Variation in skin surface lipid composition among the equidae

Comp. Biochem. Physiol. Vol. 75B, No. 3, pp. 429433, 1983

0305-0491/8353.00+ .00 © 1983 Pergamon Press Ltd

Printed in Great Britain.

VARIATION IN SKIN SURFACE LIPID COMPOSITION AMONG THE EQUIDAE SABIN W. COLTON VI and DONALD T. DOWNING The Marshall Dermatology Research Laboratories, Department of Dermatology, The University of Iowa College of Medicine, Iowa City, IA 52242, USA (Tel: 319353-5788)

(Received 22 October 1982) Abstract 1. Skin surface lipids from Equus caballus, E. przewalskii, E. asinus, E. grevyi, E. hemionus onager and a mule (E. asinus/E, caballus) were analyzed in detail. 2. In all species the surface lipid mixtures consisted of giant-ring lactones, cholesterol, cholesteryl esters and minor amounts of wax diesters. 3. In E. caballus, the lactone hydroxyacids were entirely branched chained, while in E. asinus and E. grevyi they were almost exclusively straight chained. In E. przewalskii, the onager and the mule there were both straight and branched chain hydroxyacid lactones. 4. These results are in harmony with published interpretations of the evolutionary relationships among Equus species.

INTRODUCTION Surveys have shown that lipids produced by the sebaceous glands in mammalian skin can differ widely in chemical structure among species (Nicolaides et al., 1968: Lindholm et al., 1981). However, within genera, or even families such as the Canidae, Bovidae and Equidae, where the species are morphologically and ecologically similar, the skin surface lipids seemed to contain similar compounds (Lindholm et al., 1981). Although the sebaceous lipids are believed to function as water repellants for the mammalian pelt, this would not explain the generally wide divergence in chemical composition that has evolved, since all of the sebaceous lipids are similar in physical properties and water repellancy. Investigators have therefore considered what other functions the skin surface lipids might fulfil, including pheromonal activity. Certain specialized sebaceous glands do indeed play a role in chemical communication, as in the civet cat and the musk deer, as well as in a wide range of mammals which engage in territorial marking (Mykytowycz and Goodrich, 1974; Miiller-Schwartze and Mozell, 1977). The value of such pheromonal functions would be reduced, however, if related species had similar skin surface lipid composition, as appeared to be the case in the Equidae. In a previous study we showed that the skin surface lipids of the domestic horse (Equus caballus) consist of cholesterol (14~), cholesteryl esters (38~), and lactones (48~o) formed by cyclization of to-hydroxyacids having predominantly 32, 34 and 36 carbon atoms, which we named equolides (Downing and Colton, 1980). A small proportion of the equolide molecules was saturated (16~), the majority had one double bond (78~o) and 6 ~ had two double bonds. In the monounsaturated lactones, about half had the double bond located 8 carbon atoms from the alkyl oxygen end of the molecule and half had o~-10 unsaturation. Virtually all of the lactones had a 429

methyl group in the ¢o-1 position. Similar structural features were present in the fatty acids from the cholesteryl esters. In the present study we have examined the structures of the equolides, cholesteryl esters and wax esters from additional species of Equus. N o two species were alike in the chain structures, or mixtures of structures, of their surface lipids, which were readily distinguishable and which may form a basis for interpretation of taxonomic relationships. MATERIALS AND M E T H O D S

Collection oJ sur/~we lipids Lipid collections were made by pouring 100 ml of acetone onto an area of intact skin and then scraping upwards over the area with a large beaker. With all but the common horse, the animals were immobilized chemically for necessary veterinary procedures. The recovered solvent extracts were evaporated in a rotary evaporator and the lipid residues were dried under high vacuum and then redissolved in toluene.

Lipid analyses The amounts and identity of lipid classes present in the extracts were examined by analytical thin layer chromatography (TLC). Aliquots of the lipid solutions were applied to 250/~m layers of silica gel G on 20 x 20 cm glass plates on which the adsorbent had been ruled into 6 mm-wide lanes. The chromatograms were developed successively with hexane (to 19 cm), then toluene (to 19 cm), then hexane:ether:acetic acid, 70:30:1 (to 10 cm). The dried chromatograms were sprayed with 50~o HzSO4 and heated to 220' C. The charred chromatograms were then quantified by photodensitometry (Downing, 1968; Downing and Stranieri, 1980). For isolation of individual lipid classes, aliquots of the lipid mixtures were applied as a streak across 1 mm-thick layers of silica gel G on 20 x 20 cm glass plates and the chromatograms were developed with hexane:benzene (1 : 1). When dry, the chromatograms were sprayed with 2',7'dichlorofluorescein and viewed under u.v. light. The cholesIeryl ester band, and another containing the lactones and wax diesters, were scraped from the plate and extracted with ether. The lactones and wax diesters were separated by a

430

SABIY W. COI.TON Vl and DONALD T. DOWNING Table 1. Class composition (wC;,) of the skin surface lipids of some equids Species E. E. E. E. E. E.

Cholesterol

o)-Lactones

Wax diesters

Chol. esters

14 3 I0 6 2 3

47 52 56 54 67 57

I 1 9

3~; 44 24 40 29 35

eahallus przewalskii asinus asinus/eahallus hemionus onager gret3'i

second chromatogram, which was developed twice with carbon tetrachloride, and the resolved lipids were recovered with ether. Equolides. The lactone fraction from each species was resolved by TLC on silica gel H/AgNO, into saturated and monounsaturated fractions which were then examined by gas chromatography (GLC) on a 2m x 2mm column packed with 3°~, OVl01 on Supelcoport held at 310C. Individual lactones were tentatively identified by their GLC retention times, and relative amounts were calculated from GLC peak areas. The structures of the lactone fractions were confirmed by nuclear magnetic resonance (NMR) spectrometry of their proton absorptions at 90 MHz, using a JEOL FX90Q pulse Fourier transform instrument. Cholesteryl esters. The cholesteryl ester fraction from each species was hydrolyzed with 0.5 N KOH in 95~i, methanol at 60°C for 1 hr and the liberated fatty acids were methylated by direct addition of excess BF3/methanol. The recovered cholesterol and fatty acid methyl esters were resolved by TLC. Saturated and unsaturated methyl esters were resolved by TLC of their bromomercurimethoxy derivatives (Sebedio and Ackman, 1981). The resulting fractions were analyzed by GLC on OV101. NMR spectrometry confirmed the chain structures of the predominant components. Wax diesters. These were a minor component from alI species, but hydrolysis and methylation revealed that in all cases they consisted of the diesters of long chain diols. For several species, sufficient material was obtained to allow GLC analysis of the fatty acid methyl esters and of the isopropylidene derivatives of the diols. RESULTS

The relative a m o u n t s o f the surface lipid constituents from each species are s h o w n in Table 1. The

2 4

composition o f the lactones from each species are contained in Table 2, and the compositions of the cholesteryl ester fatty acids are given in Table 3. In Fig. I are shown the N M R spectra for the m o n o u n s a t u r a t e d lactones from each o f the species examined. These provide definitive evidence that these equolides from the donkey', zebra and onager consist exclusively o f the lactones o f straight chain to-hydroxyacids, while those from the c o m m o n horse are entirely branched chained, with the methyl side chain located on the carbon atom adjacent to that bearing the hydroxyl group. The m o n o u n s a t u r a t e d equolides from the mule and Przewalski's horse each gave N M R spectra confirming a mixture o f approximately equal a m o u n t s o f straight and branched chain structures. In the donkey and zebra, both the saturated and unsaturated lactones are almost exclusively straight chained while in the c o m m o n horse both fractions are entirely branched chained. In the onager, the m o n o u n s a t u r a t e d lactones are entirely straight chained, but the saturated fraction contains both straight and branched structures. In Przewalski's horse, the unsaturated lactones contained a mixture o f straight and branched components. Thus, as summarized in Table 4, no two species revealed an overall similarity in the compositions of their equolides. These distinctions between species extended also to the compositions o f the m o n o u n s a t u r a t e d fatty acids from the cholesteryl esters, but were s o m e w h a t less clear. Thus, all species showed at least some branched chain material in these acids, which was slight in the donkey, zebra and Przewalski's horse, rather abun-

Table 2. Compositions (wtl~,~) of the saturated (s) and monounsaturated (u) lactones from some equid skin surface lipids E. eaballus E. przewalskii

Chain* 28 30i 30 31i 31 32i 32 33i 33 34i 34 35i 35 36i 36 Totals

S

7.8

U

1.4

16.4 -2.2

4.7

47.3

S

1.7

U

13.2 23.4 5.8 . . 19.3 32.5 1.8

E. asinus S

U

2.1

41.3

.

-51.2 . .

E. asinus /' caballus S

1.1

E. onager S

l.I 7.3 13.9 5.9

2.9 3.7 -

3.5 --

l 1.7 2.3

12.2 26.0

16.2 4.4 2.8

3.8

21.3 7.1

2.5

U

E. grevyi S

U

2.5 6.9

31.6 --

20.9

5.4

7.4

I.I -43.7 1.9

4.1

5.1

14.2

1.8 9.4 16

78

2. I 2

98

3.1 3

97

26

Polyunsaturates and amounts less than I";, are omitted.

74

55

45

33

67

431

Variation in skin surface lipid composition Table 3. Compositions (Wt~o) of the cholesteryl ester fatty acids from equid skin surface lipids

Chain 16i |6 18i 18 20i 20 22i 22 24i 24 26i 26 28i 28 30i 30 32i 32 34i 34 36i 36 38i 38 Totals

E. caballus E. przewalskii s u s u

5.6 -4.9 12.9 -4.7 -2.7 3.4 -2.5 -2.3 -1.8 -1.0

E. asinus s u

2.3 1.6 2.5 1.5 17.2 1.3 11.2 1.4 10.0 1.4 11.6 -8.3 1.0 6.5 -3.8 -1.4

1.6 -8.2 -3.8 --1.1 -----5.9 -15.4 -5.9

E. asinus / caballus s u

2.1 -3.8 -3.0 1.2 2.9 2.0 3.4 1.5 2.4 -3.9 -3.6 -1.9

E. onager s u

1.7 1.5 2.2 17.8 2.0 7.8 -6.0 -6.1 -4.1 -3.0

3.5 5.7 5.1 7.0 4.5 4.6 3.7 9.9 3.9 11.9 3.7 8.2 1.7

E. grevyi s u

2.5 -2.8 -4.8 -9.3 -10.0 -6.2

3.2 -2.0 -1.8 -4.2 1.0 13.8 1.3 11.8 1.1 5.8 ---1.4

1.7

1.7 47

47

7

93

57

dant in the onager, predominant in the mule and, again, the c o m m o n horse showed exclusively branched chain monounsaturated material. In the saturated fatty acids from the cholesteryl esters, the c o m m o n horse had only branched chain compounds while the zebra had only straight chain homologs. All other species showed complex mixtures of structures, including various types and degrees of branching, which precluded adequate identification of the compositions of the saturated acids. Wax diesters were not obtained in sufficient amounts to provide accurate analyses of these minor components, but differences between species were apparent. Both the donkey and zebra produced long chain diols from the diesters which were exclusively straight chained, but among the fatty acid products, only the zebra produced purely straight chain compounds. DISCUSSION The present data confirm our previous observation that for each of the equid species studied, the skin surface lipids contain similar quantities of the same lipid classes. However, the detailed analyses of these classes revealed distinct differences in the chain structures and chain length distributions among the species. These differences may be significant both in the possible biological function of the surface lipids and in elucidating evolutionary relationships among Equidae. The molecular structures of the equolides are suggestive of a function in communication, since these co-lactones combine the features of muscone and civetone, including the cyclic structure, an ethylenic bond and, in some species, a methyl branched chain. Since equolides are the predominant surface lipid for

43

41

59

5

95

46

54

both sexes, any pheromonal function may be confined to species recognition, although the possibility of subtle differences between individuals cannot be dismissed. The potential for these lipids to serve as pheromones is heightened by the present observation of distinct differences in composition between species. While it might be thought that the equolides are of too high a molecular weight to function in olfactory communication, it should be recalled that molecules almost as large have not only been recognized as olfactory pheromones but can even be smelled by the relatively insensitive human nose. Thus, in the pig, two steroids function as pheromones (Patterson, 1968a,b), and similar compounds are responsible for the smell of human axillary sweat (Labows et al., 1979). In addition to the cyclic ketones of 15 to 20 carbon atoms which are pheromones in some species, to-lactones of this size, such as ambrettolide and exaltolide, also have a strong musk-like odor for humans. It can therefore be accepted that similar compounds less than twice as large might be detected by animals having more sensitive olfaction, especially when the transmitter is a large, warm surface. Interpretations of genetic relationships among the Equidae have been based on a number of criteria, including electrophoresis of serum proteins (Kaminski et al., 1978), hemoglobin amino acid sequences (Clegg, 1974), immunochemistry, and karyotyping (Ryder et al., 1978). Cladograms based on such data do not, however, show good agreement (Kaminski, 1979). Skin surface lipid composition might be expected to be especially useful in taxonomic studies because the lipids are almost entirely synthesized de novo in the sebaceous glands and their fatty acid composition does not reflect dietary or physiological influences (Downing and Strauss, 1974, 1982). Furthermore, the lipid class composition ap-

432

SABIN W. C()LFON V| and DONALD T. DOWNIN(,

E. c a b a l l u s

E. g r e v y i

j

I

i'

l

E. a s t n u s

E. h e m i o n u s

onager

i

E.asinus/caballus

E

przewalskii

i

i

i

u'

PPM

Fig. 1. Proton magnetic resonance spectra of the monounsaturated equolides fron] five species of Equus and the mule, measured at 90 MHz. The branched chain lactones characteristic of E. caballus produced a doublet at 0.92 ppm (methyl protons) and at 3.90 ppm (protons on carbon bearing the alkyl oxygen), whereas the straight chain lactoncs found on E. ~,,rer.ri, the donkey and the onager produced no methyl proton absorption and had a triplet at 4.06 ppm for the protons adjacent to alkyl oxygen. The lactones from the mule and Przewalski's horse each produced a composite of these spectra, showing a mixture of the two types of structures. The absorptions at 1.27 ppm (methylene protons). 2.22 ppm (methylene protons adjacent to carboxyl), and 5.34 ppm (protons o n elhylcnic carbons) were the same for each structure.

parently varies slowly over evolutionary time scales, we having found no differences among racial or ethnic groups in extensive human studies, and only small differences in the lipid class composition among the Canidae, Bovidae and Equidae (Lindholm eta/., 1981). The present observations on variation within lipid classes among the equids might therefore be taken as evidence of their genetic relationships. This evidence supports the cladogram based on serum esterase studies (Kaminski, 1970; Kaminski et al., 1978), wherein two principal branches are proposed, one containing the zebras and asses, the other containing the true horses, from which the onagers

separated at an early stage and the common horse and Siberian horse separated in recent times (Kaminski, 1979). Thus, only the donkey and zebra produce equolides which are exclusively straight chained in both the saturated and unsaturated fractions. The onager produces some branched chain material, but only in the saturated lactones, while Przewalski's horse produces a mixture of straight and b.anched chain compounds in both the saturated and unsaturated fractions. Only in the c o m m o n horse are both the saturated and unsaturated fractions composed almost exclusively of branched chain compounds. This suggests that the development of

Table 4. Summary of the percentages of straight and branched chain hydrox~acids in the saturated (s) and monounsaturated (u) equolides of several Equus species E. ('ahallu.s

branched chains

E. przewalskii

E. h. ona~er

E. asinus

E.

~:'er

'i

S

LI

S

LI

S

LI

S

LI

S

LI

IO

45

3

97

33

67

1(~

7,~

2

58

35

Variation in skin surface lipid composition branched chain co-lactones was a gradual evolutionary drift, beginning before the separation of the onager from the line of modern horses, becoming extensive in the wild Siberian horse and becoming exclusive during the speciation of the domestic horse. That the lipid composition is genetically determined is supported by our observation of the composition of the equolides from the mule, which are an admixture of the straight chained compounds of the donkey with the branched chains from the horse. That the distinctions in equolide composition among the equids is subtle and not imposed by an inability of the zebra/ass branch to synthesize branched chains is indicated by the compositions of the cholesteryl ester fatty acids, which contain significant amounts of branched chains in all of the species studied. Further distinctions were apparent among the incomplete analyses of the wax diesters, where only the zebra produced exclusively straight chain fatty acid and diol moieties, Przewalski's horse produced mostly branched chains in both fractions and all other species had both straight and branched chains in both fractions. Although we have not observed significant differences in fatty chain structure among E. caballus individuals (Downing and Colton, 1980), there has been some variation in the relative proportions of saturated and unsaturated equolides. Furthermore, we have recently observed differences among human subjects in the relative amounts of wax ester fatty acid homologs which may be familial and perhaps genetic (Stewart and Downing, unpublished}. Such differences might form the basis for recognition of individuals under natural conditions, and might also be useful for analytical determination of intraspecific relationships. Acknowledgements--This study was supported in part by a grant from the United States Public Health Service (AM 22083). We thank Dr Stan Epperson (Mesker Park Zoo, Evansville, IL) and Dr Stan Jensen (Topeka Zoological Park, Topeka, KA) for providing access to the animals in their care.

REFERENCES Clegg J. B. (1974) Horse hemoglobin polymorphism. Ann. N.Y. Acad. Sci. 241, 61-69.

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Downing D. T. (1968) Photodensitometry in the thin layer chromatographic analysis of neutral lipids. J. Chromat. 38, 91-99. Downing D. T. and Colton S. W., VI (1980) Skin surface lipids of the horse. Lipids 15, 323-327. Downing D. T. and Stranieri A. M. (1980) Correction for deviation from the Lambert-Beer Law in the quantitation of thin layer chromatograms by photodensitometry. J. Chromat. 192, 208-211. Downing D. T. and Strauss J. S. (1974) Synthesis and composition of surface lipids of human skin. J. invest. Derm. 62, 228-244. Downing D. T. and Strauss J. S. (1982) On the mechanism of sebaceous secretion. Archs Derm. Res. 272, 343-349. Kaminski M. (1970) Common and species-specific serum esterases of Equidae--I. horse, donkey, zebra and their hybrids. Comp. Biochem. Physiol. 35, 631-638. Kaminski M. (1979) Biochemical evolution of the horse. Comp. Biochem. Physiol. 63B, 175-178. Kaminski M., Metenier L., Sykiotis M., Ryder O. A. and Dementoy M.-C. (1978) Common and species-specific esterases of Equidae--IV. Horse of Przewalski, Onager and Zebra hartmannae. Comp. Bioehem. Physiol. 61, 357-364. Labows J. N., Preti G., Hoelzle E., Leyden J. and Kligrnan A. (1979) Steroid analysis of human apocrine secretion. Steroids 34, 249-258. Lindholm J. S., McCormick J. M., Colton S. W., VI and Downing D. T. (1981) Variation of skin surface lipid composition among mammals. Comp. Biochem. Physiol. 69B, 75-78. Miiller-Schwartze D. and Mozell M. M. (1977) Chemical Signals in Vertebrates. Plenum Press, New York. Mykytowycz R. and Goodrich B. S. (1974) Skin glands as organs of communication in mammals. J. invest. Derm. 62, 124-131. Nicolaides N., Fu H. C. and Rice G. R. (1968) The skin surface lipids of man compared with those of eighteen species of animals. J. invest. Derm. 51, 83-89. Patterson R. L. S. (1968a) Identification of 3ct-hydroxy-5ctandrost-16-ene as the musk odour component of boar submaxillary salivary gland and its relationship to the sex odour taint in pork meat. J. Sci. Fd Agric. 19, 434-438. Patterson R. L. S. (1968b) 5ct-androst-16-ene-3-one: compound responsible for taint in boar fat. J. Sci. Fd Agric. 19, 31-37. Ryder O. A., Epel N. C. and Benirschke K. (1978) Chromosome banding studies of the Equidae. Cytogenet. Cell Genet. 20, 323-350. Sebedio J.-L. and Ackman R. G. (1981) Application of methoxy-bromomercuri adduct fractionation to the analysis of fatty acids of partially hydrogenated marine oils. Lipids 16, 461467.