and with surface lipids

Transcription

1 j. Soc. Cosmet. Chem., 35, (December 1984) Analysis of the lipid content of single hair bulbs. Comparison with the content of the sebaceous gland and with surface lipids NOEL GOETZ, HERVE BURGAUD, CLAUDINE BERREBI, and PIERRE BORE, Research Laboratories, Socigtg L'Oreal, 1 Avenue de Saint-Germain, Au/nay Sous Bois, Cedex, France. Received July 24, Synopsis The lipid content of single hair bulbs was investigated using capillary gas chromatography. In contrasto lipids of the sebaceous glands themselves, these samples exhibit a noticeable degree of hydrolysisince the free fatty acids fraction is about /3 to 1/2 of the (free + glycerides) fatty acids fraction. The hydrolysis reaction is structure selective in that it first cleaves the straight chain species. The (free + glycerides) fatty acids fraction found in the hair bulbs contains a higher proportion of saturated species than the corresponding fraction from lipids sampled on the scalp and hair. Thus, the human skin surface lipids after having been synthesized undergo an evolution which increases their content of unsaturated species. Hair bulb lipids were found to contain a higher proportion of free cholesterol compared to both the sebaceous gland and surface lipids. INTRODUCTION In previous studies (1,2), we have reported that the human skin surface lipids (SSL) undergo a change in their composition as they accumulate on the scalp and hair. The proportion of unsaturated species in the (free + glycerides) acids fraction was found to increase as a function of the time of accumulation. Evidence for this evolution was obtained by comparison of samples collected after one day with those collected after four days following cleansing of the scalp and hair by shampooing. However, the complete extent of this evolution can only be observed fully by a comparison with truly endogeneous material since the mixture collected on the surface after one day is far from being a representative sample. Indeed, it has been shown that the transit time from the site of synthesis to the surface is about 8 to 9 days (4,5). The lipid content of the sebaceous gland can certainly be considered as the ideal sample of the original sebum, but this mixture is not easily obtained. The isolation of a sebaceous gland is a very tedious procedure (6) beginning first with a biopsy. Thus, it can hardly be considered for multiple determinations over an experimental population. On the other hand, hair bulbs are "under-the-surface" samples which can be very easily collected. The lipid content of the hair follicle, however, cannot be considered identical to the original sebum, since it may be contaminated by epidermalipids and already biotransformed by bacterial enzymes. 411

2 412 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS In this study, several hair bulbs were taken from 14 subjects and subsequently analyzed. Additionally, several sebaceous glands from biopsies of a single subject were isolated and similarly investigated. As a consequence of the previously observed evolution, the above samples were expected to differ in their compositions from those of surface lipids. For such minute amounts of complex lipids, however, an adequate analytical procedure had to be developed. The gas-chromatographic method we used for samples deposited on ground glass platelets (2) performed well upon SSL quantities in the 1 to 20 g range. This approac had to be modified in order to handle samples which were smaller by approximately one order of magnitude. EXPERIMENTAL COLLECTION OF THE SAMPLES Single hairs were withdrawn from the subjects' scalps using a pair of tweezers. The soft portion of the root including the bulb was separated from the hair shaft by cutting through the brilliant, non-colored band which clearly divides the internal and external regions of each shaft. The bulbs were then dropped into a septum-capped vial (Reacti-Vial, from Pierce Eurochem B.V., Rotterdam, The Netherlands) containing diethyl ether. Three circular biopsies with a diameter of 3-4 mm were obtained from the forehead of a 58-year-old male. From these samples, ten sebaceous glands were isolated according to Kellum (6) and stored individually in septum-capped vials containing diethyl ether. The surface lipids were sampled using ground glass platelets as previously described (2). PRECHROMATOGRAPHIC TREATMENT OF THE SAMPLES The diethyl ether solutions containing the lipid mixtures were evaporated to dryness at room temperature under a stream of nitrogen. For the derivatization with diazomethane, 30 1 of the reagent (3g of diazomethane in 250 ml of diethyl ether prepared according to reference 7) were added to the dry lipids when analyzing the hair bulbs. For a sebaceous gland or a surface sample deposited on a ground glass platelet, 200 p l of the same reagent were used. The mixture was allowed to stand at room temperature for a few minutes and then was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 10 1 of diethyl ether for the hair bulbs, while 200 p l of the same solvent were used for the gland and the surface samples. The resulting solutions were then ready for injection. For the transesterification of the glycerides, 10 1 of toluene and 5 1 of reagent [Meth- Prep II, from Applied Science Labs., State College, PA, USA: 0.2N methanolic solution of (m-trifluoromethylphenyl) trimethylammonium hydroxide] were added to the vial residues remaining after evaporation of extracts from hair bulbs, while 50 1 of the reagent were used for the sebaceous gland residues and the samples deposited on ground glass platelets. The mixtures were allowed to react at room temperature for 15 minutes before injecting.

3 HAIR BULB, SEBACEOUS GLAND AND SKIN SURFACE LIPIDS 413 GAS CHROMATOGRAPHY OF THE FATTY ACIDS METHYL ESTERS A borosilicate glass capillary (45 m x 0.3 mm I.D.) was preconditioned using the BaCO 3 procedure of Grob (8-10). The tube was then dynamically wall-coated with CP Sil 58 stationary phase (from Chrompack, Middelburg, The Netherlands). The operating conditions were the following: injector and flame ionization detector temperatures 230øC; column temperature, programmed from 145øC to 230øC at 2øC ß min. - ; carrier gas, helium; inlet pressure, 1.1 bar; injected volume, 1 Ixl; split ratio, 1:10. The internal standard for absolute quantitation was n-heptadecanoic acid. Five micrograms of this compound were added to the lipid mixture prior to any derivatization operation whenever a quantitative interpretation of the chromatograms was desired. Possible substitutes to n-heptadecanoic acid as an internal standard are cis-9-hexadecenoic acid (palmitoleic acid) or 2-hydroxy-palmitic acid (2). GAS CHROMATOGRAPHY OF THE NEUTRAL LIPIDS A borosilicate glass capillary (12 m x 0.3 mm I.D.) was preconditioned using the persilylation procedure of Grob (11). The tube was then dynamically wall-coated with OV - 1 stationary phase. The operating conditions were the following: column temperature, 45øC to 145øC at 30øC ß min-, then from 145øC to 340øC at 7øC ß min- with final hold for 5 minutes; flame ionization detector temperature, 340øC; carrier gas, helium; inlet pressure, 0.5 bar; on-column injection of 1 Ixl (according to reference 12). RESULTS A typical chromatogram of the lipids contained a single hair bulb is shown in Figure 1. The sample was derivatized with diazomethane before injection. This reagent converts the free fatty acids into their methyl esters while leaving all other components in the mixture unchanged (2). The following compounds or classes of compounds were successively eluted: --free fatty acids (methyl-esters)--this fraction was not properly resolved using these chromatographic conditions; its constituents were separated on a dedicated system using a polar column (Figure 4). The major constituents in the fraction, however, can be recognized. These are: n-hexadecenoic acid, n-hexadecanoic acid, n-octadec- enoic acid and n-octadecanoic acid. --squalene --cholesterol --wax esters --cholesteryl esters--the compounds of this class partially overlap with the triglycerides; the main components are cholesteryl-n-hexadecenoate and cholesteryl-n-hexadecanoate. The pattern of the cholesteryl esters fraction, however, can be obtained free from any interference due to the triglycerides. To accomplish this, the sample is derivatized with Meth-Prep II for 15 minutes before injecting. This procedure results in the complete cleavage of the triglycerides as outlined below. --.triglycerides--this mixture is poorly resolved as a consequence of its very complex

4 414 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS C A B D 0 I 1 I i 0 m i n Figure 1. Chromatogram on an OV 1 capillary column of the lipids from a single hair bulb after derivatization with diazomethane. A: Elution range of the free fatty acids (methyl esters); B: Elution range of the wax esters; C: Elution range of the sterol esters; D: Elution range of the triglycerides; 1: Hexadecenoic acid; 2: Hexadecanoic acid; 3: Octadecenoic acid; 4: Octadecanoic acid; 5: Squalene; 6: Cholesterol; 7: Cholesteryl hexadecenoate; 8: Cholesteryl hexadecanoate; 9:n-C48 substituted triglycerides; 10:n-C50 substituted triglycerides; 11:n-C52 substituted triglycerides; 12:n-C54 substituted triglycerides. composition. The emerging peaks, however, can be assigned to linear chain substituted triglycerides with substituents totaling 48, 50, 52 and 54 carbon atoms. The same chromatographic conditions were used for the characterization of the lipids contained in a single sebaceous gland (Figure 2) or sampled from the scalp on a ground glass platelet (Figure 3). A typical chromatogram of the fatty acids fraction from a single hair bulb is shown in Figure 4. The sample was derivatized with Meth-Prep II for 15 minutes before injecting. This derivatization procedure converts the free fatty acids into their methylesters and transesterifies the acidic moleties of the glycerides into their corresponding methyl-esters. The transesterification of the wax esters and the sterol esters proceeds much more slowly and can be considered as negligible within the alloted reaction time (2). Thus, the chromatogram of Figure 4 is representative of the (free + glycerides) fatty acids fraction in the hair bulb sample. The main components in the fraction are: -- tetradecanoic acid --cis-6-tetradecenoic acid

5 C A B D min. I I I Figure 2. Chromatogram of the lipids from a single sebaceous gland after derivatization with diazomethane. For peak assignment, see Figure 1. 5 C A B D min Figure 3. Chromatogram of scalp surface lipids sampled on a ground glass platelet and derivatized with diazomethane. For peak assignment, see Figure 1.

6 416 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS min Figure 4. Chromatogram of the (free + glycerides) fatty acids fraction from a single hair bulb on a CP i Si158 capillary column. The sample was derivatized with Meth-Prep II for 15 minutes. 1: Tetradecanoic acid; 2: Cis-6-tetradecenoic acid; 3: Pentadecanoic acid; 4: Cis-6-pentadecenoic acid; 5: Hexadecanoic acid; 6: Cis-6-hexadecenoic acid; 7: Cis-9-hexadecenoic acid; 8: Heptadecanoic acid; 9: Cishepradecenoic acid (mixture of isomers); 10: Octadecanoic acid; 11: Cis-8-octadecenoic acid; 12: Cis-9- octadecenoic acid. --pentadecanoic acid -- cis-6-pentadecenoic acid -- hexadecanoic acid -- cis-6-hexadecenoic acid -- cis-9-hexadecenoic acid --heptadecanoic acid --cis-heptadecenoic acid (mixture of positional isomers) -- octadecanoic acid --cis-8-octadecenoic acid --cis-9-octadecenoic acid The same chromatographiconditions were used for the resolution of the (free + glycerides) fatty acids fractions found in single sebaceous glands (Figure 5) and in surface samples (Figure 6). The degree of hydrolysis of the triglycerides in the hair bulbs was determined after quantitation of the free fatty acids fraction and the (free + glycerides) fatty acids fraction. The former quantity is accessible from the chromatogram of the diazomethane derivatized mixture. The (free + glycerides) fatty acids fraction is quantified from the chromatogram of the mixture derivatized using Meth-Prep II. These quantitative

7 HAIR BULB, SEBACEOUS GLAND AND SKIN SURFACE LIPIDS lo min. I I Figure 5. Chromatogram of the (free + glycerides) fatty acids fraction from a single sebaceous gland on a CP Sil 58 capillary column. The sample was derivatized with Meth-Prep II for 15 minutes. For peak assignment, see Figure 4. determinations were performed upon five hair bulbs sampled from each of four subjects, as shown in Tables I and II. The distribution between saturated and unsaturated species within the (free + glycerides) fatty acids fraction has been characterized, as in previous studies (1), by the hexadecanoic acid cis-6-hexadecenoic acid ratio. This determination was performed upon five hair bulbs from each of ten subjects as shown in Table III. DISCUSSION All the hair bulbs that we investigated contained significant amounts of free fatty acids as can be seen on the chromatograms from Figure 1. This shows that the hydrolysis of the triglycerides is already taking place before the lipid mixture reaches the skin surface. The degree of hydrolysis in the hair bulbs can be estimated by ratioing the results which are presented in Tables I and II. It is evident that the degree of hydrolysis ranges between 1/3 and /2.

8 418 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 6 õ 9 2,0 3 0 min Figure 6. Chromatogram of the (free + glycerides) fatty acids fraction from surface lipids collected on the scalp and hair. The sample was derivatized with Meth-Prep II for 15 minutes. For peak assignment, see Figure 4. Table Quantitative Determination of the Free Fatty Acids Fraction in Single Hair Bulbs Subject No. Bulb No }xg 1.9 }xg 1.6 g Mean 1.0 tg 1.7 }xg 1.9 }xg 1.4 I

9 HAIR BULB, SEBACEOUS GLAND AND SKIN SURFACE LIPIDS 419 Table Quantitative Determination of the (Free + Glycerides) Fatty Acids Fraction in Single Hair Bulbs Subject No. Bulb No Ixg 2.0 Ixg 5.4 Ixg Mean 3.3 tg 3.7 Ixg 5.4 btg 2.5 II On the other hand, the sebaceous glands contain only very limited amounts of free fatty acids as can be seen on the chromatogram of Figure 2. As a consequence, the trace of the intact triglycerides is very clear, with sharp and intense peaks emerging from the broad unresolved band. These peaks have been identified as tri-n-substituted triglycerides with substituents totaling 48, 50, 52, and 54 carbon atoms. These species, however, are barely detectable in the extensively hydrolyzed surface sample shown in Figure 3. This confirms the hypothesis that the microbiological lipases which cleave the fatty acids from the triglycerides proceed in a structure-selective manner affecting, preferentially, the n-substituted functional groups. A similar conclusion has already been given in previous studies (1,2) after comparativexamination of the free fatty acids fraction and the glyceride fatty acids fraction. Squalene is a prominent component in the hair bulbs, as is free cholesterol. We have always found a larger proportion of free cholesterol in the bulbs than in both the sebaceous glands and the surface samples. Since free cholesterol is considered to be a product of the keratinizing epidermis (13), whereas the gland produces little or none of it (14), the observedifference can be attributed to the presence of epidermalipids in the bulbs. On the other hand, it has been shown that fatty acids liberated from the sebaceous glycerides are used for cholesterol esterification by cutaneous bacteria (I5). This mechanism is consistent with our observation that the proportion of free cholesterol is higher in the bulb than in the surface samples. Table III Hexadecano¾c Acid/Cis-6-Hexadeceno¾c Ratio in the (Free d- Glycerides) Fatty Acids Fraction of Hair Bulbs Bulb No. Subject No Mean

10 420 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The (free + glycerides) fatty acids fraction from hair bulbs contains the species which have already been identified in skin surface lipids. These are mainly straight-chain saturated and cis-monounsaturated compounds ranging from 14 to 18 carbon atoms. A distribution of positional isomers is found to occur for the C 6 to C 8 unsaturated chains as was reported earlier for surface samples (16). The (free + glycerides) fatty acids fraction found in the hair bulbs differs considerably, however, from the corresponding surface fraction as far as the balance between saturated and monounsaturated species is concerned. This balance can be characterized by the ratio hexadecanoic acid cis-6-hexadecenoic acid as has been discussed previously (1). The values of this ratio, determined from five hair bulbs from each of ten subjects as shown in Table III, can be compared to the corresponding values previously determined for surface samples over a population of 40 subjects (1). This comparison shows that the under-the-surface samples contain a higher proportion of saturated species than those of the surface samples. This has been illustrated in Table IV where three sets of hexadecanoic acid cis-6-hexadecenoic values are presented over an experimental population of ten subjects. The three sets of data as presented are: acid --lipids present in the hair bulbs --.lipids accumulated on scalp and hair after 1 day --.lipids accumulated on scalp and hair after 4 days From the data concerning the surface samples, it was concluded earlier (1) that the (free + glycerides) fatty acids fraction becomes more unsaturated as the mixture ages. The data collected from the hair bulbs, however, show that the extent of this evolution is far greater than what could be estimated after examining the surface samples., Table IV Comparison of the Lipids From Hair Bulbs With Surface Samples: Values of the Hexadecano¾c Acid/Cis-6-Hexadeceno¾c Ratio in the (Free + Glycerides) Fatty Acids Fractions Total scalp and hair Symptom** Subject Bulbs* 1 day** 4 days** (O = oily) * Mean values from Table III. ** See Reference 1.

11 HAIR BULB, SEBACEOUS GLAND AND SKIN SURFACE LIPIDS 421 This point was further confirmed by an examination of the lipids from several isolated sebaceous glands. For the triglyceride acid fraction in the glands, we consistently observed values of the hexadecanoic acid cis-6-hexadecenoic acid ratio greater than 5 as is illustrated by the example in Figure 5. This chromatogram also reveals the presence of noticeable amounts of cis-9-hexadecenoic acid and cis-9- octadecenoic acid (oleic acid). Such species which are unsaturated at odd-numbered carbon atoms are not products of the sebaceous gland itself (17). Most likely they are components of the structuralipids from the maturing cells' membranes which have been released in the lipid mixture either during the isolation procedure of the glands or the solubilization step. In previou studies, the degree of unsaturation of the fatty acids fraction was correlated to the rheological properties of the skin surface lipids (2,3,16) which, in turn, could be correlated to the oily hair symptom of the subjects. An increase in the degree of unsaturation resulted in a decreased viscosity of the mixture (3) and an increase in the low temperature melting fraction (16). This situation was found to be favorable to the development of the oily hair symptom. Thus, every time the measured value of the ratio hexadecanoic acid cis-6-hexadecenoic acid fell below 0.79, the hair was observed to be oily. The results from Table IV, however, clearly show that these subjects, although they bear an oily mixture on their scalp and hair, do not actually synthesize such a mixture since the "under-the-surface" samples always contained a high proportion of saturated species. CONCLUSION The examination of the lipid content of hair bulbs and sebaceous glands shows that the compositions of these "under-the-surface" samples were very different from those of the skin surface lipids present on the scalp and hair. The difference is evident not only in the degree of hydrolysis of the triglycerides but also in the balance between the saturated and unsaturated species in the (free + glycerides) fatty acids fraction. From previou studies, it was concluded that the proportion of unsaturated fatty acidic chains in skin surface lipids increases as a function of time. Using a micro-scale analytical technique, it was possible to observe that the proportion of unsaturated species increases the further away the lipid mixture is sampled from the site of biosynthesis. At the present time, the causes of this evolution are not fully understood; physicochemical processes have been shown to play a role (1), but a contribution due to microbiological activity cannot be excluded. Additionally, it was observed that the hair bulbs themselves contain a higher proportion of free cholesterol compared to both the sebaceous glands and to the surface lipids. This discrepancy possibly due to microbiological activity. This hypothesis, however, needs further experimental confirmation.

12 422 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS ACKNOWLEDGEMENTS Our sincere thanks are due to Dr. Jean Arouette who kindly provided skin biopsies from one of his patients and to D. Good for revision of this manuscript. We wish to express our gratitude to Mr. G. Kalopissis, Mr. B. Jacquet and Mr. D. Reymond for permission to publish this contribution. REFERENCES (1) P. Bore, N. Goetz, P. Gataud, and L. Tourenq, Evolution in the composition of human skin surface lipids during their accumulation on scalp and hair, Int. J. Cosmet. Sci., 4, (1982). (2) N. Goetz, G. Kaba, and P. Bore, Capillary gas chromatographic analysis of human skin surface lipids after microsampling on ground-glass platelets, J. Chromatogr., 223, (1982). (3) P. Bore and N. Goetz, A physical method for qualitative examination of human sebum, J. Soc. Cosmet. Chem., 28, (1977). (4) E. H. Epstein and W. L. Epstein, New cell formation in human sebaceous glands, J. Invest. Dermatol., 46, (1966). (5) D. T. Downing, J. S. Strauss, P. Ramasastry, M. Abel, C. W. Lees, and P. E. Pochi, Measurement of the time between synthesis and surfac excretion of sebaceous lipids in sheep and man, J. Invest. Dermatol., 64, (1975). (6) R. E. Kellum, Isolation of human sebaceous glands, Arch. Derm., 93, (1966). (7) Th. J. De Boer and H. J. Backer, Diazomethane, Org. Synth. Collect. Vol., 4, (1963). (8) K. Grob and G. Grob, A new, generally applicable procedure for the preparation of glass capillary columns, J. Chromatogr., 125, (1976). (9) K. Grob, G. Grob, and K. Grob, Jr., The barium carbonate procedure for the preparation of glass capillary columns; Further information and developments, Chromatographia, 10, (1977). (10) K. Grob, Jr., G. Grob, and K. Grob, Preparation of apolar glass capillary columns by the barium carbonate procedure, J. High Resolut. Chromatogr. Chromatogr. Commun., 1, (1978). (11) K. Grob, G. Grob, W. Blum, and W. Walther, Preparation of inert glass capillary columns for gas chromatography, J. Chromatogr., 244, (1982). (12) M. Galli, S. Trestianu, and K. Grob, Jr., Special cooling system for the on-column injector in capillary gas chromatography eliminating discrimination of sample compounds, J. High Resolut. Chromatogr. Chromatogr. Commun., 2, (1979). (13) N. Nicolaides, Skin lipids. II. Lipid class composition of samples from variouspecies and anatomical sites, JAOCS., 42, (1965). (14) J. S. Strauss, P. E. Pochi, and D. T. Downing, The sebaceous gland: Twenty-five years of progress, J. Invest. Dermatol., 67, (1976). (15) S. M. Puhvel, Esterification of (4-Z4C) cholesterol by cutaneous bacteria (Staphylococcus epidermidis, propionibacterium acnes and propionibacterium granulosum), J. Invest. Dermatol., 64, (1975). (16) P. Bore, N. Goetz, andj. C. Caron, Differential thermal analysis of human sebum as a new approach to rheological behaviour, Int. J. Cosmet. Sci., 2, (1980). (17) D. T. Downing, M. E. Stewart, P. W. Wertz, S. W. Colton, and J. S. Strauss, Skin lipids, Comp. Blochem. Physiol., 76B (4), (1983).