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Biochimica et Biophysics Acta, 431 (1976) l-8 0 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 56768

PHOSPHATIDYL-(N-ACYL)-ETHANOLAMINE

A LIPID COMPONENT OF MAMMALIAN EPIDERMIS

GM. GRAY

MRC Unit on the Experimental Pathology of Skin, The Medical School, The Uniwrsity, Birmingham 15 (U.K.)

(Received November 26th, 1975)

Summa~

A lipid present in the granular cells of mammalian epidermis was identified as phosphatidyl-(N-acyl)-ethanolamine.

The structure was deduced from the ratio of phosphorus : nitrogen : glycerol : fatty acid esters : total fatty acid (1 : 0.94 : 0.97 : 2.1 : 2.9), from analyses of the products of alkaline and acid hydrolyses and from its infrared spectrum. Conclusive evidence was obtained by a direct comparison of the chromato- graphic properties, degradation products and infrared spectrum of the isolated lipid with those of synthetic 1,2-dip~mitoyl-so-glycero-3-phospho-(~-p~mi- toyl)-ethanolamine.

The fatty acids attached to the ethanolamine were predominantly saturated (69% of total) and hexadecanoic acid was the major component (41% of total).

Phosphatidyl-(N-acyl)-ethanolamine was hydrolysed by a phospholipase C (Bacillus cereus) to diacylglycerol, inorganic phosphorus and N-acylethanol- amine. Evidence for the presence of ~-acyleth~olamine in granular cells and in stratum corneum suggested that an epidermal phospholipase C may be involved in the catabolism of phosphatidyl-(~-acyl)-ethanolamine.

Introduction

It has been shown El] that granular cells isolated from pig and human epi- dermis contained an unusual phospholipid (phospholipid X) as a major compo- nent of the total phospholipids of the cell. This phospholipid was not present in stratum corneum [l] and the small amounts found in the preparations of basal and spinous cells of the epidermis [l] could be accounted for by some contaminating granular cells,

Initial attempts at characterization were limited by lack of material but the

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presence of a diacylglycerylphosphate (‘phosphatidyl’) structure was confirmed [ 11. However, the conclusion that nitrogen was not present in the molecule and the suggestion that the ‘phosphatidyl’ structure might be attached to a monoacylglycerol were shown to be incorrect by subsequent analyses of a pure preparation of phospholipid X isolated from the total lipids of pig epidermis. Conclusive evidence is presented that phospholipid X is phosphatidyl-(N-acyl)- ethanolamine and its role in epidermal cell metabolism is discussed.

Expe~mental and Results

Analytical methods Phosphorus was determined by the method of Long and Yardley [a], acyl

ester groups by the method of Snyder and Stephens [3] and glycerol by estima- tion of formaldehyde produced by periodate oxidation [4]. Total nitrogen was determined by the method of Lang [ 51. Fatty acids isolated from phospholipid X as methylesters were analysed by gas-liquid chromatography on 10% EGSS-X (support: Gas Chrom Q 100-120 mesh, Applied Science Laboratories, State College, Pa., U.S.A.) at 18O”C, and 3% OVI (support: Gas Chrom Q 100-120 mesh, Applied Science Laboratories, State College, Pa., U.S.A.) at 190°C.

Materials 1,2-Dipalmitoyl-sn-glycerol-3-phosphoethanolami~e and phosphatidic acid

were obtained from Koch-Light (Colnbrook), ethanolamine from Sigma, and phospholipase C (EC 3.1.4.3) from Clostridium perfringens and Bacillus cereus were, respectively, from the Lister Institute (Elstree) and from Boehringer (~annheim). 1,2-Dipalmitoyl-sn-glycero-3-phospho-(N-palmitoyl)-ethanolamine (phosphatidyl-(N-palmitoyl)-ethanolamine) was synthesized as described by Bornstein [6]. N-Palmitoylethanolamine was synthesized by a standard proce- dure for N-acylation of amines [ 71.

~xtracti~~~ of lipids from epidermis Pig tails and/or ears were obtained within 1 h after death. They were washed

with cetrimide (cetyltrimethylammonium bromide, 5% w/v, aqueous solution) and water and the ears or skin from the tails were spread flat over a damp sponge sheet. Hairs were removed with clippers and slices of epidermis were taken from the skin with a Castroviejo keratotome (0.1 mm cut) [8]. The slices were minced witb scissors and homogenized in chloroform/meth~ol (1 : 1, v/v; 10 ml/g of tissue). The mixture was filtered and the residue re-extracted twice with chloroform/methanol (2 : 1, v/v; 5 ml/g tissue). The extracts were com- bined and washed twice with 0.2 volumes of 0.1 M KC1 [9]. The aqueous upper phases were discarded and the chloroform phase was evaporated to dry- ness below 40°C under vacuum. The lipids were redissolved in dry chloroform and stored at -20°C. A total of 18 g lipid containing 210 mg of lipid phos- phorus was obtained from approximately 800 g of epidermis.

Isolation of phospholipid X The total lipids in chloroform were applied to a column of silica gel 60

(210 g, 70-230 mesh; Merck, Darmstadt) in chloroform and were separated

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with the sequence of solvents described by Vance and Sweeley [lo]. Solvent fractions from the column were collected and monitored by thin-layer chro- matography on silica gel H (Merck) with chloroform/methanol/4 M aqueous ammonia (65 : 25 : 4, by vol.) as the solvent system. Lipids were detected by spraying the thin-layer chromatography plate with 50% (v/v) HzS04 and heating for 20 min at 160-180°C. Phospholipid X was eluted from the column with acetone/methanol (9 : 1, v/v) along with some acidic phospholipids and cho- lesteryl sulphate [ 11. All solvent fractions containing phospholipid X were combined (phosphorus = 8 mg) and the total lipid from these fractions was re- covered and redissolved in chloroform/methanol (19 : 1, v/v). The solution was put on to a column of silica gel 60 (16 g, 230--400 mesh) saturated with am- monia [ 11. Phospholipid X was eluted slowly by chloroform/methanol (19 : 1, v/v). The recovered phospholipid X was still slightly contaminated by one other compound, which was removed by passage through another silica gel 60 column (5 g) previously saturated with ammonia and washed with chloroform. The lipid was put on to the column in chloroform and chloroform/acetone (1 : 1, v/v), acetone and acetone/methanol (9 : 1, v/v) were passed through the col- umn. Phospholipid X was eluted with acetone/methanol (9 : 1, v/v). Recovery of phosphorus (2.4 mg) was approximately 70% of the total phospholipid X present in the original lipid extract. Only one component was detected when a sample of phospholipid X was chromatographed on silica gel H thin-layer plates in chloroform/methanol/water (65 : 25 : 4) (Solvent a), chloroform/methanol/ 4 M aqueous ammonia (65 : 25 : 4) (Solvent b), chloroform/methanol/acetic acid/water (80 : 24 : 6 : 1) (Solvent c) and tetrahydrofuran/methylal/methanol/ 4 M aqueous ammonia (10 : 6 : 4 : 1) (Solvent d).

Characterization of phospholipid X Phospholipid X contained fatty acid ester groups in a molar ratio to phos-

phorus of 2.1 : 1. A molar ratio to phosphorus of 2.9 : 1 was obtained for the total fatty acid methyl esters extracted with hexane from a sample of phospho- lipid X treated with 0.8 M methanolic HCl for 18 h at 80°C in a sealed tube. Phospholipid X was hydrolysed with mild alkali (chloroform/methanol/l M aqueous NaOH, 1 : 8 : 1 by vol.) for 1 h at 37°C after which time all the phos- phorus became water-soluble. An unusual property of this phosphate ester was its complete solubility in chloroform as well as in water. Paper chromatography [ll] of the phosphate ester in butan-1-al/acetic acid/water (5 : 4 : 1) (Sol- vent e), and phenol/ethanol/acetic acid/water (50 : 22 : 3 : 3) (Solvent f) showed, after spraying chromatograms with acid/molybdate [ 121 and exposing to ultraviolet light, only one phosphorus-containing component. Its retention values relative to sn-glycerol-3-phosphate in the two solvent systems were re- spectively 2.5 and 2.3. Periodate oxidation of the water-soluble phosphate ester produced 1.1 mol of formaldehyde per mol of phosphorus. Periodate oxidation of the products of acid hydrolysis (4 M HCl, 110°C for 60 h in a sealed tube) of the water-soluble phosphate ester produced 1.94 mol formaldehyde/mol phos- phorus.

Treatment of intact phospholipid X with glacial acetic acid (2 h, 100°C) produced another lipid as the major product (80% of total phosphorus) which was chromatographically similar to phosphatidic acid in thin-layer chromato-

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OC I

4ooo I I I 1 -~LL__~~__1

3000 2000 1000 1200 600 400

Wavenumber (cm’1

Fig. 1. The infrared spectra of (a) 1,2-dipiilmitoyl-sn-glycero-3-phospho-(~-paln~it~yl)~thanol~ine and (b) phospholipid X. The infrared spectrum of each phospholipid deposited as a thin film on a NaCl disc was recorded in a Perking-Elmer spectropbotometer model 180.

graphy with solvents (a)-(d). Its fatty acid ester to phosphorus ratio was 2.1 : 1. Mild alkaline hydrolysis of this lipid product gave one water-soluble phosphate ester chromato~aphic~ly indistin~ishable from sn-glycerol-3-phos- phate in Solvents e and f.

The infrared spectra of intact phospholipid X (Fig. 1) showed strong absorp- tion bands at 1650 and 1540 em-‘, which were indicative of an amide group. Nitrogen in phospholipid X was confirmed and determined by the method of Lang [ 51 and its molar ratio to phosphorus was 0.93. Phospholipid X was hydrolysed with 6 M HCl at 105°C for 3 h in a sealed tube. Fatty acids were extracted with hexane and the aqueous solution was evaporated to dryness in a stream of N,. Excess HC1 was removed under vacuum for 18 h over NaOH pellets. The residue was redissolved in water. It reacted with ninhydrin and was identified as eth~ol~ine by ion-exchange chromatography in an amino acid analyser, by paper chromatography in buts-l-al/acetic acid/water (12 : 3 : 5, by vol.) and in phenol/water (4 : 1, w/v) and by gas-liquid chromatography on 10% carbowax 20 M terephthalic acid (support: Gas-Chrom Q 100-120 mesh) at 190°C after N-acetylation and conversion to the trimethylsilyl derivative

n31. The experimental evidence indicated that phospholipid X was a phosphatidyl-

(N-acyl)-ethanolamine. This was confirmed by a comparison of the infrared spectra of phospholipid X and synthesized phosphatidyl-(iV-palmitoyl)-ethanol- amine (Fig. l), which were virtually identical. The natural and the synthetic phospholipids co-chromatographed as one compound in thin-iayer chromato- graphy Solvents aad. Their water-soluble phosphate esters obtained after treatment of the fipids with mild alkali were indistin~ishable by paper chro-

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matography in Solvents e and f. The fatty acids attached to the glycerol (0-acyl) of phospholipid X obtained after treatment of phospholipid X with mild alkali and those attached to the ethanolamine (N-acyl), obtained after treatment of the partially deacylated, water-soluble phosphate ester with 0.8 M methanolic HCl for 18 h at 80°C in a sealed tube, were analysed by gas chromatography (Table I). The N-acyl fatty acids were predominantly saturated (69% of total) and hexadecanoic (palmitic) acid was by far the major component (40% of total).

The possible production of artifacts in the course of isolating the lipids from epidermis was considered previously and discounted [ 13 . The identification of phospholipid X as phosphatidyl-(N-acyl)-ethanolamine raised a particular pos- sibility that the compound could be formed from a base-catalysed aminolysis [14] during the isolation of lipids. This was not supported by the facts that lipid extracts were isolated exclusively under neutral or slightly acid conditions and that, depending on their source, the lipid extracts contained different amounts of phosphatidyl-(N-acyl)-ethanolamine which did not increase during the isolation procedures.

Action of phospholipase C on phospholipid X A sample of phospholipid X (-40 pg phosphorus) was sonicated in 1.0 ml of

10 mM Tris/maleate buffer (pH 7.2) for 10 min to give a slightly milky opales- cent solution. Phospholipase C (8 units/l0 ~1) from B. cereus (400 units */mg) was added and the mixture incubated at 37°C. After 2 h a further 8 units of phospholipase C were added and the mixture was incubated for a further 18 h. The enzyme was inactivated with 3 ml chloroform/methanol (2 : 1). Analysis of the aqueous and chloroform phases indicated that 75% of the total lipid phosphorus was water-soluble and 90% of that was present as inorganic phos- phorus. Thin-layer chromatography of the chloroform-soluble material in

TABLE I

FATTY ACIDS IN O-ACYL AND N-ACYL POSITIONS OF PHOSPHATIDYL-(N-ACYL)-ETHANOL-

AMINE OF PIG EPIDERMIS

Values are expressed as percentages of total fatty acids.

Fatty acid

designation

O-Awl N-Awl

14 : 0 1.5 2.3

15 : 0 1.8 4.4

16 : 0 16.7 40.9

16 : 1 1.4 9.7

17 : 0 1.9 trace

18 : 0 12.8 12.9

18 : 1 27.9 14.8

18 : 2 14.2 6.2

19 : 0 - trace

20 : 0 3.9 2.9

22 : 0 6.5 5.9

Unidentified 5.4 -

* 1 unit Of enzYme will hydrolyse 1 pm01 phosphatidylcholine per min at 37°C.

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chloroform/methanol/water (90 : 10 : 1) and light petroleum/diethyl ether/ acetic acid (30 : 70 : I) indicated two major compounds whose chromatograph- ic properties corresponded to diacylglycerol and ~-p~mitoyleth~clamine. Minor compounds were monoacylglycerol, free fatty acid and intact phospho- lipid X. The whole of the remaining chloroform-soluble material was separated into components by thin-layer chromatography on silica gel H with a chloro- form/methanol/water (90 : 10 : 1) solvent system. The lipids were visualized with Iz vapour and the component corresponding to the ~-palmitoylethanol- amine marker was recovered from the silica gel by extracting with chloroforms methanol (2 : 1, v/v). The solvent was removed with a stream of N, and the lipid residue was hydrolysed with 6 M HCl for 3 h at 100°C. The hydrolysate was extracted with hexane. Fatty acids were the only components in the hexane extract and ethanolamine, identified by paper chromatography, the sole water- soluble compound. Under similar conditions phosphatidic acid (40 E;rg phos- phorus) was hydrolysed completely by phospholipase C to diacylgIy~ero1 and inorganic phosphorus and phosphatidylethanolamine to diacylglycerol and phosphorylethanolamine. No inorganic phosphorus was released by the enzyme from the latter phospholipid. Synthetic phosphatidyl-(N-palmitoyl)-ethanol- amine gave the same products as phospholipid X. Phospholipase C from Cl. ~e~~~~~ge~s (13 units/mg) was inactive towards phospholipid X and syntlletic phosphatidyl-(~-palmitoyl)-eth~ol~ine in 10 mM Tris/maleate buffer (pH 7.2) and in 0.1 M borate buffer (pH 7.4), both with added Ca2+ (10 mM), in either an aqueous system or a diethyl ether/water (1 : 2) system.

As N-acylethanolamine was a product of the action of phospholipase C on phospholipid X, its possible presence in the lipids from different cell popula- tions of pig epidermis was investigated. The ~hromatographic properties of N- palmitoyiethanolamine on thin-layer plates of silic gel H and on columns of silica gel were very similar to those of ceramides. Samples from the fractions containing ceramides, which were previously isolated from the total lipids of (a) basal and spinous cells, (b) granular cells and (c) stratum corneum of pig epidermis [I] , were hydrolysed with 6 M HCl at 100°C for 3 h. The hydrolysate was extracted with hexane to remove fatty acids. Chloroform~methanol (2 : 1, v/v) was added to the hydrolysate and the mixture shaken. The chloroform phase was discarded and the aqueous phase evaporated to dryness in a stream of N2. Excess HCl was removed under vacuum over NaOH pellets. The residue was redissolved in water and a sample analysed by paper chromatography in Solvents e and f. Ethanol~ine hydrochloride was included for reference. Com- pounds were detected with ninhydrin spray. No ethanolamine was detected in the hydrolysis products of lipid from basal and spinous cells but those from the granular cells and from the stratum corneum did contain ethanolamine. From this evidence it is reasonably certain that N-acylethanolamine is a component of the neutral lipid fractions isolated from granular cells and from the stratum corneum of pig epidermis.

Discussion

Phosphatidyl-(N-acyl)-ethanolamine has been identified in plant tissues [ 151 and in a Butyrivibrio strain tentatively identified as B~~yrivibr~o fibris~~vens

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[ 161. The identification of phosphatidyl-(N-acyl)-ethanolamine in pig epidermis is, I believe, the first report of the occurrence of this compound in a mam- malian tissue, though a closely related lipid, phosphatidyl-(N-acyl)-serine has been identified in sheep erythrocytes [ 171. Phosphatidyl-(N-acyl)-ethanol- amine was also detected in human and rat epidermis [18] and its presence in skin may be widespread.

How is this phospholipid produced in mammalian epidermis? It was shown previously [l] that (a) gross changes occur in the lipid compositions of epi- dermal cells in their passage from the basal layer to the outer surface of the stratum corneum, the major changes taking place during the cell’s passage through the granular layer of the epidermis as various catabolic activities in- crease, (b) all phospholipids were catabolised as the cells passed through the granular layer, since the stratum corneum cells did not contain phospholipids, and (c) phosphatidyl-(N-acyl)-ethanolamine was present in the granular cells but not in the basal and spinous c&s. Therefore one possibility is that the N- acyl compound is an intermediate in the catabolism of phosphatidylethanol- amine. Hazlewood and Dawson [15] showed that a Butyriuibrio organism could carry out the intermolecular transacylation reaction: 2 phosphatidyl- ethanolamine -r phosphatidyl-(N-acyl)-ethanolamine plus lysophosphatidyl- ethanolamine. The organism also 0-deacylated phosphatidyl-(N-acyl)-ethanol- amine to lysophosphatidyl-(N-acyl)-ethanolamine. ,4 similar transacylation reaction may occur in granular cells of epidermis though lysophosphatidyl- ethanolamine was not detected in the phospholipids of these cells. O-Deacyla- tion may also occur because the phospholipid, phospholipid Y, present in granular cells [l] is, on the basis of the identification of phospholipid X, lyso- phosphatidyl-(N-acyl)-ethanolamine. Other lysophospholipids were not detected in granular cells even though phospholipid catabolism must involve phospho- lipases and phospholipase A has been reported in epidermis [ 191. Furthermore, a relationship between the loss of sphingomyelin and the increase in ceramide in the upper layers of the epidermis noted previously [l] suggested a phospho- lipase C activity. The evidence that phosphatidyl-(N-acyl)-ethanolamine was hydrolysed by a phospholipase C (from B. cereus) to diacylglycerol, inorganic phosphorus and N-acylethanolamine and that N-acylethanolamine was a com- ponent of the lipids isolated from granular cells and from stratum corneum gives further support for the presence of a phospholipase C in epidermis and also sug- gests a catabolic pathway for phosphatidyl-(N-acyl)-ethanolamine.

If not a catabolic intermediate, phosphatidyl-(N-acyl)-ethanolamine might be synthesized for a particular function associated with the final stages of epider- ma1 cell differentiation or, less probably, it may be a natural artifact from an unusual substrate-enzyme interaction made possible by the general degenera- tion and catabolism of the subcellular membrane systems within the granular cell.

Acknowledgments

I wish to thank Miss Anne Tabiowo and Mrs. Jacqueline Willis for expert technical assistance and Dr. J.K. Brown, Chemistry Department, University of Birmingham, for the infrared spectra.

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