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D T Downing

Publications and source records attributed to D T Downing.

At least 55 records · Page 3Linked to original sources
PubMed1989

Free sphingosines in porcine epidermis.

Sphingosines and phytosphingosines serve as intermediates in the synthesis of ceramides and glucosylceramides, which are prominent components of mammalian epidermis. In the present study, we have investigated the possibility that free sphingoid bases also may be present in epidermal tissue. Samples of pig epidermis were trypsinized to separate the stratum corneum from the unkeratinized portion of the epidermis. After drying, the lipids were extracted and analyzed by thin-layer chromatography using ninhydrin to detect free amino groups. Both the stratum corneum and the unkeratinized epidermal material contained a ninhydrin-positive material with the same mobility as the sphingosine standard. Quantitation of the chromatograms by photodensitometry indicated that free sphingosine bases account for 0.44% by weight of the total stratum corneum lipid and 0.09% of the lipid in the viable portion of the epidermis. To further identify this material, it was treated with 1-fluoro-2,4-dinitrobenzene, which resulted in the production of an intensely yellow N-2,4-dinitrophenyl derivative with the same mobility as N-2,4-dinitrophenylsphingosine on thin-layer chromatography. Oxidation of the isolated dinitrophenyl derivative with lead tetraacetate produced a mixture of aldehydes which were analyzed by gas-liquid chromatography. This analysis indicates that the free sphingoid bases from the stratum corneum consist of a mixture of mainly 16- through 20-carbon sphingenines and sphinganines, the most abundant components being d17:0, d17:1, d18:1 and d20:1. The production of these free sphingosine bases may be significant in regulating epidermal differentiation.

Animals↗
PubMed1989

Beta-glucosidase activity in porcine epidermis.

One of the final steps in epidermal differentiation is the conversion of glucosylceramides to ceramides, which presumably is mediated by a beta-glucosidase activity. In the present manuscript, it is demonstrated that pig epidermis contains beta-glucosidase activity which is 3.3-times greater than alpha-glucosidase and 5-times greater than beta-galactosidase. This beta-glucosidase was found to be maximally active between pH 3.0 and essentially inactive at pH 9.0. In a standard assay, a disk of epidermis (8 mg dry weight) was submerged in 1 ml of 50 mM acetate buffer (pH 4.7) containing 150 mM NaCl and 15 mM p-nitrophenyl-beta-D-glucopyranoside at room temperature. Reaction was stopped by addition of 4 ml of 100 mM (pH 9.0) borate buffer and the supernatant was transferred to a separate tube. The nitrophenylate anion was then measured spectrophotometrically at a wavelength of 405 nm. Under these conditions, product formation was linear for at least 90 min and an apparent Km of 244 microM was estimated for the synthetic substrate. When the amount of epidermis in the assay was varied, the formation of product per unit of time remained proportional to the amount of epidermis. The level of beta-glucosidase activity was enhanced slightly by the inclusion of sodium taurocholate.

Animals↗
PubMed1989

omega-Hydroxyacid derivatives in the epidermis of several mammalian species.

1. omega-Hydroxyacids from the acylceramides and acylglucosylceramides of mammalian epidermis were examined by thin-layer and gas-liquid chromatography to determine their degree of unsaturation and chain-length distributions. The species examined included human (Homo sapiens), pig (Sus scrofa), mouse (Mus musculus) and rat (Rattus rattus). 2. Within a species, the omega-hydroxyacids from the acylceramide and acylglucosylceramide were essentially identical. 3. The human omega-hydroxyacids proved to be mainly saturated with C30:0 being the major entity. 4. The pig contained similar saturated, monoenoic and small amounts of dienoic omega-hydroxyacids, with C30:0, C32:1 being the major entities. 5. The mouse and rat contained C32:0 and C34:1 as the major components.

Animals↗
PubMed1989

Integral lipids of mammalian hair.

1. It has been demonstrated that hair contains lipids which cannot be removed by extensive extraction with chloroform-methanol mixtures. These integral lipids can be extracted only after the hair has been subjected to alkaline hydrolysis. 2. Integral hair lipids include cholesterol sulfate (0.7-2.9 mg/g hair), ceramides (0.6-1.4 mg/g), cholesterol (0.3-1.4 mg/g), fatty alcohols (trace-0.2 mg/g) and fatty acids (2.3-4.0 mg/g). 3. One of the major integral hair lipids, representing 38.4-47.6% of the total fatty acids, is the anteisobranched 18-methyleicosanoic acid. 4. The species examined included human (Homo sapiens), pig (Sus scrofa), dog (Canis familiaris), sheep (Ovis ammon aries) and cow (Bos taurus).

Animals↗
PubMed1989

Molecular models of the intercellular lipid lamellae in mammalian stratum corneum.

Intercellular lipid lamellae in the stratum corneum constitute the barrier to water diffusion and may also play a role in cohesion between corneocytes. The lamellae arise from stacks of lamellar disks that are extruded from the granular cells and then fuse edge-to-edge to form sheets. It has been proposed that each lamellar disk is formed from a flattened vesicle, and therefore consists of two lipid bilayers in close apposition. In the present study, electron microscopic examination of ruthenium-tetroxide-fixed stratum corneum from mouse, pig, and human skin revealed that the double bilayer pattern persists in the intercellular lamellae. In addition, distinctive patterning of the intercellular lamellae has led us to propose novel molecular arrangements of the intercellular lipids. These include interlamellar sharing of lipid chains to produce lipid monolayers between pairs of bilayers. The pattern reflects the provenance of the intercellular lamellae from lamellar granule disks and the nonrandom orientation of the lamellar lipids.

Animals↗
PubMed1989

Changes in the relative amounts of endogenous and exogenous fatty acids in sebaceous lipids during early adolescence.

Skin surface lipid samples were collected from the scalps of 40 males, aged 9-15, and the lipid class composition of each was analyzed by quantitative thin layer chromatography. The ratio of wax esters/[cholesterol + cholesterol esters] (WE/[CH + CE]) increased with age. The wax ester, cholesterol ester, triglyceride, and free fatty acid classes were isolated from each sample and the fatty acid compositions were determined by capillary gas chromatography of fatty acid methyl esters (FAME) prepared from each lipid class. The concentrations of most of the different types of fatty acids were found to be correlated with the WE/[CH + CE] ratio. Those straight chain fatty acids that are thought to be synthesized mainly within the sebaceous glands, such as 14:0, 14:1, 16:1, and 18:2 delta 5, 8 tended to increase with increasing WE/[CH + CE], while fatty acids which circulate in the blood, such as 18:0, 18:1, and 18:2 delta 9, 12, tended to decrease with increasing WE/[CH + CE]. For the majority of straight chain fatty acid types, the data could be fitted to the equation y = a + b/[x + 1], which can be derived from simple assumptions concerning the origins of the various sebum components. The FAME from the wax esters were separated into saturated and monounsaturated fractions and analyzed by capillary gas chromatography to determine the concentrations of the different types of branched chain FAME present. In the wax esters, straight chain fatty acids tended to increase with increasing WE/[CH + CE], while terminally branched (iso and anteiso) fatty acids tended to decrease. Other branched chain fatty acids increased up to a WE/[CH + CE] ratio of about 2 and then decreased at higher ratios.

Adolescent↗
PubMed1989

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.

Animals↗
PubMed1989

The role of the corneocyte lipid envelopes in cohesion of the stratum corneum.

Treatment of isolated stratum corneum with certain detergents results in complete disaggregation of the corneocytes within hours at 45 degrees C without agitation. This is prevented by prior heating of the tissue to 80 degrees C or by solvent extraction of the intercellular lipids. In the present study, electron microscopy revealed that the heated or solvent-extracted tissue was characterized by cell-to-cell contacts that appeared to involve the chemically bound hydroxyceramides which constitute the corneocyte lipid envelope. It is proposed that the irreversible bonding between corneocytes that results from heating or lipid extraction results from interdigitation of the sphingosine chains belonging to those hydroxyceramides that are bound to the corneocyte protein envelope by the omega-hydroxyl function of the 30- and 32-carbon hydroxyacid moieties. Similar interdigitation of adjacent envelopes might be involved in natural stratum corneum cohesion, limited mostly to the periphery of corneocytes where the absence of intercellular lamellae allows the appropriate cell-to-cell contact.

Animals↗
PubMed1989

The origin of alkanes found in human skin surface lipids.

Lipids extracted from human skin contain variable amounts of paraffin hydrocarbons. Although the composition of these alkanes strongly resembles petroleum waxes, it has been proposed that they are biosynthetic products of human skin. To investigate this question, skin surface lipids from 15 normal subjects were analyzed for the amount and composition of alkanes, using quantitative thin-layer chromatography and quartz capillary gas chromatography. The alkanes were found to constitute 0.5% to 1.7% of the skin lipids. Subjects differed greatly in the chain length distribution of their alkanes between 15 and 35 carbon atoms, and in the relative amounts of normal alkanes (like those in petroleum waxes) and branched chain alkanes (like those in petroleum lubricating oils). In 6 subjects, the alkane content of cerumen from each ear was examined to investigate whether alkanes arrive at the skin surface by a systemic route or by direct contact with environmental surfaces. No trace of alkanes was found in 11 of the 12 cerumen samples. Using a tandem accelerator mass spectrometer for carbon-14 dating, a combined sample of the skin surface alkanes was found to have a theoretical age of 30,950 years, similar to that of a sample of petrolatum. These analyses indicate that the alkanes found on the surface of human skin are mixtures of a variety of petroleum distillation fractions that are acquired by direct contamination from the environment.

Adult↗
PubMed1989

Preparation of model membranes for skin permeability studies using stratum corneum lipids.

Liposomes were prepared from stratum corneum lipids consisting of epidermal ceramides (55% by weight), cholesterol (25%), free fatty acids (15%), and cholesteryl sulfate (5%). Multiple lamellae were formed by air-drying the liposomal suspensions on hydrophilic filter disks, and water permeabilities through these filter-supported lamellae were measured using a diffusion cell. Ultrastructure of the lipid lamellae was characterized by scanning and thin-section electron microscopy. Water flux data and ultrastructure of the model membranes are discussed in relation to stratum corneum, the horny layer of the epidermis that constitutes the epidermal barrier.

Animals↗
PubMed1989

Covalently bound lipids of human stratum corneum.

In the present study, we demonstrate that human stratum corneum contains covalently bound lipids accounting for 1.4% of the dry weight of the tissue. The major component (53.3% of the total by weight) is a ceramide (CER-A) consisting of 30 through 34-carbon omega-hydroxyacids amide-linked to sphingosine. The other bound lipids in human stratum corneum include fatty acids (12.7%), omega-hydroxy acids (9.4%), and a second, more polar, omega-hydroxyacid-containing ceramide (CER-B, 24.8%). The predominant omega-hydroxyacids in both ceramides, as well as the free hydroxyacid fraction, are the 30-carbon saturated and 32- and 34-carbon monoenoic species. The bound fatty acids consist largely of 14 through 22-carbon saturated species, but significant proportions of monoenoic species and linoleic acid are also present. Psoriatic scale contains a similar total concentration of the same covalently bound lipids, but the proportions of the individual bound lipids are different from those found in normal stratum corneum. It is suggested that the principal function of the covalently bound lipids in human stratum corneum is the formation of a lipid envelope on the outer surface of the keratinized cells.

Animals↗
PubMed1989

Changes in sebum secretion and the sebaceous gland.

New data have shown that sebum secretion is highest in young adults and then declines steadily throughout life in both men and women. This parallels the decline in androgen levels, but this cannot be the sole factor because there is so much overlap in sebaceous gland activity between men and women. The increased occurrence of dry skin in the elderly has been shown to be unrelated to the sebum secretion rate. This is not unexpected, as children, with even less sebum, rarely have this problem. Seborrheic dermatitis, which is also much more common in the elderly, is also unrelated to sebum secretion rate.

Aging↗
PubMed1988

Effect of epidermal acylglucosylceramides and acylceramides on the morphology of liposomes prepared from stratum corneum lipids.

Epidermal acylglucosylceramides (AGC) and acylceramides (AC) cause aggregation and stacking of stratum corneum lipid liposomes formed from a lipid mixture containing epidermal ceramides (40%), cholesterol (25%), palmitic acid (25%), and cholesteryl sulfate (10%). This demonstrates the ability of these sphingolipids to hold adjacent bilayers in close apposition and their roles in the assembly of lamellar structures in the epidermis. However, AGC and AC in their hydrogenated form also caused aggregation and stacking of the stratum corneum lipid liposomes. This throws into doubt the proposed structural specificity of linoleate in the function of AGC and AC as molecular rivets in the assembly of the epidermal lamellar granules and the stratum corneum intercellular lamellae, respectively.

Animals↗
PubMed1988

Isolation of corneocyte envelopes from porcine epidermis.

Sheets of porcine stratum corneum were dispersed into individual corneocytes after 4 h in a solution consisting of 8 mM N,N-dimethyldodecylamine oxide and 2 mM sodium dodecylsulfate in phosphate-buffered isotonic saline, at 45 degrees C. With continued detergent treatment and moderate sonication, most of the cells lost their keratin contents and were then separated from the remaining intact cells by centrifugation in cesium chloride solution of density 1.280. Electron microscopy showed that the cell envelopes retained both the crosslinked protein envelope and its attached lipid envelope. The dry weight of envelopes was approximately 7% of the estimated dry weight of the original stratum corneum, while the corneocytes surviving intact also amounted to 7% of the starting weight. Mild alkaline hydrolysis of the corneocyte envelopes allowed the extraction of hydroxyceramides amounting to 10% of the dry weight of the envelopes. The procedure therefore provides isolated corneocyte envelopes suitable for studying both the protein and lipid components of this compound sheath.

Animals↗
PubMed1988

Hydroxyacid derivatives in human epidermis.

The principal objective of this investigation was to examine the omega-hydroxyacid derivatives in the cornified layer of human skin. Toward this end, sunburn peelings were collected, and the lipids were extracted with chloroform/methanol. Comparison with authentic standards by thin layer chromatography (TLC) indicated the presence of both N-(omega-acyloxy)acylsphingosine (acylceramide) and omega-acyloxy fatty acid (acyl acid), which accounted for 1.7% and 2.0% of the total lipid, respectively, as estimated by photodensitometry of the charred chromatograms. Each of these lipids was isolated by preparative TLC, hydrolyzed, and the resulting fragments were further analyzed by TLC and by gas liquid chromatography (GLC) of appropriate derivatives. In both of these lipids, the predominant omega-hydroxyacid proved to be the 30-carbon saturated species (56-59% of the total), while linoleate was the major ester-linked fatty acid. Linoleate was more abundant in the acyl acid (38.0%) than in acylceramide (21.4%). These findings represent the first demonstration of acyl acid in human skin and support the proposition that acyl acid, like the other linoleate-rich omega-hydroxyacid derivatives, functions as a molecular rivet in maintaining epidermal structure and function.

Ceramides↗
PubMed1988

Integral lipids of human hair.

It has long been recognized that hair is coated with nonpolar lipids originating in the sebaceous glands, and recently it has been shown that hair also contains cholesterol sulfate and small amounts of ceramides, similar to those found in the keratinized portion of the epidermis. In the present study, it is demonstrated that significant amounts of several additional lipids are tightly associated with hair in such a way as to be highly resistant to solvent extraction. These integral hair lipids included cholesterol sulfate (3.3 mg/g of extracted hair), cholesterol (0.6 mg/g), fatty alcohols (0.2 mg/g) and free fatty acids (4.3 mg/g). The principal fatty acid, comprising 40% of the total fatty acids, was identified as 18-methyl-eicosanoic acid by cochromatography with authentic standard on gas-liquid chromatography (GLC) and by mass spectrometry (MS).

Fatty Acids↗
PubMed1988

Fusion patterns of liposomes formed from stratum corneum lipids.

Stratum corneum lipid liposomes formed from epidermal ceramides (40%), cholesterol (25%), palmitic acid (25%), and cholesteryl sulfate (10%), when exposed to hypertonic medium, form flattened liposomes. Epidermal acylglucosyl-ceramides (AGCs) and acylceramides (ACs) cause aggregation and fusion of these flattened vesicles. This could serve as a model to study (a) the fusion of membranous disks in the intercellular space of the stratum corneum and (b) the roles of AGCs and ACs in the assembly of lamellar structures in the epidermis.

Animals↗