Skin lipids: their biochemical uniqueness.

Two key words characterize the uniqueness of skin lipids: complexity and perversity. Each suggests a function. Complexity manifests itself in the large number and variety of both saturated and unsaturated fatty chains synthesized by human skin. Functionally, this allows each individual to have a distinct odor or chemical fingerprint. Perversity manifests itself when one compares the lipids synthesized by skin with those synthesized by internal tissues. For example, skin makes odd instead of only even chains, branched instead of only straight chains, free instead of only esterified acids, places double bonds in unusual positions in the fatty chains, extends chains to extreme lengths, and accumulates intermediates in the synthesis of a biologically valuable compound such as cholesterol. Functionally, these products may pose metabolic problems to potential pathogens and thus contribute to the survival of only compatible microorganisms.

Sebum composition: a comparison of thin-layer chromatography and infrared reflectance spectroscopy.

Thin-layer chromatography and infrared reflectance spectroscopy were compared as methods for measuring sebum composition. Solutions of varying known concentrations of free fatty acids and triglycerides were analyzed by both methods. Skin surface lipid samples were similarly analyzed. Infrared spectroscopy produced more variable data than thin-layer chromatography with photodensitometry. This was particularly true at low concentrations of free fatty acids. It is concluded that thin-layer chromatography with photodensitometry is a more accurate method for the measurement of sebum composition.

Possible genetic control of the proportions of branched-chain fatty acids in human sebaceous wax esters.

The composition of human sebum varies among individuals with respect to the proportions of various types of fatty acids in the ester lipid classes. Iso-branched fatty acids with even numbers of carbons (iso-even) are particularly variable. However, in 13 identical twin pairs, intrapair differences in the proportions of iso-even fatty acids in sebaceous wax esters were found to be very small, while interpair differences were as large as in the nontwin population. Intrapair differences in iso-even fatty acids were also quite small in 4 of 8 fraternal twin pairs. Heritability estimates suggested that proportions of iso-even fatty acids in human sebum are controlled, at least in part, by genotype.

Estimation of sebum production rates in man by measurement of the squalene content of skin biopsies.

Previous studies have shown that lipids are synthesized at a constant rate in sebaceous glands and secreted onto the skin surface an average of 8 days after synthesis. As a result, measurement of the sebum content of a skin specimen of known surface area should allow calculation of the sebum production rate which had been occurring in vivo. In the present study, such measurements have been made on scalp skin specimens from 14 men. The amount of sebum in 4-mm punch biopsies was calculated from the squalene content of the specimens, as measured by quantitative thin-layer chromatography of lipid extracts. The average sebum production rate for the group was found to be 1.45 mg/10 sq cm/3 hr.

Measurement of sebum secretion rates in young children.

Sustainable rates of lipid secretion were measured on the foreheads of 24 children, ages 6-8. The method of measurement involved absorption of skin surface lipid into bentonite clay and subsequent separation and measurement of the various lipid classes by quantitative thin-layer chromatography. In 9 of the children, sebum secretion appeared to be virtually nonexistent, judging by the low amounts of lipid recovered and the low percentages of wax esters and squalene, which are purely sebaceous lipid classes. An effect of age on sebum secretion rates was observed with median rates of wax ester secretion, being 7, 28, and 48 micrograms/10 cm2/3 h for 6-, 7-, and 8-year-olds, respectively. These values are well below those of most young adults. The relation between wax ester secretion rate and skin surface lipid composition on the forehead was investigated and found to conform to the formula: microgram wax esters/10 cm2/3 h = 28 X wax esters/(cholesterol + cholesterol esters), with a correlation coefficient (r) of 0.94.

Measurement of sebum output using a lipid absorbent tape.

A sebum absorbent tape is introduced as a reproducible and convenient method for estimation of sebaceous gland output. We have tested the reproducibility of this method by serial measurements of sebum excretion rates (SER) of 10 individuals over a 6-week period, and in addition we have correlated this method with the conventional hexane extraction technique. The sebum absorbent tapes gave consistent values for the SERs, and within subjects variation over the 6-week period was statistically nonsignificant. A coefficient of variation for the tapes was calculated as 16.25 +/- 6.78% based on these serial measurements. Furthermore, the amount of total lipid collected using this technique (n = 16) correlated well with the hexane extraction technique, r = 0.89. Free fatty acids (r = 0.87), triglycerides (r = 0.92), wax and cholesterol esters (r = 0.83), and squalene (r = 0.88) also showed a good correlation. Cholesterol occasionally suffered from incomplete separation on thin-layer chromatograms; however, a sample cleanup procedure was developed for tape extracts that removed interfering materials and allowed complete separation of all sebum components.

Variation in sebum fatty acid composition among adult humans.

Quartz capillary gas chromatography was used to analyze the wax ester fatty acids of 4 sebum samples collected at 2-week intervals from each of 10 adult human subjects. Marked differences in wax ester fatty acid composition between subjects were apparent. The greatest variation was present in the even-carbon-numbered iso-branched acids, which ranged from 1-22% of the monounsaturated acids and from 1-13% of the saturated acids. The anteiso chain structures formed 3-7.5% of the unsaturated acids and 3-13.5% of the saturated acids. Fatty acids bearing one or more methyl branches at other positions in the chain made up 12-22% of the saturated acids, but were not present in the unsaturated fatty acid fraction. This and other features of the composition of the unsaturated fatty acids indicate that the delta 6-desaturase that produces the monounsaturated fatty acids of human sebum requires a substrate having a straight chain of at least 12 carbon atoms extending from the carboxyl group. The differences in fatty acid composition between subjects and the constancy of composition for each of the subjects over the 2-month period indicate that the synthesis of each of the types of chain structure is under genetic control.

Skin surface lipid composition, acne, pubertal development, and urinary excretion of testosterone and 17-ketosteroids in children.

Fifty-two children, age 5-10, from acne-prone families, were studied for a period of 1 year to examine the interrelationship between sebum, acne, pubertal development, and urinary steroid excretion. In each of the subjects, 30 boys and 22 girls, the composition of forehead skin lipid was determined 4 times yearly by thin-layer chromatography, with measurement of triglycerides, diglycerides, free fatty acids, wax esters, squalene, cholesterol, and cholesterol esters. Twice yearly, examination was made of the presence or absence of acne, pubertal maturation and the 24-hour urinary excretion of testosterone as determined by radioimmunnoassay, and of total 17-ketosteroids, dehydroepiandrosterone, androsterone, and etiocholanolone, as determined by paper chromatography. The relative amount of sebaceous lipids was positively correlated with age of the subjects (wax esters p less than .001, squalene p less than .05), as was the triglyceride-diglyceride component (p less than .05). No significant correlation was seen with the fatty acids. Acne, primarily comedonal, occurred in 27/52 subjects (15 girls, 12 boys) and was associated with higher sebum values. One-half of the children with acne had no signs of pubertal development. A significantly positive correlation was observed between the relative amount of sebaceous lipid and the urinary excretion of 17-ketosteroids, androsterone, and etiocholanolone in both sexes, and of testosterone and dehydroepiandrosterone in boys. The development of acne in children is an early pubertal event, often evident before other signs of pubertal maturation, and it is associated with an increase in sebum and in the urinary excretion of androgenic steroids.

Proportions of various straight and branched fatty acid chain types in the sebaceous wax esters of young children.

The fatty acid composition of sebaceous wax esters was examined in 20 children, ages 6-8, using quartz capillary gas chromatography. The results were compared with a previously reported study of wax ester composition in 10 adults, in which the same analytical techniques were used. The children's wax esters showed more variability in the proportions of all fatty acid chain types than was found in adults. The most striking difference between the 2 age groups was in the proportions of iso-branched fatty acids with odd numbers of carbon atoms. These ranged from 1-26% of fatty acids in the children's wax esters but were not more than 3% of fatty acids in the adults' wax esters. Some of the variability in wax ester fatty acid composition in children seemed to be related to variation in rates of sebum production. The highest percentages of terminally branched (iso-even, iso-odd, and anteiso) and the lowest percentages of internally branched fatty acids were found in the children with the lowest rates of sebum secretion. However, even in children with similar rates of sebum secretion there were considerable differences in wax ester fatty acid composition, especially with respect to iso-even fatty acids. The results suggest that sebum fatty acid composition is influenced both by genetic factors and by limitations on the supply of precursors for terminally branched, but not internally branched, fatty acids.

Effect of aging on sebaceous gland activity and on the fatty acid composition of wax esters.

Using fused-silica capillary gas chromatography, we investigated sebum samples from 55 healthy individuals to discover the effects of aging on the sebaceous gland activity and on the fatty acid composition of wax exters. The sebaceous gland activity, which was expressed by the ratio of wax esters/[cholesterol + cholesterol esters] (WE/[C + CE]), showed a distinct change from infancy through maturity to senescence; the curve of the ratio made a peak in our subjects's 20s. Using the fatty acid analyses, we found an interesting relationship between C16:1 straight and C16:1 iso-branched chains, each of which occupied a large proportion in the fatty acids of wax esters; the former increased in proportion from infancy toward the 20s, with a correlation with aging (r = 0.788, p less than 0.01), and decreased thereafter until our subject's 50s (r = -0.611, p less than 0.01). In contrast, the proportion of the latter followed an entirely reversed course with advancing age. The percentages of C16:1 straight chain components were correlated positively with the WE/[C + CE] ratio (r = 0.642, p less than 0.01), while there was found to be a negative correlation between the proportion of C16:1 iso-branched chain components and the WE/[C + CE] ratio (r = -0.556, p less than 0.01). The results suggest that more active sebaceous glands in lipid production excrete lipids with a higher proportion of C16:1 straight chain fatty acid and a lower proportion of C16:1 iso-branched chain fatty acid. As well as the sebaceous gland activity, the fatty acid composition in sebum wax esters is affected by advancing age in Japanese.

Age-related changes in sebaceous wax ester secretion rates in men and women.

Sustainable rates of sebaceous wax ester secretion were measured on the foreheads of 109 men and 167 women, aged 15-97. Each measurement was made after first depleting the cutaneous sebum reservoir by overnight absorption of lipid into a layer of bentonite clay. Lipid was then absorbed for 3 h into fresh clay in which two 2-cm cloth disks were embedded. The absorbed lipid was extracted from the disks with ether and analyzed for wax esters by thin-layer chromatography. For both men and women there was a wide range of wax ester secretion rates at all ages. Rates were highest in the 15- to 35-year-olds and appeared to decline continuously throughout the adult age range. Values of log(wax esters) were better correlated with age than the untransformed values of wax ester secretion. The equations of best fit of log(wax esters) vs age suggested that sebum secretion declines about 23% per decade in men and 32% per decade in women.

The effect of marked inhibition of sebum production with 13cis-retinoic acid on skin surface lipid composition.

Sebum production and skin surface lipid composition have been measured during the oral treatment with 13cis-retinoic acid of a group of patients with severe cystic acne. Two hundred and thirty-eight paired samples have been analyzed and compared. A marked, drug-induced decrease in sebum production was accompanied by a decrease in the concentration of wax esters, a slight decrease in the squalene concentration and in increase in the cholesterol concentration in the skin surface lipids. These changes were consonant with changes in the relative contributions of sebum and epidermal lipid to the surface film, except that at very low levels of sebum production there might be preferential preservation of squalene synthesis.

Changes in the relative amounts of endogenous and exogenous fatty acids in sebaceous lipids during early adolescence.

Skin surface lipid samples were collected from the scalps of 40 males, aged 9-15, and the lipid class composition of each was analyzed by quantitative thin layer chromatography. The ratio of wax esters/[cholesterol + cholesterol esters] (WE/[CH + CE]) increased with age. The wax ester, cholesterol ester, triglyceride, and free fatty acid classes were isolated from each sample and the fatty acid compositions were determined by capillary gas chromatography of fatty acid methyl esters (FAME) prepared from each lipid class. The concentrations of most of the different types of fatty acids were found to be correlated with the WE/[CH + CE] ratio. Those straight chain fatty acids that are thought to be synthesized mainly within the sebaceous glands, such as 14:0, 14:1, 16:1, and 18:2 delta 5, 8 tended to increase with increasing WE/[CH + CE], while fatty acids which circulate in the blood, such as 18:0, 18:1, and 18:2 delta 9, 12, tended to decrease with increasing WE/[CH + CE]. For the majority of straight chain fatty acid types, the data could be fitted to the equation y = a + b/[x + 1], which can be derived from simple assumptions concerning the origins of the various sebum components. The FAME from the wax esters were separated into saturated and monounsaturated fractions and analyzed by capillary gas chromatography to determine the concentrations of the different types of branched chain FAME present. In the wax esters, straight chain fatty acids tended to increase with increasing WE/[CH + CE], while terminally branched (iso and anteiso) fatty acids tended to decrease. Other branched chain fatty acids increased up to a WE/[CH + CE] ratio of about 2 and then decreased at higher ratios.

In vitro effects of skin lipid extracts on skin bacteria in relation to age and acne changes.

Skin lipid extracts of 185 persons of different age were tested in vitro against single representatives of skin bacteria: Staphylococcus aureus, S. epidermidis, Propionibacterium acnes, P. granulosum and Corynebacterium sp. in order to find differences in their inhibitory effects. Percentages of the extracts inhibiting the first 4 bacteria were nearly the same (about 20%) while corynebacteria were inhibited by only 3 extracts. Extracts showing these properties were isolated mainly from adolescents with acne changes, young adults, about 20 yr old, and from children 8--10 yr old, but not from elderly people. The age-related differences in growth inhibition may be related to known differences in relative composition of the sebum constituents according to age. Forty-two extracts of acne patients and healthy adults and adolescents showed an inverse effect in vitro: they enhanced bacterial growth. Growth enhancement was demonstrated only for P. granulosum.

Sebum: analysis by infrared spectroscopy. II. The suppression of fatty acids by systemically administered antibiotics.

Various tetracyclines and erythromycins have been used rather empirically for the systemic treatment of acne vulgaris for more than a decade. It has been impossible to accurately evaluate clinically the numerous derivatives and to compare their effectiveness with other antibiotics such as clindamycin. As a substitute for this approach, this present study ranks these antibiotics, when given orally, as to their effectivensss in suppressing the fatty acids in sebum in normal volunteers. As a group, the tetracyclines were more effective than the erythromycins in decreasing the fatty acids, but clindamycin was significantly more potent than either.

The effect of 13-cis-retinoic acid on sebaceous glands.

The drug, 13-cis-retinoic acid, which has been demonstrated to have a marked effect on nodulocystic acne, probably has several mechanisms of action. This article summarizes the effects on the sebaceous glands, and the accompanying changes in cutaneous lipids that result from 13-cis-retinoic acid therapy. These changes in lipid composition support the concept that linoleate may be of importance in the pathogenesis of acne.

A modified photometric quantification of skin surface lipids sampled using the cigarette paper method.

In severe cases of acne conglobata the use of substances which considerably reduce the sebum excretion rate, such as 13-cis-retinoic acid, is reasonable. To monitor the effect of such preparation a noninvasive and accurate method for measuring skin surface lipids (SSLs) is required. A new modified photometric quantification of SSLs sampled using the cigarette paper method based on reaction with phosphovanilic reagent was introduced. Our overall results indicate that a 3-h SSLs assessment measured photometrically is as accurate as the gravimetric technique; it is reproducible in situations with low and high levels of SSLs.

The lipometre: an easy tool for rapid quantitation of skin surface lipids (SSL) in man.

The purpose of this study is to present a rapid, simple, and accurate method for quantitation of SSL in man. The technique which is derived from the works by Schaefer and Kuhn-Bussius, employs a series of clearn ground glass plates, and an apparatus, the Lipometre, for the determination of the variation of light transmission across the plates as a function of the fat deposited on them. Results on 32 adults, males and females, show that the first four iterative samplings, at constant pressure, on the same forehead area follow an identical physical law independently of whether the skin is fatty or dry. This law affords a precise definition of the Casual-Level, its quantification and its correlation with skin patterns. It is consequently possible, either as an investigative or a routine technique, to predict the total amount of SSL with good accuracy within 2 min. The SSL, for non-pathological skins, fall within the range 150 microgram/cm2 to 900 microgram/cm2 which is in good agreement with values quoted in the literature.

Effect of cyproterone acetate-ethinyl estradiol treatment on the proportions of linoleic and sebaleic acids in various skin surface lipid classes.

The effect of high-dose cyproterone acetate-ethinyl estradiol treatment on rates of sebum secretion and on the proportions of linoleic acid (18:2 delta 9,12) and sebaleic acid (18:2 delta 5,8) in the skin surface lipids of three female acne patients was examined. Changes in rates of sebum secretion were evaluated indirectly by measuring the ratio of wax esters/(cholesterol + cholesterol esters) in the subjects' skin surface lipid. In two of the subjects, this ratio indicated a reduction of sebum secretion rates to the childhood range. Concomitantly, there was an increase in linoleic acid and a decrease in sebaleic acid in all lipid classes. In the third subject, in whom there was only a small reduction in sebum secretion rate, the proportion of linoleic acid in the cholesterol esters more than doubled, but the changes in the other lipid classes were small or nonexistent. The results indicate that the proportions of linoleic acid and sebaleic acid in sebum are influenced by sebum secretion rates.

Continuous-flow system for determination of diffusion coefficients: use of a natural membrane.

A natural membrane was employed in an automated diffusion system. A mature male Mongolian gerbil sebaceous gland pad was excised and mounted into a suitable retainer so that the external surface was oriented toward the concentrated aqueous drug solution. Aqueous solutions of benzoic acid and the three commonly used parabens were studied. The gerbil sebaceous pad effectively prevented any diffusion of these drug solutions within 15 hr. Water by itself, however, was transported through the skin even against a pressure gradient. Although no apparent diffusion of these compounds occurred, a significant amount of drug was retained by the sebaceous pad. An expression for membrane-water partition coefficients could be calculated. Based upon thicknesses of natural and synthetic membranes, theoretical approximations of diffusion rates were found using lag time calculations.