Comp. Biochem. Physiol. Vol. 86B, No. 4, pp. 671-673, 1987 0305-0491/87 $3.00 +0.00 Printed in Great Britain © 1987 Pergamon Journals Ltd

POLAR LIPID COMPOSITION OF MAMMALIAN HAIR

M~auc A. Wtx, PHILIP W. WERTZ and DONALD T. DOWNING Marshall Dermatology Research Laboratory, Department of Dermatology, University of Iowa College

of Medicine, Iowa City, IA 52242, USA (Tel: 319-335-8080)

(Received 20 May 1986)

Abstract--1. The types and amounts of polar lipids from the hair of monkey (Macaccafascicularis), dog (Canis familiaris), pig (Sus scrofa) and porcupine (Erethizon dorsatum) have been determined by quantitative thin-layer chromatography.

2. The polar lipid content of the hair samples ranged from 0.6 to 1.6 wt%. 3. Lipid compositions included ceramides (57-63% of the polar lipid by weight), glycosphingolipids

(7-9%) and cholesteryl sulfate (22-29%). Several minor components (4-7%) remain unidentified. 4. The results suggest that cholesteryl sulfate may be an important determinant of the cohesiveness of

hair.

INTRODUCTION

Although much progress has been made regarding the chemistry, biochemistry and physiology of the nonpolar (principally sebaceous) lipids associated with hair (Nicolaides et al., 1968; Lindholm et al., 1981; Stewart, 1986), the polar lipids have been largely ignored. A preliminary survey of the polar lipids of various keratinized tissues (Birkby et al., 1982) indicated that hair contained ceramides and glycosylceramides; however, these components were not quantitatively measured and one other major highly polar component remained unidentified.

The principal goals of the present investigation were to complete identification of the polar hair lipids and to quantitate the lipids from several mammalian species.

MATERIALS AND METHODS

Isolation of polar lipids from hair Hair was collected from a monkey, dog and pig by means

of electric clippers. Porcupine (Erithizon dorsatum) hair and quills were clipped from a freshly killed animal with scissors. Each hair sample was weighed and nonpolar lipids were then extracted into hexane. The remaining hair lipids were then collected by successive extractions with chloro- form:methanol (2:1, l : l and finally 1:2). Each solvent mixture was in contact with the hair for 2 hr at room temperature. The chloroform:methanol extracts were then combined and taken to dryness. Examination by TLC indicated that the hexane extract contained only nonpolar lipids, but a small amount of nonpolar lipid, principally cholesterol, was also present in the chloroform: methanol extracts. This residual nonpolar lipid was removed by preparative TLC on 0.5 mm thick silica gel H (E.M. Re- agents, Darmstadt, FRG.) with hexane:ether:acetic acid (70:30:1) as the mobile phase. In this solvent system, the polar lipids remained on the origin. This region of silica gel was scraped from the plate and the polar lipids were eluted from the silicic acid with chloroform:methanol:water

Send reprint requests to: Philip W. Wertz, Ph.D., Marshall Dermatology Research Laboratory Department of Dermatology University of Iowa College of Medicine Iowa City, IA 52242, USA.

(50: 50:1). The polar lipid fraction was dried, weighed and redissolved at a concentration of 25mg/ml in chloro- form: methanol (2:1).

Thin-layer chromatography All TLC employed 20 x 20 cm glass plates coated with

silica gel. For analytical TLC, a 0.25 mm thick layer of silica gel G (E.M. Reagents) was scored into 6 mm wide lanes. Reference materials included synthetic cholesteryl sulfate and a range of ceramides (Wertz and Downing, 1983a), glucosylceramides (Wertz and Downing, 1983b) and acyl- glucosylceramides (Wertz and Downing, 1983c) previously prepared from pig epidermis. One sample or standard was applied per lane, and the chromatograms were then developed with an appropriate solvent. Chloro- form: methanol: water (40:10:1) resolved the ceramides as a group from the glycosylceramides and cholesteryl sulfate, while chloroform: methanol: acetic acid (190: 9:1) resolved the individual ceramides. After development, the plate was air-dried prior to spraying with 50% sulfuric acid. The lipids were then charred by slowly heating on a hot plate to 220°C. After completion of charring, the chromatograms were quantitated by photodensitometry (Downing, 1968).

For preparative TLC, a 0.5 ram thick layer of unscored silica gel H was employed. Samples were applied in a thin line 2 cm from the bottom of the plate. After development, the lipid bands were located by spraying the plate with 2',7"-dichlorofluorescein (100 mg/l of ethanol) and visual- izing under u.v. light.

Chemical method~ To saponify a sample, the lipid was dissolved in l M

KOH in 90% methanol and heated at 65°C for l hr. The sample was then neutralized with 2 M HCI, and the lipid products were extracted into chloroform. Acid catalyzed methanolysis was achieved by heating the sample at 65°C with I M HCI in 90% methanol overnight.

RESULTS AND DISCUSSION

Table 1 summarizes the polar lipid content of hair from the four species under study. This polar lipid content (0.6-1.6%) was considerably smaller than the 8-10% polar lipid reported for stratum corneum (Gray and Yardley, 1975; Yardley and Summerly, 1981) but was comparable to the lipid content of hoof (Ueta et aL, 1971; Wertz and Downing, 1984). This

671

672 MARK A. Wlx et al.

Table 1. Polar lipid content of hair (wt %) Polar lipid content of hair (wt%)

Species Common name wt% lipid Macaca fascieularis Monkey 0.7 Canis familiaris Dog 1.3 Sus scrofa Pig 0.6 Erithizon dorsatum Porcupine 1.6

Table 2. Polar lipid composition of hair. Results (presented as wts% of total polar lipids) were obtained by quantitative TLC (Downing,

1968) Polar lipid composition of hair

Monkey Dog Pig Porcupine Ceramides 56.5 63.1 56.8 60.7 Cholesteryl sulfate 28.9 22.2 28.6 24.1 Glycosylceramides 8.3 7.0 9.2 7.9 Others 6.3 7.7 5.4 7.3

lower lipid content suggests that there is probably insufficient lipid to form an effective permeability barrier as is found in stratum corneum. Nevertheless, the lipid present in these hard keratinized tissues may be functionally significant in other ways.

Figure 1 is a densitometer record of a chro- matogram of the polar lipids from monkey hair, showing the ceramides, glycosylceramides (GSL) and the major highly polar component. Several minor unidentified components were also present. The ma- jor component at Rf 0.18 was isolated by preparative TLC and shown to be cholesteryl sulfate. This assign- ment was based on the following observations: 1. Its TLC mobility was identical to that of authentic cholesteryl sulfate. 2. Like authentic cholesteryl sul- fate, it produced a brilliant magenta color on brief heating with sulfuric acid. 3. It was nonsaponifiable. 4. After acid hydrolysis, the only lipid product ob- tained was cholesterol as judged by TLC.

With this major component identified, the class composition of the polar hair lipids from the different mammals can be summarized as shown in Table 2. There are several points that emerge from these results. First, the similarity of the lipid composition among these different species is striking. This great similarity suggests there may be a functionally significant role for the polar lipids in hair. Such fundamental significance may have resulted in the conservation of lipid composition in spite of other evolutionary divergence influencing hair density (dog vs pig) or gross structure (monkey vs porcupine). Second, the high proport ion of cholesteryl sulfate is noteworthy. In stratum corneum, where cholesteryl sulfate accounts for 5-12% of the polar lipid (Long et al., 1985; Ranasinghe et al., 1985), it has been

0 .2 0 . 4 0 . 6 0 . 8 Fig. 1. TLC record of polar lipids from monkey hair. The chromatogram was developed with chloroform:meth- anol:water (40:10:1). Numbers indicate Rf values.

GSL = glycosphingolipids.

Table 3. Detailed composition (wt %) of the polar lipids from mon- key hair determined by densi-

tometry Polar lipids of monkey hair

Ceramide 1 0.3 Ceramide 2 19.3 Ceramide 3 4.3 Ceramide 4 8.4 Ceramide 5 6.4 Ceramide 6 14.2 Cholesteryl sulfate 28.9 Glucosylceramides 8.3 Others 9.9

implicated in the cohesiveness of the horny cells. Cholesteryl sulfate hydrolysis has been shown to accompany normal desquamation (Long et al., 1985; Ranasinghe et al., 1985), and failure to hydrolyze it in recessive X-linked ichthyosis leads to the abnormal retention of horny cells on the skin surface (Williams and Elias, 1981). Recently, horse hoof has also been shown to contain a high proport ion of cholesteryl sulfate (Wertz and Downing, 1984). Perhaps, choles- teryl sulfate serves as an intercellular cement in these keratinized tissues.

The chromatographic record shown in Fig. 1 indi- cated that the ceramides are structurally heteroge- nous, although this chromatographic system did not resolve the individual structural types. Use of a less polar mobile phase (chloroform-methanol-acetic acid, 190:9:1) revealed six chromatographically dis- tinct fractions which were essentially identical to the ceramides found in epidermis as judged by com- parison on TLC. When these different ceramide fractions were taken into account, a complete polar lipid composition was determined for monkey hair, and this is shown in Table 3. These results represent the first quantitative analysis of the polar lipids from hair. It is clear from these data that cholesteryl sulfate is actually the single most abundant polar lipid in hair. By analogy with the role of this highly polar lipid in stratum corneum, it can be postulated that cholesteryl sulfate is an important determinant of the cohesiveness of hair.

Acknowledgements--This work was supported in part by grants from the United States Public Health Service (AM22083 and AM32374) and by a grant from Richardson Vicks, Inc., Wilton, CT, U.S.A.

REFERENCES

Birkby C. S., Wertz P. W. and Downing D. T. (1982) The polar lipids from keratinized tissues of some vertebrates. Comp. Biochem. Physiol. 73B, 239-242.

Downing D. T. (1968) Photodensitometry in the thin-layer chromatographic analysis of neutral lipids. J. Chromat. 38, 91-99.

Gray G. M. and Yardley H. J. (1975) Different populations of pig epidermal cells: isolation and lipid composition. J. Lipid Res. 16, 441-447.

Lindholm J. S., McCormick J. M., Colton S. W., 6th and Downing D. T. (1981) Variation of skin surface lipid composition among mammals. Comp. Biochem. Physiol. 69B, 75-78.

Long S. A., Wertz P. W., Strauss J. S. and Downing D. T. (1985) Human stratum corneum polar lipids and de- squamation. Archs Derm. Res. 277, 284-287.

Polar lipids of hair 673

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.

Ranasinghe A. W., Wertz P. W., Downing D. T. and Mackenzie I. C. (1985) Lipid composition of cohesive and desquamated corneocytes from mouse ear skin. J. invest. Derm. 86, 187-190.

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. 823-832. Springer, Berlin.

Ueta N., Kawamura S., Kanagawa I. and Yamakawa T. (I 971) On the nature of so-called ungulic acid. J. Biochem. 70, 881-883.

Wertz P. W. and Downing D. T. (1983a) Ceramides of pig epidermis: structure determination. J. Lipid Res. 24, 759-765.

Wertz P. W. and Downing D. T. (1983b) Glucosylceramides of pig epidermis: structure determination. J. Lipid Res. 24, 1135-1139.

Wertz P. W. and Downing D. T. (1983c) Acylglucosyl- ceramides of pig epidermis: structure determination. J. Lipid Res. 24, 753-758.

Wertz P. W. and Downing D. T. (1984) Cholesteryl sulfate: the major polar lipid of horse hoof. J. Lipid Res. 25, 1320-1323.

Williams M. L. and Elias P. M. (1981) Stratum corneum lipids in disorders of cornification. Increased cholesterol sulfate content of stratum corneum in recessive X-linked ichthyosis. J. clin. Res. 68, 1404-1410.

Yardley H. J. and Summerly R. (1981) Lipid composition and metabolism in normal and diseased epidermis. Pharmac. Ther. 13, 357-383.