Skin surface lipids of the mole Scalopus aquaticus

Comp. Biochem. PhysioL Vol. 86B, No. 4, pp. 667-670, 1987

0305-0491/87 $3.00 + 0.00 © 1987 Pergamon Journals Ltd

Printed in Great Britain

SKIN SURFACE LIPIDS OF THE MOLE

SCALOPUS AQUATICUS DONALD T. DOWNING and MARY ELLEN STEWART Marshall Research Laboratories, Department of Dermatology, University of Iowa College of Medicine, Iowa City, IA 52242, USA (Tel: 319-335-8080)

(Received 20 May 1986) Abstract--l. Skin surface lipids of the mole Scalopus aquaticus were found to consist principally of squalene (70%), wax esters (15%), and sterol esters (5%), together with small amounts of triglycerides, free fatty acids, free fatty alcohols, and free sterols. 2. Analysis of the fatty acids occurring free and as wax esters and sterol esters showed these to consist of approximately equal amounts of saturated and monounsaturated compounds. 3. The saturated fatty acids consisted predominantly of odd-carbon anteiso and even-carbon straightchain compounds, with minor amounts of even-carbon iso-branched chains. 4. The unsaturated fatty acids had double bond positions that would have been produced by A9-desaturation of Cl4, Ci6 and Cl8 straight chain saturated precursors. 5. Both the free and the esterified fatty alcohols had chain structures corresponding with those of the fatty acids but of somewhat greater average chain length. 6. Discovery of a major proportion of squalene in the sebum of this animal extends the number of non-human species that have this characteristic to four, all of which inhabit a damp environment, suggesting that squalene conveys some biological advantage under these conditions.

INTRODUCTION

Lipid analysis

N o n p o l a r lipids on the skin and fur of mammals are predominantly products of the sebaceous glands and are species-specific in composition (Stewart, 1986). F o r example, triglycerides are the major component of human sebum, but have not been found in the sebum of any other species. Also, human sebum contains 12% squalene, and this lipid has been found in sebum from only 4 of the 60 or more species that have been examined for this (Nicolaides et al., 1968; Lindholm et aL, 1981). It has intrigued us that the three-non-human species that produce squalene in their sebum are either aquatic (otter and beaver), or are indigenous to a d a m p (tropical rain forest) enviroument (kinkajou) (Lindholm and Downing, 1980). It seems, therefore, that squalene may be advantageous to mammals that frequent such habitats, though not obligatory, since it is absent from the sebum of the muskrat (Lindholm et al., 1981). In the present study, we examined the sebum of a mole (Scaiopus aquaticus), another m a m m a l that has a d a m p environment, and have found that it also contains a high proportion of squalene. MATERIALS AND METHODS

Collection of skin lipids The mole (95g) was trapped in Iowa City in April 1986. The intact dead animal was held by the forefeet and dipped in acetone up to the neck. The acetone was then evaporated, leaving 55 mg of crude extract. Correspondence to: Donald T. Downing, PhD., 270 Medical Laboratories, University of Iowa College of Medicine, Iowa City, IA 52242, USA. 667

Analytical thin-layer chromatography (TLC) (Fig. 1) indicated that the skin surface lipids of the mole had several of the same constituents as human sebum, probably including squalene, cholesteryl esters, and wax esters, together with small amounts of triglycerides, free fatty acids, free alcohols, and free cholesterol. The amounts of these constituents were determined by photodensitometry of analytical thin-layer chromatograms (Downing, 1968). The individual constituents were then isolated by preparative TLC and identified by chemical and physical methods, as follows. Squalene. The crude lipid extract was applied to a 20 × 20 cm TLC plate coated with a I mm thick layer of silica gel G and the chromatogram was developed with hexane. The chromatogram was then sprayed with 2',7'-dichlorofluorescein and viewed under u.v. light. The bands corresponding to squalene and all of the material remaining at the origin were scraped from the plate and extracted with ether to recover the lipids. The material corresponding to squalene gave a single peak on gas chromatography, having a retention time identical to authentic squalene when chromatographed on an OVI01 column. Also, the material had an infra-red spectrum identical with that of squalene, so that identification was considered conclusive. Wax esters. The whole of the material recovered from the origin of the first preparative TLC was applied to a second chromatogram, which was developed with hexane:ether:acetic acid (70:30:1, v/v). Visualization with the fluorescent indicator and u.v. light allowed the bands corresponding to monoesters (wax esters plus sterol esters), triglycerides, free fatty acids, free alcohols and free sterols to be marked and scraped from the plate. The lipids were recovered by elution with ether, the solvent was evaporated and the lipid residues weighed. The fraction containing the monoesters was then refractionated on a column of Mg(OH)z, using HPLC equipment, and the separate fractions corresponding to wax esters and sterol esters (Stewart and Downing, 1981) were recovered and evaporated.

668

DONALD T. DOWNING and MARY ELLEN STEWART

0 . 0

0 ( ~

~: HUMAN "

.

I

e

e

®

=

fl

e.

O.OO

.

.

.

. . . . . .

-o. zoo.~ 19. O 2.

5 0--~-.0

"-

lt',O. O

~

~

r

150 ;. O

:

i

iL

OOO

i

m o , . 6oo_~

MOLE

O m I . 2 oo...~

0 o Ooo....~

1 2 4 5 6

25. ? 41.0 51,3 ~31.7 74. 1 02.8

4248.76 7714.1;3 3557.465 ~5S5. 539 2074.699 1025.492 7 124,7 42420.98 8 1,56.1 1936~9.0 ............................................

TOTnL

O. 4

0

25024~,

t U U t t.I

U II I.i

I

|. 6 ?-.9 1.3 1.3 0.0 0.3 16.4 74.9

L-..

]

~

!

......... i ! :

o.oe

19. 0

i

!50. O

ilOO. O

150. O

*

i

DISTANCE (ram) Fig. 1. Photodensitometer records of thin layer chromatograms of human and mole surface lipids, developed in adjacent lanes of a plate with hexane (to 20 cm), then toluene (to 20 cm), then hexane:ether:acetic acid (70:30:1, twice to 8 cm), then charred by spraying with 5 0 0 H2SO4 and heating to 220°C. The wax esters were then subjected to saponification with 1 M KOH in 9 5 0 methanol at 70°C for 1 hr, followed by addition of BCl3/methanol. Water was added and the products were recovered in chloroform. Analytical TLC of the products showed only two constituents, corresponding in chromatographic mobility to methyl esters and fatty alcohols. These were separated by preparative T L C with hexane:ether:acetic acid (70:30:1, v/v). The recovered alcohols were then converted into fatty acids by oxidation with chromic acid in acetone and the methyl esters of the resulting acids were prepared by treatment with BCl3/methanol. These methyl esters and those of the original wax ester fatty acids were then analyzed by gas chro-

matography on a 50m x 0.22 mm quartz column wall coated with OV101 silicone, at 160°C and at 220°C, The two mixtures of methyl esters derived from the wax esters were then subjected to preparative TLC on silica gel H containing 10% AgNO 3. Development with toluene produced bands corresponding to saturated and monounsaturated fatty acid methyl esters. These were recovered and analyzed by gas chromatography as before. Individual fatty acid methyl esters were identified by use of standard mixtures as well as by regression analysis of their equivalent chain lengths (Green et al., 1984). Sterol esters. The sterol ester fraction separated from the wax esters by Mg(OH)2 chromatography was saponified

Skin surface lipids o f mole with 1 M K O H and the liberated fatty acids were converted to methyl esters. The resulting mixture of methyl esters was then separated f r o m the sterols by T L C and analyzed by G L C before and after isolation of the saturated and m o n o unsaturated esters. Free fatty acids. These were converted to methyl esters with BCl3/methanol and analyzed by G L C before and after resolution of the saturated and m o n o u n s a t u r a t e d esters. Free fatty alcohols. These were converted directly to fatty acids by CrO3 in acetone and the acids methylated with BC13/methanol. The resulting methyl esters were analyzed by G L C before and after AgNO3 separation o f the saturated and unsaturated esters. Free sterols. Thin-layer c h r o m a t o g r a p h y indicated that the sterols consisted predominantly of cholesterol.

RESULTS T h e series o f c h e m i c a l a n d p h y s i c a l p r o c e d u r e s c o n f i r m e d t h e i d e n t i t y o f t h e m o l e s k i n s u r f a c e lipids as s q u a l e n e , w a x m o n o e s t e r s , s t e r o l e s t e r s , free f a t t y acids, free f a t t y a l c o h o l s , a n d free sterols. T h e relative proportions of these constituents, obtained by quant i t a t i v e T L C (Fig. 1), a r e s h o w n in T a b l e 1. T h e c o m p o s i t i o n o f t h e free f a t t y a c i d s a n d free f a t t y a l c o h o l s a r e s h o w n in T a b l e 2. T h e c o m p o s i t i o n s o f the fatty acids and fatty alcohols of the wax esters, and the fatty acids from the sterol esters, are shown in T a b l e 3.

669 Table 1. Skin surface lipid composition of the mole Sealopus aquaticus (wt%) Squalene Wax esters Sterol esters Triglycerides Free fatty alcohols Free fatty acids Free sterols Polar lipids Total

70 15 5 1 1 1 3 2 98

DISCUSSION T h e p r e s e n t s t u d y r e v e a l s a n o t h e r m a m m a l in w h i c h t h e s k i n s u r f a c e lipids c o n t a i n a m a j o r p r o p o r tion of squalene. This provides another example of association of this sebum constituent with a damp h a b i t a t , c u r i o u s l y e m p h a s i z e d in t h i s i n s t a n c e b y t h e species name. However, the nature of any biological advantage that squalene might convey remains obscure. T h e o c c u r r e n c e o f w a x m o n o e s t e r s in t h e m o l e s u r f a c e lipids p r o v i d e s a s e c o n d s i m i l a r i t y w i t h h u man sebum, although the composition of the wax ester fatty acids and fatty alcohols maintains the now expected diversity of sebum composition among m a m m a l s . A n y p o s s i b l e f u n c t i o n o f w a x e s t e r s in

Table 2. Composition of the fatty acids and fatty alcohols of mole skin surface lipids* Free fatty acids Saturated Monounsaturated (40%) (60%) No. of carbons

i

14 15 16 17 18 19 20 21 22 23 24 25

a

1

2

44

1

--

8 --

n

n5

10

6

1 20 1

-16

16

5

n7

n9

Free fatty alcohols Saturated Monounsaturated (60%) (40%) i 1

8

14

a 6

3

--

--

22

24

n

n5

4

1

2

--

n7

n9

25

5

1

3

8

16

1

2 --

6

9

10

1

--

2

22

15

3

1

1

11 --

1

4

2

3 2

1

2

2

2 5

2

I

9

*wt%; 1 = iso-branched; a = anteiso-branched; n = normal; n5, n7, n9 = position of double bonds from the methyl terminus of the fatty chains. Table 3. Composition of the fatty acids and fatty alcohols from the esters of mole skin surface lipids* Wax ester fatty acids Saturated Monounsaturated (40%) (60%) No. of Carbons 12 13 14 15 16 17 18 19 20 21 22 23 24

i

2 2 1

a 1 -63 -8

n

n5

n7

14 1 3

21 -25

16

1

4

9

6

10

n9

Wax ester fatty alcohols Saturated Monounsaturated (50%) (50%) i

2 3 2 1 -1

a 1 -11 -21 -1 -7 -1

n

n5

1 -12 2 28

7

2 2

8 8 -17 -4

n7

2 -18 -14 -3

n9

1 -3 -2

Cholesteryl esters Saturated Monounsaturated (60%) (40%) i

a

1 2 1

9 -4

1

I1 --7

n

4 1 17 2 17 3 3 3

n5

n7

4

3

2

l0

4

7

1 I

I

n9

36 --8 4 2 2 8

* = wt%; i = iso-branched; a = anteiso-branched; n = normal; n5, n7, n9 = position of double bonds from the methyl terminus of the fatty chains.

670

DONALD T. DOWNINGand MARY ELLEN STEWART

sebum is also unknown, although it is perhaps noteworthy that such compounds are resistant to bacterial degradation on the skin surface. The wax esters in human sebum, for instance, seem to be completely unaffected by the bacterial hydrolysis that can result in almost total hydrolysis of triglycerides in the pilosebaceous follicle (Nicolaides et al., 1970). It may be that both the diversity and the species specificity of sebum composition have the effect of maintaining resistance to development of bacteria capable of utilizing these materials as nutrients. Minimization of bacterial colonization would not only be an advantage in itself, but would allow the preservation of the sebum for whatever biological function it might have. One possible function that seems not to have been proposed is the action of sebum as a seal around the hair shaft, allowing continurus outgrowth of the hair while at the same time preventing both the ingress of bacteria and the egress of water.

Acknowledgement--This study was supported in part by a grant from the United States Public Health Service (AM22083).

REFERENCES

Downing D. T. (1968) Photodensitometry in the thin layer chromatographic analysis of neutral lipids. J. Chromat. 38, 91-99. Green S. G., Stewart M. E. and Downing D. T. (1984) Variation in sebum fatty acid composition among humans. J. invest. Derm. 83, 114-117. Lindholm J. S. and Downing D. T. (1980) Occurrence of squalene in surface lipids of the otter, the beaver and the kinkajou. Lipids 15, 1062-1063. 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. Nicolaides N., Fu H. C. and Rice G. R. (1968) The skin surface lipids of man compared with those of eighteen species of mammals. J. invest. Derm. 51, 83-89. Nicolaides N., Ansari M. N. A., Fu H. C. and Lindsay D. G. (1970) Lipid composition of comedones compared with that of human skin surface in acne patients. J. invest Derm. 54, 487-495. Stewart M. E. (1986) Sebaceous gland lipids. In Biology of the Integument 2, Vertebrates (Edited by Bereiter-Hahn J., Matoltsy A. G. and Richards K. S.), pp. 824-832. Springer, Berlin. Stewart M. E. and Downing D. T. (1981) Separation of wax esters from steryl esters by chromatography on magnesium hydroxide. Lipids 16, 355 359.