Br.J.Derm.

(1972)87, 333.

Department of Dermatology, The, General Infirmnry Leeds; St James's (University) Hospital, and Harrogate General Hospital and Medical Research Council, Mineral Metabolism Unit, The General Infirmary, Leeds {Mr Bulusu)

AGE AND SEX VARIATTOX IN SKIN SURFACE LIPID COMPOSITION AND SEBUM EXCRETION RATE
J. A. COTTERILL. \\\ .1. CUXLIFFE, B. WILLIAMSON ANII U BULUSU Summary.The sebum excretion rate and skin surface lipid composition were determined in 193 male and female control subjects aged between 4 and 60 years. The sebum excretion rate, squalene and wax ester content of surface lipid were maximal in males aged 26-40. The percentages of surface lipid wax esters were greater in males than females at all ages, but the prepubertal peak of wax esters in males may be due to an epidermal contribution from sterol esters. There was no significant age or sex variation in the sum of the percentages of triglycerides and free fatty acids, but the degree of hydrolysis varied considerably with age. The free fatty acid content was greatest in the first decade in both sexes, when the sebum excretion rate is at the lowest levels. Cholesterol content of surface lipid rose to maximal levels at puberty in both sexes. IT ii^mijortant to identify factors controlling the bioclieinical oompositiou of sehuin and skin surface lipid since the composition of sebum may be important ill the pathogenesis of acne (Strauss and Pochi, 1905). It seemed possible that useful information on such factors could bo obtained by an investigation of a control population free of acne, but at present limited data only is available (Nikkari, 19r)o: Ramasastry e/a/.. 1970). Forehead skin surface lipid composition was therefore determined in a large group of subjects free of acne. Forehead skin surface lipid was sampled by absorbent papers so that the rate of sebum exeretion could be measured simultaneously. SUBJECTS AND METHODS One liundred and ninety-three subjects aged 4 to 60 years were investigated. These subjects had no evidence of clinical acne. A past history of acne, or acne scarring, clinically excluded subjects from further study. None of the subjects was receiving any systemic or topical therapy at the time of the investigations. The following age groups were investigated: 1. Range 0-10 years: 17 males (age range 4-10 years, mean 7 7) and 8 females (age range 7-10 years, mean 8-6). 2. Range 11-15 years: 35 males and 49 females. Twenty-five of the female subjects had established menstrual bleeding and had a mean age of 13 9. The remaining 24 female subjects were awaiting the menarche and had a mean ag(^ of 12-4. 3. Range 16-25 years: 9 males and 27 females. 4. Range 26^0 years: 8 males and 11 females. 5. Range 41-60 years: 6 males and 23 females.
Accepted for publication May 19th. 1972.

334

J . A. COTTERILL, W. .1. C i r N L I F F E , IJ. WILLIAMSON AND L. UULUSU

Tlic .schuin excretion rate was determined from the forehead. iKsing al)sorljent by the method of Strauss and Poehi (1961) as moditied by Cunliffe and .Shustei- (iy(i!)a). From the same samples the composition ua.s dctcrrniiu'd wcmiqiiaTititativel;' for sijualene, wax and sterol esters, triglyeerides. free fatty acidw and cliolestero! by thin-la_\'er chromatograpliy (Cottt'rill ft al.. 1071a). All samples were analysed w ithin 24 hours of collettlon to avoid any possible changes in the composition of the sample due to storage (Downing. 1970). tSiniace lipid was collected at the wainc time of day to avoid any changes pi-{diiccd by circadian variation, either in sebum excretion rate (Burton ef al., 1970) or skin surface lipid composition (Cotterill ft al.. 1971b).

RESULTS The results are shown in Table I and Fig. 1. 2.

Excretion Rate

Males aged between O-Ht and II I'> have a significanth- lower sebum excretion ratethan males aged between 10 and 25 (/> < ()-(IOI): 2(i-40 (/> < 0-001): and 4l-()() [p < O-(HH. Fig. 1). The rate of sebum excretion is signiHeantly lower in males aged 11-15 when compared to females aged 11 15 with established menstrual bleeding ( / j < 0 001). These females also have a signilicantly higher sebum excretion rate than females aged 11-1 o awaiting tlie menareho [p < 0-01), After the age of 15 the sebnm excretion rate was greater in males than females, and continued to increase in both sexes, reaching a maximum between the ages of l(i and 40. Thereafter, the rate declined in both sexes, particularly in females (Fig. 1). Squalene Although the squalene content varietl little w itii age and sex. there were certain similarities betweeii tbe shape of the graphs for squalene and sebum excretion rate (Fig. 1). In both sexes the lowest squalene eontent was in the 11-15 age group (prepiibertal in females). This was followed by an increase, reaching maximum levels between the ages of 20-40 in males; the squalene content tliereafter decreased in the 41-()0 age group. The squalene content in the group of males aged 2()-40 was significantly greater than in the group of males aged I I-Ii5 {p < 0-025). There were no sex clifferences in forehead surface lipid squaleue content. Wax and Sterol Esters The jiereeiitages of surface lipid wax and sterol esters showed no significant variation with age, but maximal levels were achieved in the groups of males aged respeetively 0-10 and 26-40. Wax esters in males were generally greater than tho.se in females (Fig. 1). Itut these differences were not statistically signifieant. Triglycerides and Free Fatty There was a elear reciprocal relationship between the percentage of free fatty acids and triglycerides (Fig. 2). The sum of the percentages of the trigh'cerides and free fatty acids was higher in females than males, but the differences were not statistically signifieant, and there was no signifieant variation with age. The degree of lipolysis to free fatty aeid.s, however, varied considerably with age. The

AGE AND 8EX VARIATION IN SKIN SURFACE LIPID fOMPOSlTlO.V

335

p. ' X S

336

J . A. COTTERILL, W. J . CUNLIFFE, B. WILLIAMSON AND L. BULUSU

free fatty acid content fell and the triglyceride content rose in both sexes at puberty, and remained at these pubertal levels thereafter. The changes in surface lipid free fatty acids and triglyceridcs were statistically significant only in males, however.
Cholesterol

Peak values for cholesterol occurred in both sexes between the ages of 11 and 15 (Fig. 2). Males aged between 41 and 00 had a signifioantl\' lower percentage of surface lipid cholesterol than males aged U-lo {p < O-Ol).
MALES & FEMALES 16-25years

sebum excretion rate

52 -

mean r: males

triglycerides squalene 7o wax and sterol esters free fatty acids

06

cholesterol '*
0-10 1 - 5 16-25 26-40 11 group 1 No acne n m IV Mild Moderate Severe acne acne acne

years
FIG. 1.Age and sex variation in sebum excretion rate and in the [jorceiitages of surface lipiti s([ualcn(' iiiiil \va\ ami storol esterw.

Fio. 2.- -Age and sex variation in the pcrct^ntages of surface lipid triglyoondos, free fatty acids and cholesterol.

Females aged 11-15 with established menstrual bleeding had a significantly greater percentage of surface lipid cholesterol than females aged 16-25 {p < O-OOI); 26-40 [p < 0-005); and 41-60 [p < 0-005). The differences in the percentage of cholesterol in the 2 different groups of females aged 11-15 were not significant {p > 0-05), but females with an established menstrual cycle had a significantly greater percentage of surface lipid cholesterol than males aged 11-15 [p < 0-05). Menstrual Cycle There was no significant variation in either the sebum excretion rate or forehead skin surface lipid composition in relation to the menstrual cycle. Correlation Between Sebum Excretion Rate, and Forehead Skin Surface Lipid (Jomposition There was no significant correlation between the sebum excretion rate and the forehead surface lipid constituents studied in cither sex, with the exception of a significant correlation between sebum excretion rate and surface lipid squalene content in females (correlation coefficient 0-20, p < 0-05).

AGE AND SEX VARIATION IN SKIN SURFACE LIPID COMPOSITION

337

DISCUSSION In this study the results for tlie sebum excretion rates showed a distribution similar to that reported by CuuHffe and Shuster (1969b). The finding by these workers of a greater rate of sebum excretion in females up to the age of 15 years is confirmed, and is likely to be due to the earlier onset of puberty in females. The significantly lesser sebum excretion rate in females awaiting the menarche compared to the post-menarche group is in keeping with this hypothesis, although the mean age of the latter group was 1 -4 years greater than that of the former. After the age of 15, the sebum excretion rate is greater in males than females, and the rate rises to maximal levels in both sexes between the ages of 26 and 40. declining thereafter, but to a greater degree in females. Although the distribution of results was similar to that of Cunliffe and Shiister (l9G9b), the absolute values for the sebum excretion rates were higher in both sexes in this series. Both the age groups investigated and the technique used to determine the sebum excretion rate were similar to those of Cunliffe and Shuster (1909b) in Newcastle. It seems possible that regional variations in the rate of sebum excretion may occur, and this possibility merits further investigation. iSebaceous gland activity iu man is androgen-mediated (Strauss and Pochi, 1963), wliilst oestrogens reduce sebaceous gland size, either by inhibiting the synthesis of endogenous androgen (Strauss et al.. 1962) or possibly by inhibiting the sebaceous gland (Kbling, 1963). There is, however, comparatively little data on the effect of these hormones on skin surface lipid composition in man. From studies in the rat it is known that surface lipid composition is determined by the secretory activity of the sebaceous glands, which in turn are stimulated by androgens, whereas oestrogens have no specific primary effect on surface lipid composition in this animal, although they may produce se(;ondary changes by an effect on the secretory aetivity of the sebaceous gland (Nikkari and Valavaara, 1970). Pituitary hormones are also necessary for normal sebaceous gland function in the rat (Ebling ft al, 1969). However, there are some diHiculties in extending tfiese studies to man because human skin surface lipid is rich in triglycerides, their hydrolysis products and squalene, all of which are present in negligible amounts in surface lipid of other animals (Nicolaides et al.. 1968). In addition, the investigations which have been made in man are often difficult to interpret because of differing sites and methods of surface lipid collection. In this study the forehead was used as the sampling site because forehead surface lipid is mostly sebaceous in origin, with an epidermal contribution of less than 10% (Greene W a?., 1970). A knowledge of the origin of the various surface lipid fractions may facilitate interpretation of changes in surface lipid composition. Squalene and wax esters are predominantly sebaceous in origin (Ramasastry et al., 1970), whilst triglyceride is derived from both epidermis and sebaceous glands. Free fatty acids are derived from triglycerides by lipolysis, either by skin micro-organisms (in particular Corynebacterium acnes) (Freinkel and Shen. 1969) or by esterases present in the sebaceous ducts (Nicolaides and Wells, 1957). Cholesterol esters and cholesterol are predominantly epidermal in origin (Freinkel and Kazuo, 1969; Green et al., 1970). Studies in man have indicated that both the squalene content (Nieolaides and

338

J. A. COTTERILL, W. J. CUNLIFFK, B. WILLIAMSON AXD L. BULUSU

Kothman, 1955) and wax ester content (Ramasastry et al., 1970) of surface lipid increase at puberty and may therefore be androgen-dependent. In addition, it is known that testosterone will stimulate wax ester synthesis in the isolated mouse prejnttial gland (Sansone et al., 1971). From a study of surface lipid composition of the female ba(k during the menstrual cycle it appears that oestrogens, and possibly luteotrophic hormone, or both, influence the surface lipid cholesterol content, whilst the triglyceride content appears to be dependent on both oestrogens and progesterone (MacDoTiald and Clarke. 1970). Surface lipid cholesterol is primarily epidf^rmal in origin (Green et al., 1970) and both androgens and oestrogens are known to stiinulafe epidermal mitosis (Bullough et al., 1967). Tn the present study maximal levels of surface lipid cholesterol occurred in both sexes at puberty. Females with established menstrual bleeding had a greater percentage of surface lipid cliolesterol than pre-menarche females, and significantly greater levels than males aged 11-15. These results support the hypothesis that oestrogens are important in cliolesterol synthesis by the female skin, whilst the peak percentages of cholesterol in males around puberty may be androgen-dependent. The decrease in the cholesterol content of surface lipid after puberty may reflect increasing dilution of epidermal lipid by an increased volume of sebaceous lipid. Can the variation in forehead skin surface lipid composition be explained, as in tlie rat, by changes in the secretory activity of the sebaceous glands^ (Nikkari and \"alavaara. 1970). The sebum excretion rate in man is an indirect measure of sebum excretion (Cunliffe and Shuster, 19(>9b). Because squalene and wax esters are predominantly sebaceous in origin (Ramasastry ei al., 1970) the content of these fractions iu surface lipid mav be expected to correlate with the rate of sebum ex<Tetion. Indeed, the percentage of surfa(e lipid squalene is maximal in both sexes between the ages of 26 and 40. when the sebum excretion rate is also maximal. The interpretation of the wax ester data is more difficult. The thin-layer chromatographic technique employed (('otterill et al., 1971a) does not satisfactorily resolve wax and sterol esters, and these lipid fractions are determined together. Because, in the normal adult, sterol esters are present in surface lipid in negligible amounts, and usually less tiian 3% (Downing f/a/., 1969), it is usually not necessary to use specialized thin-layer chromatographic techniques to separate sterol and wax esters. However, it was shown after the inception of the present investigation that sterol esters, which are predominantly epidermal in origin (Freinkel and Kazuo, 1969), are present in appreciable percentages in prepubertal surface lipid and to a level of almost 15% in children aged 4-S (Ramasastry et al., 1970). In the present study the wax esters iu males showed 2 peaks. The prepuberta! peak is likely to be due to a significant sterol ester contribufion, whilst the peak in the 2(i-40 years age group may reflect maximal sebaceous activity and a true inerease in the synthesis of wax esters at this time. The lack of congelation between sebum excretion rate and wax ester content in females may be explained by changes in sterol esters masking real changes in surface lipid wax esters. There was no signifieant age or sex variation in the sum of the percentages of surfaee lipid triglycerides and free fatty acids, which suggests that this fraction forms a similar proportion of both sebum and epidermal lipid. There was a clear reciprocal relationshiji between the percentages of free fatty acids and triglycerides, and this observation is in agreement with that of earlier workers (Nicolaides and Wells, 1957; Downing et al.. 1969) and supports the hypothesis that free fatty

AGE AND SEX VARIATION IN 8KIN SURFACE LIPID COMPOSITION

339

acids are derived exclusively by hydrolysis of triglycerides. There was, however, considerable variation with age in the degree of hydrolysis of triglyterides. Skin surface fret' fatty acid content is maximal in botli sexes before tho onset of ]>uberty and falls to adult levels between tho ages of 11 and Io in both males and ietnales. Corresponding reeiprocal inereases in the percentages of surface lipid triglyeeridea are also found at this time. The surface lipid changes, however, were statistically significant only in males because of wide individual variation in the degree of hydrolysis of triglycerides in propubertal females. It is possible that the increased aebiim excretion rate found in the older age groups studied may explahi the changes in the degree of hydrolysis of triglycerides. An increased rate of sebum excretion may allow for less time for hydrolysis of triglycerides to free fatty acids by Iipases present in the skin mit-roflora or by sebaceous duet esterases. However, the deerease in the percentage of surface lipid free fatty acids in males aged Il-f5 preceded any significant increase in sebum excretion rate towards adult levels. Aa changes in skin surface lipid composition towards the adult ]>attern arc largely complete by the age of 9 years in American children (Kamasastry et aL, 1970), it could be that the degree of hydrolysis of triglycerides to free fatty acids is androgendependent and may be an earlier feature of puberty than an inerease in sebum excretion rate. Differing pathways of androgen metabolism within the skin may explain some of our findings. It is known that 17/y-hydroxysteroid dehydrogenase {M/i-KSD) activity is increased in the 20-30 age group and is found more frequently in male than in female skin biopsies ((.'alman et al., 1970). Although the significance of this finding is not clear, it may be associated with the post-pubertal enlargement of sebaceous glands and their functional maturity at this age (('alinan et al., 1970). This observation may be relevant also to the present findings of age and sex variation in both seb^^m excretion rate and skin surface lipid (omposition. While some of the age and sex changes in snrfaco lipid composition may he explained in terms of variafion in the sebum exerotlon rate, the alternative hypothesis that primary changes in sebaceous lipid composition may have a secwidary effect on the sebum excretion rate must also be considered. Jt has been claimed that squalene, and to a lesser extent free fatty acids, influences the fluidity of sebum {Lewis and Hayward, 1971). Either an increase in squalene content or a deerease in the percentage of free fatty aeids leads to an inerease in lipid fluidity. The surface lipid squalene content was maximal in both sexes when the sebum excretion rate was also maximal. In addition, the free fatty acid content of surface li])id fell at puberty in females wiion the sebum excretion rate had increased significantly compared to prepubertal levels. Thus it is possible that primary ehanges in sebum eomposition may influence the sebum excretion rate. However, in males aged 11-15 the deerease in free fatty acid content compared to prepuhertal levels was not associated with any significant increase in sebum excretion rate. Moreover, studies in patients with acne and subjects with a previous history of acne indieate that primary changes in surfaee lipid eomposition within the physiologieal range are unlikely to alter significantly the rate of sebum excretion in man (Cotterill ct al.. 1972). We conclude tiiat marked age and sex variations in both sebum excretion rate and skin surface lipid composition oeeur and that these changes may reflect altei'ation, not only in endocrine function, but also in peripheral response to hormonal stimulation during life.
26

340

J. A. COTTERILL, W. J. CUNLIFFE, B. WTLLIAMSON ANB L. BULUSU

We are grateful to the Wellcumo Trust, tin- Leeds Re(jional Hospital Board and the Board of Govoniors of Unitod Leeds Hos|)itals foi' financial .support. We also thank Mr K. A. Forster, Chief Technician, Miss J. Day, Technician, and Mr3 L, Lane for secretarial help. REFERENCES BuLLOUGH, W. S., LAURENCK, E . B . . IVERSON, O . H . & ELGIS, K . (HI67) The Vertebrate Epidermal Chalone. Nature, Lond., 214, 578. BLTRTON, J , L . , CLTNLIFFK. W . J . & SHUSTER, S . (1970) Circadian Rhythm in Seburn Excretion. Br. J. Derm..S2. 491.
CALMAN, K . C , MUIR, A . V., MII.NE. .]. A. & YOITNC. H . (1970) Survey of the Distribution of Steroid

Dehydrogenases in Sebaceou.s Glands of Human Skin. Br. J. Derm., 8 2 . 567. CoTTEKiLL, J. A,, Ci^NLiFFK, W, J . & WILLIAMSON, B, (1971a) A Semi quantitative Method for the Biochemical Analysi.s of Sebum. Br. J. Derm., 85, 35. CoTTKRiLL, J, A.. CTI;N-LIFFK. W . J . & WILLIAMSON, B . (1971b) Variation in Forehead Skin Surface Lipid Composition with Time and Order of Collection. (Presented at the First Meeting of the European Society for Dermatological Research, Holland, 1971). CoTTERii.1,, J . A., CT'NLIFFE, W . J., WiLLiAMsox, B, & Hni-VHiT, L. (1972) The Pathogencsis of Aene Vulgaris. (To be published.) CtrNLiFFE, W. J . & SHI-STRR. S. (1969a) The Rain of Sobiim Excretion in Man. Br.J. Derm.,Si. fi97. CiiNLiFFE, W. .1. & SHCHTER, S. (l9G9b) Pathogenesis of Acne. Lnncet. i. 685. Dow>;i>r(i, D. T., STBAr.ss, ,1. S. & Pot:Hl, P. E. (1969) Variability in the Chemical Composition of Human Skin Surface Lipitls. ./. invest. Derm., 53, U22. D. T. (1970) Lipolysis by Human Skin Surface Debris in Organic Solvents. ./. invest. ,..54, 395. fi, F. .J. (196li) Hormonal Control of Sebaceous Glands in Experimental Animals, Adv. Biol. Skin. 4, 200. fi, F. J., EBLINO, E . & SKINNKR, .1. (1969) The Influence of Pituitary Hormones on the Response of the Sebaceous Glands of thn Male Hat to Testosterone. ./. Endocr., 45, 245. FREINKBL, R . K . & KAZI'O, A. (19(i9) Esterification of Cholesterol. ./. invent. Derm., 52, 148. FREINKEL, R . K . & SHEX, Y . (1909) The Origin of Free Fatty Acids in Sebum I I Assay of the Lipases of the Cutaneous Bacteria and Effects of pH. J. inveM. Derm., 53, 422. GREENE, R . S., DOWNING, D . T . , P<)<H[. P. E. & STRAt^ss, J . S. (1970) Anatomical Variation in the Amount and Composition of Human Skin Surface Lipid. ./. inveM. Derm., 54, 240. LF:WIS, C . A . & HAYWARP, B . J. (1971) Human Skin Surfaee Lipid.s. Mod. Trends Derm., 4, 89. MAt DONALD, I. & CLARKE, G . (1970) Variations in the Lcv*^!.-^ u\' Cholesterol and Triglyceride in the Skin Surface Fat During the Menstrual Cycle. Br. /. Derm., 83, 473. NicoLAiDKS, N. & RoTHMAM, S. (1955) The Site of Sterol and Squalene Synthesis in the Human Skin. ./. invest. Derm,., 24, 125. NicoLAiDEs, N. & WELLS, G . C. (1957) On the Biogenesis of Free F a t t y Acids in Human Skin Surface Fat. J. invest. Derm., 29, 423. NicOLAiDEs, N., Fi', H. C. & R U E , G . K . (1968) The Skin Smfacc Lipids of Man Compai-ed with thoae of Eighteen Species of Animals. ./. invest. Derm., 5 1 , 83. NiKKARi, I. (1965) Composition anil Secretion of the Skin Surface Lipids of the R a t ; E f f e c t s ^ Dietary Lipids and Hormones. Scand. ./. din. lub. Invest.. 17, Suppl. 85, I. NiKKARi, I, '& VALAVAABA, M . (1970) The Influence of Age, Sex Hypophy-sectomy and Various Hormones on the Composition of the Skin Surface Lipids of the R a t . Br../. Derm.. 83, 459.
RAMASASTRY, P . . DOWNING, D . T . , POCUI, P. E. & STRAFSS, .). S. (1970) Chemical Composition of

Human Skin Surfaee Lipids from Birth to Puberty.

J. invest. Derm., 54, 139.

SANSONE. G . , DAVIDSON, W . , CIMMINOS, B . & REISNKR, R . M . (1971) Sebaceous Gland Lipogeneaia

Induced by Testosterone; Karly Metabolie Events. J. invest. Derm., 57, 144. STRAI'SS, J . S . & PocHi, P . E. (1961) The Quantitative Gravimetric Determination of Sebum Production. J. invest. Derm., 36, 293. STRAUSS, J . S., KLIGMAN, A. M. & POCHT, P . E. (1962) The Effect of Androgens and Estrogens on Sebaceous glands. J. invest. Derm.. 39, 139. STRAI'SS, J . S. & PocHi, P . E. (1963) The Human Sebaceous Gland; its Regulation by Steroidal Hormones and its Vne aw an E n d Organ for Assaying Androgenicity in vivo. Recent Prog. Horm. Res.. 19, 385. STRAUSS, J . S. & POCHI, P. E. (1965) Intracutaneous Injection of Sebum and Comedones. Archa Dewn., 92, 443.