would be expected that prepuberal children, who also have very inactive glands, would secrete CE with a large proportion of very-long-chain fatty acids. To test this conjecture, skin surface CE from young children were isolated and analyzed. Sebum was extracted from the hair of 38 children, aged six to nine. To obtain a measure of sebaceous lipogenesis, the class composition of the lipid was determined by quantitative thin-layer chromatography and the ratio of wax esters/[cholesterol + cholesterol esters] (WE/[CH + CE]) was calculated. CE were then isolated from the lipid and hydrolyzed. The freed fatty acids were converted to fatty acid methyl esters (FAME) and analyzed by capillary gas chromatography to determine the proportion with more than 18 carbons. FAME from five of the subjects were then separated into saturated and monounsaturated fractions and analyzed again by gas chromatography to identify chain types. Ratios of WE/[CH + CE] ranged from 0.08 to 2.8 in the subjects. The proportion of CE FAME with more than 18 carbons ranged from 15 to 72%, with the highest proportion being found in the children with the lowest WE/[CH + CE]. The saturated FAME were mostly iso- or anteiso-branched, whereas the monounsaturated FAME were mostly straight-chain extension products of 16: 1 delta 9 or 18: 1 delta 9.

Child↗
PubMed1990

Sphingolipid metabolism in organotypic mouse keratinocyte cultures.

Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine. These results indicate that considerable diversity of ceramide structures occurs among mammalian species and that cultured keratinocytes may only partially reproduce the in vivo complement of ceramides. Using labeled serine in keratinocyte cultures, we have also demonstrated the de novo synthesis of ceramides and the transfer of label from glucosylceramides to ceramides during terminal differentiation of lifted cultures. The covalently bound corneocyte lipid envelope, which has recently been characterized in pig and human epidermis, was also present in mouse epidermis and was reproduced by the lifted cultures. Very-long-chain omega-hydroxyceramides were the dominant bound lipid and labeling studies in culture indicated that they were derived from ceramides synthesized in the viable epidermis.

Animals↗
PubMed1990

Free sphingosine in human epidermis.

The aim of the present study is to determine the concentration and composition of free long-chain bases in human stratum corneum. Samples of foot callus obtained from a local chiropodist were extracted at room temperature with chloroform: methanol mixtures (2:1, 1:1, and 1:2). The extracts were concentrated, filtered to remove nonlipid debris, and dissolved in chloroform: methanol, 2:1, at a concentration of 25 mg/ml. Small portions of extract were treated with 2,4-dinitrofluorobenzene to convert free amino groups to N-dinitrophenyl derivatives, which were subsequently separated from other components by thin-layer chromatography (TLC) and quantitated by photodensitometry at a wave-length of 355 nm. The N-dinitrophenylsphingosines were then isolated by preparative TLC and oxidized with lead tetraacetate in benzene to produce aldehydes, which were analyzed by gas-liquid chromatography. It was found that callus lipid contained 3.39 +/- 0.57 mg free sphingosine, 1.60 +/- 0.72 mg dihydrosphingosine and traces of phytosphingosine per g lipid. The sphingosines and dihydrosphingosines ranged from 16 through 20 carbons in length, with 18:1 as the predominant constituent. These findings are similar to those recently reported for pig epidermis and support the possible role of free sphingosine as a regulator of epidermal differentiation.

Bony Callus↗
PubMed1990

Metabolism of linoleic acid in porcine epidermis.

The time course of linoleic acid metabolism in porcine epidermis has been studied in order to better evaluate the lipid transformations that accompany differentiation in this tissue. One day after intradermal injection of [U-14C]linoleic acid, most of the radioactivity in the epidermis was associated with phospholipids, triglycerides, and free fatty acids. Within 3-7 days, a portion of the radiolabel was selectively transferred to acylglucosylceramide and acylceramide fractions, after which the specific activities of all lipids fractions decreased. The results suggest that neither the lipoxygenation of linoleate nor the hydrolysis of acylceramide to form acylacid and free sphingosine are major routes of linoleate metabolism in normal epidermis. The principal pathway of linoleate metabolism in normal epidermis appears to be transfer from phospholipids to acylglucosylceramide to acylceramide. The triglyceride fraction, although minor in amount, attains an initially high specific activity, and may serve as an intermediate in linoleate transfer.

Animals↗