Integral lipids of human hair

878

Integral Lipids of Human Hair Philip W. Wedz* and Donald T. Downing The Marshall Dermatology Research Laboratories, Department of Dermatology, University of Iowa College of Medicine, Iowa City, Iowa 52242

It has long been recognized that hair is coated with nonpolar lipids originating in the sebaceous glands, and recently it has been shown that hair also contains cholesterol sulfate and small amounts of ceramldes, similar to those found in the keratinized portion of the epidermis. In the present study, it is demonstrated that significant amounts of several additional lipids are tightly associated with hair in such a way as to be highly resistant to solvent extraction.

These integral hair iipids included cholesterol sulfate (3.3 mg/g of extracted hair), cholesterol {0.6 mg/g), fatty alcohols {0.2 mg/g) and free fatty acids (4.3 mg/g). The principal fatty acid, comprising 40% of the total fatty acids, was identified as 18-methyl-eieosanoic acid by cochromatography with authentic standard on gas- liquid chromatography (GLC) and by mass spectrometry (MS). Lipids 23. 878-881 I1988).

cross-linked protein envelope to form what appears like a plasma membrane (8,9). It is actually an integral part of the cell envelope and can be removed from the cell surface only after saponification (8,9).

The purpose of the present investigation was to determine whether human hair, a keratinized epidermal appendage, contains covalently bound lipids as found in the stratum corneum. To answer this question, one pooled sample of hair clippings from several people and samples from three individuals were obtained. After extensive extraction with chloroform]methanol mixtures to remove all of the extractable lipids, the hair samples were saponified and reextracted. Additional lipids recovered in this way included major amounts of cholesterol sulfate (3.3 mg/g extracted hair) and free fat ty acids (4.3 mg/g) and small amounts of cholesterol, fatty alcohol and several unidentified components. The major fat ty acid, comprising 40% of the total fat ty acids, proved to be 18-methyl-eicosanoic acid.

Human scalp hairs consist of a central medulla of loosely connected keratinized cells surrounded by more-tightly- packed, 80-100 w-long cortical cells with 6 to 10 outer layers of flattened, rectangular cuticle cells (1). Late in the sequence of differentiative events leading to formation of the mature cuticle and cortex cells, a multi- layered material is deposited between the bounding membranes of the individual cells to form membrane complexes (2-4). Although it is chemically undefined, the intercellular material (3,4) has been referred to as a cement substance (3). The chemical nature of the cell periphery in hair, like that of the cornified epidermal cell, also has been somewhat of a puzzle because hair does not contain the usual membrane-forming phospho- lipids (5).

Associated with the hair follicles are sebaceous glands that provide abundant nonpolar lipids to the skin surface and the hair (6,7). While these sebaceous lipids may serve to lubricate and waterproof, they are not membrane-forming lipids. Recently, it has been demonstrated that hair from several species of mammals contains cholesterol sulfate and several series of ceramides similar to those found in the keratinized portion of the epidermis, or stratum corneum, and it has been postulated that these lipids are of structural significance in hair (5). However, the amounts of these polar lipids were low, and it was uncertain as to whether they provided enough material to account for the observed membranes.

More recently, the occurrence of significant amounts of covalently bound lipid in the stratum corneum has been demonstrated (8). This material consists mainly of an unusual r ester-linked to a

*To whom correspondence should be addressed at 270 Medical Laboratories, Dept. of Dermatology, University of Iowa College of Medicine, Iowa City, IA 52242 USA Abbreviations: ECL, equivalent chain lengths; GLC, ga~liquid chromatography; MS, mass spectrometry; TLC, thin-layer chromatography.

MATERIALS AND METHODS

Preparation of delipidized hair. Samples of human hair were repeatedly rinsed with distilled water. They were then extracted, first with methanol and then with chloroform]methanol 2"1, 1:1 and 1:2, for 2 hr each, at room temperature. Each of the chloroform/methanol extractions was then repeated for 24 hr. Finally, the hair was extracted with chloroform in a Soxhlet extractor, con- tinuously for 24 hr. The extracted hair was thoroughly dried en vacuo and weighed. One large sample (100 g) of pooled hair from several individuals and smaller (3.5-8.9 g) hair samples from three individuals were processed by this method.

Saponification and recovery of constitutive lipids. Extracted hair samples (3-5 g after extraction) were saponified by heating for 2 hr at 60 C in 200 ml 1 M NaOH in 90% methanol. The reaction was cooled to room temperature, and 88 ml of water and 360 ml of chloroform were added. The mixture was shaken and the phases were separated in a separatory funnel. The lower chloroform layer was transferred to a flask, and the upper phase and hair residue were acidified by addition of 50 ml of 6 N HC1. The acidified upper phase was extracted with an additional 360 ml of chloroform, and the combined chloroform extracts were taken to dryness with a rotary evaporator. The dried residue was dissolved in 5 ml chloroform:methanol, 2:1, and washed with 1 ml of 2 M KC1 containing 0.1 M HC1. The lower chloroform phase then was filtered through a solvent-resistant, 0.5-micron millipore filter, and the filtrate was collected and taken to dryness in a tared glass tube. The wt of the dried lipid was determined, and chloroform:methanol was added to produce a solu- tion containing 20 mg lipid per ml.

Thin-layer chromatography (TLC}. Analytical TLC was done on 0.25-mm layers of Silica Gel G (E.M. Rea- gents, Darmstadt, West Germany} on 20 • 20 cm glass plates. Samples were applied 2 cm from the bottom

LIPIDS, Vol. 23, No. 9 ('1988)

879

INTEGRAL LIPIDS OF HAIR

edge of the plate, which was developed first to 9 cm with chloroform/methanol/acetic acid/water (40:10:1:1), and then to 20 cm with hexane/ether/acetic acid (70:30:1). After drying, the plate was sprayed with 50% sulfuric acid and charred by slowly heating to 220 C. The charred chromatograms were quantitated by photodensitometry (10) with a Shimadzu model CS-930 photodensitometer. Known amounts of stearic acid, stearyl alcohol, ch~ lesterol and cholesterol sulfate were used as standards.

Fat ty acids, fat ty alcohols and the sterol fraction were isolated by preparative TLC on 0.5-mm Silica Gel H (E.M. Reagents) using a mobile phase of hexane/ether/acetic acid {70:30:1). Cholesterol sulfate was isolated using chloroform/methanol/acetic acid/water (40:10:1:1) as the mobile phase.

The isolated fractions, as well as the solvolysis product derived from cholesterol sulfate, were checked for purity by TLC with solvent systems of hexane/ether/acetic acid (70:30:1), chloroform]methanol/acetic acid (190:9:1), chloroform]methanol]water (40:10:1) and chloroform/methanol]acetic acid/water (40:10:1:1). Each fraction was judged to be chromatographically pure.

Acetylated sterols, acetylated fatty alcohols and fatty acid methyl esters were purified by preparative TLC using a mobile phase of toluene.

Chemical procedures. The isolated fat ty acid fraction was converted to methyl esters by treatment with 10% BC13 in methanol at 50 C for 1 hr. The reaction mixture was dried under nitrogen.

Cholesterol sulfate was solvolyzed overnight by treatment with 10% BCI 3 in methanol at 55 C. The sample was dried under nitrogen.

The free sterol, fat ty alcohol and the sterol obtained after solvolysis of cholesterol sulfate were acetylated by treatment with acetic anhydride in pyridine at room temperature for 2 hr. Excess reagents were removed by evaporation under a gentle s t ream of nitrogen.

Gas-liquid chromatography (GLC). The fat ty acid methyl esters were examined by GLC on a 50 m CP- SIL 88 quartz capillary column (Chrompack, Inc., Bridge- water, NJ). An initial temperature of 160 C was maintained for 5 min, after which the temperature was in- creased linearly at a rate of 5 C/min, until 220 C was reached. The final temperature was maintained until no further peaks eluted. The fat ty acid methyl esters also were examined isothermally at 160 C and 175 C on the CP-SIL 88 column, as well as at 200 C on a nonpolar 25-m BP-1 quartz capillary column (Scientific Glass Engineering, Inc., Austin, TX).

Standard fat ty acid methyl ester mixtures included 16:0, 18:0, 18:1, 18:2, 18:3, 20:0 (15A, NuChek Prep, Elysian, MN); 14:0, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3 (CE1-62, NuChek Prep); 20:0, 20:1, 20:4, 22:1, 22:6 (I.r209, Applied Science, State College, PA); and fat ty acids isolated from wool wax (ll).

GLC of fractions isolated by argentation-TLC was used to confirm the identification of saturated and monoenoic fat ty acid methyl esters.

The acetylated sterols derived from both the free sterol fraction and the sterol sulfate fraction were examined by GLC with the CP-SIL 88 column at 220 C and on BP-1 at 290 C. Authentic cholesterol acetate and cholestanol acetate were used as standards.

The fat ty alcohol acetates also were examined by

GLC with the CP-SIL 88 column, both isothermally at 160 C and with the temperature program described above. Standard fat ty alcohol acetates included C14:0, C16:0, C18:0 and C20:0.

Mass spectrometry. Fat ty acid methyl esters were separated on a 25-m CP-SIL 5 quartz capillary column operated with a temperature program, starting at 150 C and increasing 5 C/min, until a final temperature of 250 C was attained. Mass spectra were recorded with a Nermag R 10-10 C mass spectrometer with an ionizing voltage of 70 eV. A range of mass/charge from 40 through 600 was examined.

RESULTS

Composition of constitutive hair lipids. A typical densit~ meter tracing of a thin-layer chromatogram of the lipids released by saponification of previously extracted hair is presented as Figure 1. As described below, the most prominent components were identified as free fat ty acids and cholesterol sulfate, and minor components included cholesterol and fatty alcohols. Some lipid material appearing between Rf 0.2-0.4 was not identified and variable amounts of origin material not thought to be lipid were sometimes present. The quantitative results of such TLC analyses are summarized in Table 1.

E

A

B C

' 0 : 2 0'.4 Rf 0 : 6 0 : 8 '

FIG. 1. Densitometer tracing of a thin-layer chromatogram of the constitutive iipids of hair. The chromatogram was developed to Rf 0.30 with chloroform/methanol/water/acetic acid {40:10:1:1) and then to the top with hexane/ether/acetie acid {70:30:1). The charred chromatogram was scanned with a Shimedzu model CS-9~0 photo. denm'tometer. Peak identification: A, cholesterol sulfate; B, unidentified; C, cholesterol; D, fatty alcohols; E, fatty adds.

TABLE 1

Constitutive Lipids of Human Hair Mg lipid per g extracted hair

Fatty Cholesterol Fatty Sample acid sulfate Cholesterol alcohol

A 4.7 3.8 0.7 0.1 B 4.2 3.2 0.4 0.2 C 3.2 1.8 0.5 0.1

Pool 5.2 4.2 0.6 0.3 Mean 4.3 3.3 0.6 0.2 SD 0.7 0.9 0.1 0.1

Weights of lipid were estimated by quantitative TLC.

LIPIDS, Vol. 28, No. 9 (1988)

880

P.W. WERTZ AND

Identification of cholesterol sulfate. The identifica- t ion of cholesterol su l fa te was based on i ts chro- matographic and chemical properties. The isolated material behaved like authentic cholesterol sulfate in te rms of bo th i ts mobil i ty on TLC in several solvent sys t ems and the pink-violet color produced on heat ing with sulfuric acid prior to charring. Also, the products produced by hydrolysis and hydrolysis plus acetylat ion of the isolated lipid behaved during TLC and charr ing like authentic cholesterol and cholesterol acetate respec- tively. Finally, the acetyla ted product gave rise to a single major {96.6% of the total} peak when examined by GLC with either a polar (CP-SIL 88) or nonpolar (BP-1) column. This major component comigra ted with authentic cholesterol acetate. Several minor components were not identified.

Identification of free steros The isolated free sterol was identified as cholesterol by essential ly the same criteria used to identify the sterol released by solvolysis of the sterol sulfate fraction. GLC of the sterol ace ta te gave one major peak (96.9%) tha t cochromatographed with cholesterol ace ta te on bo th the polar and the nonpolar columns.

Identification of fatty alcohols. The f a t t y alcohols had the same mobil i ty as authent ic f a t t y alcohols on TLC with mobile phases of hexane/ether/acetic acid (70:30:1) and chloroform/methanol/acetic acid (190:9:1). Like the free sterol, acetylat ion yielded a single less polar product tha t migra ted on TLC like authent ic f a t t y alcohol acetate.

Examina t ion by GLC revealed a complex mixture. Al though the major components account ing for 63.3% of the to ta l were identified as member s of the s t ra ight- chain sa tu ra ted series by compar ison with s t andards and construct ion of plots of carbon number vs log retent ion t ime f rom isothermal chromatograms , the

TABLE 2

Composition of Alcohol Acetates

ECL Chain % ECL Chain % 12.00 12:0 0.1 20.30 21:0br 0.2 13.00 13:0 0.1 20.38 21:0br 0.2 14.00 14:0 4.7 20.41u 20:1 6.0 15.00 15:0 2.1 20.50u 20:1 1.5 15.53 16:0br 0.3 20.56 21:0br 1.4 16.00 16:0 10.6 20.71 21:0br 4.0 16.30 17:0br 0.2 21.00 21:0 1.8 16.40 17:0br 0.1 21.08 22:0br 0.3 16.53 17:0br 0.5 21.43 22:0br 0.2 16.70 17:0br 2.6 21.57 22:0br 2.5 17.00 17:0 1.1 22.00 22:0 5.2 17.53u 17:1 0.6 22.28u 22:1 2.7 18.00 18:0 17 .0 22.33u 22:1 2.6 18.40u 18:1 0.4 22.43 23:0br 0.7 18.50u 18:1 0.2 22.49 23:0br 0.2 18.54 19:0br 0.9 22.59 23:0br 0.4 18.71 19:0br 1.2 22.72 23:0br 1.8 19.00 19:0 2.0 23.00 23:0 1.7 19.49 20:0br 0.1 23.36 24:0br 0.7 19.55 20:0br 2.3 23.59 24:0br 1.8 20.00 20:0 11.6 24.00 24:0 5.3 Monounsaturated species, are designated by a u following the ECL value, as well as by the N:I designation of the chain struc- ture. It is not known if any of the unsaturated chains also are branched. For the saturated species, branching is indicated by a br at the end of the chain assignment.

D.T. DOWNING

remainder consisted of components with fractional equivalent chain lengths (ECL). The f a t ty alcohol aceta tes were identified as either sa tura ted (86%) or unsa tura ted (14%) after fractionation of the acetates on silver nitrate- impregna ted silicic acid. This information is summarized in Table 2.

Identification of fatty acids. The f a t t y acids were conver ted to methy l es ters and examined by GLC and by G L C - m a s s spec t romet ry (MS). A representa t ive ch roma tog ram is shown in Figure 2, and the composi- t ion is summar ized in Table 3.

The major f a t t y acid was identified as 18-methyl- eicosanoic acid on the basis of i ts GLC behavior and i ts mass spectrum, presented as Figure 3. ECL values of 20.74 and 20.67 were determined from isothermal chromatograms obtained with CP-SIL 88 and BP-1, respec- tively. A value of 20.72 would be expected for 18-methyl- eicosanoic acid (12). Fur thermore , on bo th the polar and nonpolar columns, the f a t t y acid methy l ester in quest ion cochromatographed with the C-21 anteiso methyl-branched f a t ty acid methy l es ter derived f rom wool wax (11).

21ai

FIG. 2. Gas-liquid chromatogram of methyl esters prepared from the fatty add fraction of hair constitutive lipids. The methyl esters were chromatographed on a CP~SIL 88 quartz capillary column with a temperature program as expl~ned under Materials and Methods. The stralght~,hMned saturated species and the 21~wbon anteiso- branched component are indicated.

TABLE 3

Composition of Covalently Bound Fatty Adds Chain Wt %

14:0 0.8 15:0 1.1 16:0 18.3 16:1 1.9 17:0br 5.5 17:0 1.9 18:0 7.0 18:1 3.9 18:0br 8.4 20:0 2.3 20:0br 3.5 21:0 anteiso 40.5 others 4.9 Several saturated species indicated by br, appear to be branched.

LIPIDS, Vol. 23, No. 9 (1988)

INTEGRAL LIPIDS OF HAIR

74

O

c 3 OCH3

II ~ ............ I ,, , M

C7 199 M-43

I. I ,,I ,L,I ,I,.I , I w , , , ~ s , L"~'-,-,s , f ' I"" I ' I l ;---r"I'-T"T'-;"T"'"'T "~"T'"F'-I ' l ' ! ' I: ' I ' ~ ' I:' I 'III ' I IL' I II I' '-'I]"-"II ' I"~II ' ~h"r"J-"--l"r I II' I"' I ' I ' 1 1 I '""l

881

40 bO flO 1 0 0 120 1 4 0 l b O IBO 2 0 0 2 2 0 2 4 0 2bO 2 8 0 3 0 0 3 2 0 3 4 0 3bO 3B0 400

FIG. 3. Electron impact mass spectrum of methyl-18-methyl~icosanoate.

The identification was confirmed by the mass spec- t rum (Fig. 2). The spectrum included a molecular ion at m/e 340, consis tent with the formula C22H4402. The spectrum also included a number of features characteristic of f a t ty acid methyl esters. These included a base peak at m/e 74, and prominent f ragments at m]e 87 and 143. Also, characteristic chain-degradation fragments (C3-C8) were present. Of part icular significance are the peaks at M-29 and M-31 (13). M-31 is present in all methyl esters and is thought to represent formation of an acylium ion. The M-29 represents loss of ethyl, and is more prominent than M-31 only in cases of anteiso me thy l branching. In the present spectra , M-29/M-31 -- 2.2.

DISCUSSION

The present results demonst ra te tha t human scalp hair contains const i tut ive lipids tha t are not removable by exhaust ive extract ion with chloroform/methanol mixtures. These lipids, tha t together represent 0.7-1.3% of the weight of hair, were recovered only after t r ea tment with alkaline methanol. The major lipid class recovered in this way is f a t ty acid, which is present at a level of 4.3 mg per g of hair. This f a t ty acid is likely a t tached to the cell surface through ester or thioester linkages. Cholesterol sulfate is the second most abundant of the hair const i tut ive lipids and const i tutes 3.3 mg of each g of hair. The nature of the s t rong interaction of this lipid with hair is uncertain; however, it may be present in the form of an insoluble salt ra ther than a covalent adduct. Cholesterol and fa t ty alcohol, both relatively minor const i tut ive lipids, could be esterified to acidic groups at the cell surface.

Recently, covalently bound lipids have been identified in mammalian epidermis (8,9,14). In contras t to the present findings, the major bound lipid found in the epidermis is an unusual ceramide consisting of a long- chain {30-34 carbon) r acid in amide linkage with sphingosine. This is the predominant bound lipid in the s t r a tum corneum, where it is thought to com- prise a lipid envelope on the exterior of each keratinized cell. By analogy, the constitutive lipids of hair also may be bound to the cell surface to make a pliable but environ- mentally resistant lipid coat or envelope.

Perhaps the most striking result of the present inves- t igat ion was the finding tha t 40% of the fa t ty acid bound to hair is 18-methyl-eicosanoic acid. This anteiso methyl-branched fa t ty acid is present in small amounts in human sebum and vernix caseesa {15) and in wool

The molecular ion ~M) and other significant fragments are indicated.

wax (11), but is not abundant in any source other than hair. Shorter 13-17 carbon anteiso fa t ty acids are major lipid components of some microorganisms (16-18), where it is thought tha t they are used, instead of unsa tura ted fa t ty acids, to make fluid membranes. I t seems feasible tha t the 21-carbon anteiso fa t ty acid in hair also may serve to fluidize membranes. In this capacity, the branched-chain acid would have an advantage over unsa tura ted f a t ty acids in being resis tant to oxidative damage on long exposure to the atmosphere.

A C K N O W L E D G M E N T S

This study was supported in part by Grants AR 32374 and AR 01610 from the United States Public Health Service.

REFERENCES

1. Chapman, R.E. (1986) in Biology of the Integumen~ (Bereiter- Hahn, J., Matoltsy, A.G., and Richards, K.S., eds.) pp. 293-311, Springer-Verlag, Berlin, West Germany.

2. Birbeck, M.S.C., and Mercer, E.H. (1956) Nature 178, 985-986. 3. Rogers, G.E. {1959) Ann. N.Y. Acad. Sci. 83, 378-399. 4. Parakkal, P.F., and Matoltsy, A.G. {1964) J. Invest. DermatoL

43, 23-34. 5. Wix, M.A., Wertz, P.W., and Downing, D.T. (1987) Comp.

Biochen~ Physiol. 86B, 671-673. 6. Nicolaides, N., Fu, H.C., and Rice, G.R. (1968) J. Invest.

Dermatol. 51, 83-89. 7. Stewart, M.E., Downing, D.T., and Strauss, J.S. {1983) De~

matol. Clin. 1, 335-344. 8. Wertz, P.W., and Downing, D.T. {1987) Biochim. Biophys.

Acta 917, 108-111. 9. Swartzendruber, D.S., Wertz, P.W., Madison, K.C., and

Downing, D.T. {1987)J. Invest. DermatoL 88~ 709-713. 10. Downing, D.T. (1968)J. Chromatogr. 38, 91-99. 11. Downing, D.T., Kranz, F.H., and Murray, K.E. {1960) Aust.

J. Chem. 13, 80-94. 12. Nicolaides, N., and Apon, J.M.B. (1976) Lipids 11, 781-790. 13. Abrahamsson, S., Stallberg-Stenhagen, S., and Stenhagen,

E. (1963) Prog. Chem. Fats Other Lipids 7, 1-164. 14. Wertz, P.W., and Downing, D.T. (1986) Biochem. Biophys.

Res. Commun. 137, 992-997. 15. Nicolaides, N., Fu, H.C., Ansari, M.N.A., and Rice, G.R.

(1972) Lipids 7, 506-517. 16. Zuneda, M.C., Guillenea, J.J., Dominguez, J.B., Prado, A.,

and Goni, F.M. {1984) Lipids 19, 223-228. 17. Song, Y., Sawa, T., Tsuvhiya, M., Kondo, S., Hamada, M.,

and Umezawa, H. {1981) J. Antibiot. Tokyo 34, 980-983. 18. Kaneda, T., and Smith, E.J. {1980) Can. J. Microbiol. 26,

893-898.

[Received April 13, 1988; Revision accepted June 25, 1988]

UPIDS, VoL 23, No. 9 (1988)

Documents

Integral lipids of human hair