Comp. Biochem. Physiol. Vol. 93B, No. 2, pp. 265-269, 1989 0305-0491/89 $3.00 + 0.00 Printed in Great Britain © 1989 Pergamon Press plc

og-HYDROXYACID DERIVATIVES IN THE EPIDERMIS OF SEVERAL MAMMALIAN SPECIES

PHILIP W. WERTZ and I~ONALD T. DOWNING Marshall Dermatology Research Laboratories, Department of Dermatology, University of Iowa College

of Medicine, Iowa City, IA 52242, USA (Tel: 319 335 8076)

(Received 12 August 1988)

Abstract--l. co-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 co-hydroxyacids from the acylceramidc and acylglucosylceramidc were essentially identical.

3. The human oJ-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 og-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.

INTRODUCTION

Recently, several unusual co-hydroxyacid derivatives have been identified in mammalian epidermis. These include acylglucosylceramide (Gray et al., 1978; Wertz and Downing, 1983a; Abraham et aL, 1985), acylceramide 0Nertz and Downing, 1983b) and acyl- acid (Bowser et al., 1985; Wertz and Downing, 1988). Acylglucosylceramide consists of a long-chain o~-hydroxyacid (Wertz and Downing, 1983a) amide- linked to sphingosine with a fl-D-glucosyl group attached to the primary hydroxyl group of the sphin- gosinc (Gray et al., 1978) and linoleic acid estcrified to the co-hydroxyl group (Abraham et al., 1985). The acylccramidc is structurally analogous but lacks the sugar, and acylacid consists simply of linoleic acid ester-linked to the to-hydroxyl of the co-hydroxyacid. These lipids arc thought to be important for the normal formation of the epidermal barrier to water loss (Wertz and Downing, 1982; Nugtercn et al., 1985).

Each of the several mammalian species which have been examined has been found to contain epidermal co-hydroxyacid-derived lipids, as judged by thin-layer chromatography. These species included human (Wertz et al., 1987), pig (Wertz and Downing, 1983a,b; Bowser et aL, 1985), rat (Wertz et al., 1983; Hansen and Jensen, 1985), horse (Equus caballus; Wertz, 1986) and mouse (Wertz, 1986). In addition, acylglucosylceramides and acylceramides have be~n found in bird epidermis, but not in fish or amphibians (Wertz, 1986). In the case of the mouse, which is one of the most commonly-used animals in cutaneous biology, no detailed analyses of the a~-hydroxyacids have been published. In the case of the pig, conflicting

Correspondence to: Philip W. Wertz, 270 Medical Labora- tories, Department of Dermatology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.

CBP(B) 93/2--E

reports have been published concerning the occurrence of saturated chains. In one series of reports, saturated o~-hydroxyacids were found to predominate in the porcine O-acylsphingolipids (Wertz and Downing, 1983a,b), while in a report from a different laboratory, monocnoic and dienoic hydroxyacids but no saturated species at all wcrc detected (Bowser et aI., 1985). The purpose of the present investigation was to clarify these points.

MATERIALS AND METHODS

Preparation of epidermal lipids Epidermis was separated from freshly-kiUed pigs by

pressing a 65°C aluminum cylinder against the intact skin for 30 sec (Hedberg et al., 1988). A circular sheet of epidermis could then be loosened by scraping the edges with a spatula and peeled from the dermis.

Neonatal mice were killed by treatment with ethyl ether vapor. They were then immersed in 65°C water for 20 sec, after which the entire epidermis could be peeled.

Several whole rat skins were excised and hair was re- moved by treatment with depilatory wax (Wertz et al., 1983). The skins were then placed epidermis down on a 70°C hot plate for 2 min, and the epidermis was scraped from the dermis with a spatula.

The contents of an epidermal cyst were obtained at the time of surgery as previously described (Wertz et al., 1987).

All materials were dried/n vacuo prior to extraction. Each sample was then extracted with chloroform :methanol, 2:1, h l and 1:2, for 2hr each at room temperature. The combined extracts from each sample were taken to dryness, and the lipid residue was dissolved in chloroform:methanol, 2:1, at 25 mg/ml. Acylceramide and acylglucosylceramide were isolated by

preparative thin-layer chromatography (TLC) as previously described (Wcrtz and Downing, 1983a,b).

Chemical procedures Isolated acylceramide or acylglucosylceramide samples

were treated with 10% BCI 3 in methanol to cleave both the ester and amide linkages in one step. The resulting

265

266 PHILIP W. WERTZ and DONALD T. DO~VNING

¢o-hydroxyacid methyl esters were isolated by preparative TLC on 0.5-ram thick silica gel H (Merck, Darmstadt, West Gehnany) with a mobile phase of hexane: ethyl ether:acetic acid, 70:30:1. The isolated hydroxyacid methyl esters were acetylated by treatment with acetic anhydride:pyridine, 1 : 1, for 2 hr at room temperature. Excess reagent was evapo- rated under a gentle stream of nitrogen. The ~o-O-acetyl fatty acid methyl esters were purified by preparative TLC with toluene as the mobile phase.

Argentation TLC

A mixture of 3.5 g AgNO 3 and 35 g silica gel H was slurried in approximately 82 ml water, and the slurry was spread as a O.5-mm thick layer on 20 x 20 era glass plates. The plates were dried in a 110°C oven, cleaned by develop- ment with freshly-distilled ethyl ether and reactivated at 110°C prior to use.

The mixture of co-O-acetyl fatty acid methyl esters was applied as a thin streak 2 cm from the bottom of the plate. After development to the top with hexane:ethyl ether:acetic acid, 70:30:1, the plate was air dried. The dry plate was sprayed with an ethanolic solution of 2',7'-dichloro- fluorescein (1 mg/ml), and the plate was again allowed to dry. Lipid bands were then visualized under ultraviolet light, and the appropriate regions of silica gel were scraped from the plate. The lipids were recovered from the silica gel by elution with chloroform:methanol, 2:1.

Each fraction was checked for purity and rechromato- graphed on AgNO3-impregnated silica gel if necessary. Pure fractions were redissolved in equal volumes of chloroform:methanol, 2:1, to permit their quantitative comparison. Aliquots of each sample were applied on a 0.25-ram thick layer of silica gel G (soft layer adsorbasil plus 1, Alltech Associates, Deerfield, IL, USA), and the plate was developed to the top with toluene. After drying, the plate was sprayed with 50% sulfuric acid and slowly heated on a hot plate to 220°C. After charring was complete, the plate was cooled and the charred spots were quantitated" by photodensitometry (Downing, 1968) with a Shimadzu model CS 930 photodensitometer.

Gas-liquid chromatography (GLC)

For the quantitative evaluation of chain-length distribu- tions, a Varian 3700 gas chromatograph (Variafi Associates, Inc., Palo Alto, CA, USA) equipped with a probe injector, a flame ionization detector and an electronic integrator (Varian model CDS-Iil) was used. A glass column (6ft long x 0.085 in inside diameter) packed with 3% OV-101 on 80/100 mesh Supelcoport (Supelco, Inc., Bellefonte, PA, USA) was operated at 275°C with 25 ml/min of helium carder gas. The injector and detector temperatures were both 300°C.

GLC-mass spectrometry

oJ-O-Acetyl fatty acid methyl esters were separated on

a 0.2 ram x 25 m capillary column with a stationary phase of 5% phenyl methyl silicone. Chemical ionization (NH3) mass spectra were recorded with a Nermag 10-10C mass spectrometer. A range of mass/charge of 60 through 700 was examined.

RESULTS

Representative structures of epidermal acyl- ceramides and acylglucosylceramides are presented in Fig. 1. The relative proportions of the saturated, monoenoic and dienoie og-hydroxy acids from these sphingolipids are summarized in Table 1, and de- tailed compositions are given in Table 2. Within a given species, it appears that each of these sphingoli- pids contains essentially the same profile of og-hydroxyacids; however, each of the four species examined appears to be unique in terms of the degree of unsaturation of its to-hydroxy acids (Table 1). The human is the most highly saturated, followed in order of decreasing saturation by the pig, mouse and rat. Only the pig contains detectable dienoic species. Although the present results with rat and human are in good agreement with earlier findings (Wertz et al., 1983; Wertz et al., 1987), it should be pointed out that this is the first study in which the og-hydroxy acids from the mouse have been examined. Also, the results with the porcine hydroxyacids are in accord with our own previous results (Wertz and Downing, 1983a,b), but are not in agreement with those of Bowser et al. (1985), who found no saturated species at all in pig acylglucosylceramide.

Figure 2 illustrates the GLC profiles obtained for the porcine saturated, monoenoic and dienoic og-hydroxy acids analyzed as og-O-acetyl fatty acid methyl esters). As can be seen, the major entities include C30:0 and C32:1, arid although the dienes represent only a minor fraction of the pig co-hydroxy acids, the major component of this fraction is C34:2. As indicated above, the human acylceramide contains mainly saturated og-hydroxy acids with only a small proportion of monoenes and no dienoic species. The chain-length distributions of these human co-hydroxy acids, given in Table 2, are almost identical to those from the pig. This is in contrast to the situation with the rodent og-hydroxy acids, where the profiles appear to be shifted by 2 carbons toward longer chain-lengths. The contrast between the co-hydroxy acids from the pig and mouse is shown in Fig. 3.

In preliminary experiments, the og-O-acetyl fatty acid methyl esters were subjected to electron impact

Acylglucoaylceramlde

Acylceramide 14o

Fig. 1. Representative structures of epidermal acylgiucosylceramide and acylceramide.

Epidermal to-hydroxyacid derivatives

Table 1. Distribution of saturated, monoenoic and dienoic m-hydmxyacids among acylceramides and acylglucosylceramides from several mammalian species

Acylceramide Acylglucesyleeramide Mouse Rat Pig Human Mouse Rat Pig

Saturated 49.2 36.9 64.5 84.1 55.4 35.2 59.1 (2.8) (0.7) (5.1) (5.1)

Monoenoic 50.8 63.1 30.0 15.9 44.6 64.5 34.0 (5.1) (0.5) (5.1) (4.3)

Dienoic ND ND 5.5 ND ND ND 6.9 (1.1) (0.8)

Results are presented as weights percent. For mouse and pig, the reported values represent the means from three samples, and the standard deviations are given in parentheses. The results for rat and human represent single samples.

Table 2. Compositions of co-hydroxyacids from acylceramides and acylglucosyleeramides of several mammalian species

Acylceramide Acylgiucosylceramide Chain Mouse Rat Pig Human Mouse Rat Pig

28:0 0.6 0.5 6.8 9.0 0.9 1.3 5.5 29:0 0.3 0.1 4.7 5.6 0.2 0.6 3.8 30:0 6.6 7.4 38.6 39.5 7.0 3.9 33.1 30:1 ND 2.6 6.1 0.7 ND 2.0 7.3 31:0 2.9 1.0 2.8 10.5 3.5 0.8 4.0 31:1 ND 1.1 2.4 0.5 ND 0.6 3.0 32:0 29.8 24.4 11.6 19.5 ~ .7 23.2 12.7 32:1 5.1 8.0 15.1 6.0 5.0 8.9 18.0 32:2 ND ND 2.1 ND ND ND 2.8 33:0 2.1 0.5 ND ND 3.0 0.5 ND 33:1 1.6 4.3 0.8 4.0 1.5 4.3 2.0 34:0 6.6 3.0 ND ND 7.0 4.8 ND 34:1 34.1 39.4 5.6 4.7 28.3 39.4 3.8 34:2 ND ND 3.4 ND ND ND 4.0 35:1 1.9 0.7 ND ND 0.9 1.0 ND 36:1 8.1 7.1 ND ND 2.0 8.6 ND

Results are presented as weights percent. ND indicates that the component was not detected.

267

C30:0 SATURATED

G32:1 MONOENOIC

C34:2 DIENOIC

C) 1'2 2'4 3'6 4'8 Time, min.

C 3 t ~

llA'IrIJRATIED

SATURATED

MONOENOIC

C34:1 MOUU

() 1 '2 2'4 3'6 4'8 Time, rain.

Fig. 2. GLC of oJ-O-acetyl fatty acid methyl esters derived Fig. 3. Comparison of oJ-O-acetyl fatty acid methyl esters from pig epidermis, derived from pig and mouse epidermis.

268 PHILIP W. WERTZ and DONALD T. DOWNING

Table 3. Chemical ionization mass spectroscopy of co-O-acetyl fatty acid methyl esters

Parent Chemical formula of co-hydroxy to-O-acetyl fatty acid Calculated Observed Intensity

acid methyl ester +NH 4 M + 18 M + 18 (% base peak)

C28:0 C3tH~O(N 514.5 515 86 C30:0 C33H6sO4N 542.5 543 100 C30:1 C33H6604N 540.5 541 100 C32: I C3sHT004N 568.5 569 75 C32:2 C35H6sO4N 566,5 567 59 C34:2 C37HraO4N 594.5 595 58

mass spectroscopy. In these experiments, no molecu- lar ions were observed (data not shown); therefore, the possibility that chemical ionization mass spectro- scopy might prove more suitable was explored. It was found that chemical ionization using NH3 yielded relatively simple mass spectra, the major feature of which was a prominent M + 18 peak. The intensity of the observed peak at M/Z = M + 18 and its intensity expressed as a percent of the base peak (i.e. the most intense peak in the spectrum) are summarized in Table 3. In the spectra of the most abundant components, the M + 18 peak, which arises from the addition of NH4 to the parent molecule, was the base peak. In the spectra of several of the less abundant species where the signal-to-noise ratio was less favorable, there were some relatively abundant low M/Z ions. In all cases, however, the M + 18 ion was of impressive intensity.

It should be noted that for molecules of the size dealt with in the present study, it becomes signifi- cant that the atomic weights of nucleides are not whole numbers. For instance, on the basis of C m: = 12.00000: H l = 1.00783; O m6 = 15.99492; and N 14= 14.00307. The weights of the molecular ions calculated on this basis also are given in Table 3, and since the spectrometer used in this study is not a high-resolution instrument, the measured M/Z values are rounded off to the nearest integer. In effect, this adds one mass unit to the nominal molecular weight of each of the molecules studied.

DISCUSSION

It has been proposed that acylglucosylceramides and acylceramides serve as molecular rivets to hold together the stacks of unit membranes which occur in the epidermal lamellar granules and within the intercellular spaces of the stratum corneum respectively (Wertz and Downing, 1982, 1983b; Wertz, 1986). Within this context, the oJ-hydroxyacyl chain is thought to span the hydrophobic portion of one bilayer while the ester-linked linoleate is inserted into an adjacent membrane, thus riveting the two membranes together. Measured from the carbonyl carbon to the hydroxy-terminal carbon atom, C30:0 and C32:1, the major entities in the pig O- acylsphingolipids, are 3.7 nm in length. In contrast, the major co-hydroxyacids from the rat and mouse are C32:0 and C34:1, which are 3.95 nm long. This implies that the membranes found in the stratum corneum and lamellar granules of the rat or mouse would be slightly thicker than those in the pig or human. Unfortunately, this small difference in thickness cannot be distinguished by the electron microscopic methods most commonly used to study

epidermal ultrastructure; however, resolution of this problem may be amenable to the X-ray or neutron diffraction methods, which are just beginning to be applied to the study of this sort of membrane system (King and White, 1986).

Mouse skin has been much used in studies of percutancous absorption (Bond and Barry, 1988), chemical carcinogenesis (Hecker, 1987) and the growth and differentiation of keratinocytes in culture (Marcelo and Tong, 1983). Since many of these studies are related to either the formation of the epidermal permeability barrier or the transport of substances across that barrier, it is desirable to know as much as possible about the barrier components. The present results concerning the co-hydroxyacids from mouse acylglucosylceramide and acylceramide are particularly relevant, since these sphingolipids appear to be uniquely associated with the formation and integrity of the permeability barrier (Wertz, 1986).

In previous studies of pig acylceramide (Wertz and Downing, 1983b) and acylglucosylceramide (Wertz and Downing, 1983a), our laboratory found mainly saturated and monoenoic og-hydroxyacids with small amounts of dienes. These assignments were based upon argentation TLC and GLC comparisons with authentic co-hydroxyacids of known composition. However, in a more recent publication (Bowser et al., 1985), it was reported that pig acylglucosyl- ceramide contains only monoenoic and dienoic co-hydroxyacids, and these assignments were based on electron impact mass spectroscopy of individual co-hydroxyacid derivatives isolated by high-pressure liquid chromatography (HPLC). This more recent investigation would appear to be more rigorous; however, it seemed possible that saturated species could have been lost during the HPLC isolation. Such losses could have resulted either from difficulties in detection or from the relatively poor solubility of saturated co-hydroxyacids and many of their derivatives. Also, it was not clear that molecular ions were observed in the mass spectra, which makes correct interpretations difficult. Alternatively, our earlier assignments may have been in error. In order to resolve this conflict, we subjected our co-O-acetyl fatty acid methyl esters to GLC combined with chemical ionization mass spectroscopy using NH 3 . This procedure produces much less fragmentation and is less suitable for the identification of specific functional groups, but it reliably produces an intense M + 18 peak and is therefore much superior to electron impact for the elucidation of molecular weights. Our present results, including those obtained by the chemical ionization method, confirm our original assignments.

Epidermal c0-hydroxyacid derivatives 269

Acknowledgements--This work was supported in part by a grant from the United States Public Health Service (AM32374).

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