COMPOSITION OF HUMAN SEBUM
Sebum is synthesized in sebaceous glands, which are part of the pilosebaceous units of the skin (1). Sebaceous glands are epidermal appendages found in all regions of the skin, except the palmar and plantar surfaces; however, the greatest density of glands is found in the scalp and facial areas.
As sebocytes move from the basal layer at the periphery of the gland toward the lumen, they synthesize neutral lipids, which accumulate as lipid droplets, and eventually all of the carbon-based components of the cell are converted into lipid. The composition of sebum is species-specific. Human sebum, as it is synthesized in the gland (2), consists of squalene (15%), wax esters (25%), cholesterol esters (2%), triglycerides (57%), and cholesterol (1%). The small proportion of free cholesterol and cholesterol esters are thought to be derived from cholesterol in the basal sebo- cyte plasma membrane. Differentiating sebocytes do not express the enzymes of the cholesterol biosynthetic pathway beyond those leading to the production of squa- lene (3). This mixture of sebaceous lipids is liquid phase at skin tempertature.
As it flows out through the follicle and over the skin surface, the triglycerides undergo at least partial hydrolysis to liberate free fatty acids (4). The first investi- gation of the free fatty acids derived from human sebum was conducted by Weit- kamp et al. in 1947 (5). Using fractional distillation, fatty acids ranging from 7 through 22 carbons in length were detected. Shorter chains may have been present, but the 7-carbon entity was the shortest that could be detected. In addition, series of D6- and D9-monoenes were identified, with the D6 series dominating. The 16- and 18-carbon species were the most abundant. Small proportions of
1,2-diglycerides and 1,3-diglycerides are also produced through lipase action (4). The esterases responsible for sebaceous triglyceride hydrolysis are bacterial (6) and probably also of epidermal origin (7).
Of the fatty acids released from sebaceous triglycerides lauric acid (C12:0) and sapienic acid (C16:1D6) are the most potent antimicrobials (8; Drake DR and Wertz PW, unpublished observations). Sapienic acid is the single most abundant fatty acid in human sebum and is not found in abundance in any other known source, whereas lauric acid is a relatively minor sebaceous fatty acid. These two fatty acids both have potent antimicrobial properties, especially against gram-positive bacteria.
Frequently, alkanes are found in lipid samples collected from the human skin surface. Carbon dating has proven this material to be derived from petroleum (9), although it is not certain if it is simply surface contamination, as opposed to an internalized contaminant that is delivered to the skin surface through the sebaceous secretions.
In addition to the sebaceous lipids synthesized in the gland, hydrophobic materials from the circulation may partition into the sebaceous glands. This
includes the antioxidants vitamin E (10,11) and coenzyme Q10 (11). Sebum secretion appears to be the major route for delivery of these antioxidant vitamins to the skin surface. This may be of particular significance for defense against reactive oxygen species and protection of the linoleate containing acylceramides in the stratum corneum (12).
The major fatty acids in human sebum range in length from 12 carbons through 20 carbons, with the 16- and 18-carbon species predominating (13,14). In prepubertal children, some longer fatty chains are found (15). As noted, C16:1D6 is the most abundant fatty acid in human sebum. C18:1D8 is produced from C16:1D6 by chain extension. An unusual characteristic of human sebum is the presence of various methyl branched fatty acids (13,14). These include saturated iso- and anteisomethyl branched chains, but also various other internal and multi- methyl branched chains. The methyl branching pattern of saturated fatty acids varies among individuals, but is invariant with time for a given individual (14). This indicates genetic control.
Saturated and monounsaturated fatty acids predominate in the sebaceous esters with only small proportions of dienes being present (4). Among the wax ester fatty acids, the saturated-to-monounsaturated fatty acid ratio is approxi- mately 40 to 60; whereas in the cholesterol ester and triglyceride fractions this ratio is 65:35 and 70:30, respectively (13,16). The dienoic fatty acids include linoleic acid (C18:2 D9,12), derived from the diet, and an isomer thereof (C18:2 D5,8), which is synthesized in the gland (17). The proportion of C18:2 D9,12 relative to C18:2 D5,8 is decreased in acne. This is consistent with the suggestion that comedogenesis is initiated by a localized essential fatty acid deficiency (18).
MEASUREMENT OF SEBUM SECRETION Cigarette Paper Method
One of the most widely used methods for measurement of sebum secretion rates is the cigarette paper method of Strauss and Pochi (19). In this method, the forehead was first cleansed to remove surface lipid. A previously extracted cigarette paper was then placed against the skin of the forehead and held in place with an ace bandage. After a three-hour collection period, the cigarette paper was removed. Lipids were extracted from the paper using ethyl ether and quantitated either by weighing (19) or by quantitative thin-layer chromatographic analysis (4,20).
Subsequently, bentonite has been used to adsorb sebum on the forehead (21). After washing the forehead with soap and water and swabbing with an ethanol-soaked gauze pad, a thin layer of bentonite gel was applied to the forehead. In initial studies, two 1.8-mm diameter circular disks of Dacron mesh were pressed into the bentonite, and these were covered with additional bentonite. At three hours interval thereafter, the Dacron disks and adhering bentonite were replaced and sampling continued to 24 hours. The amount of sebum adsorbed per disk over three hours decreased steadily for about the first 12 hours, after which the rate of sebum secretion became constant. The excess sebum secreted during the initial 12 hours of collection was interpreted as a reflection of a follicular reservoir. Only after this reservoir was depleted was the sustainable sebum secretion rate
measurable. This sustainable secretion rate is equal to the rate of synthesis in the glands. In later studies, bentonite was applied to the forehead, and a rectangular piece of Dacron mesh large enough to cover most of the forehead was pressed into the gel and covered with additional bentonite. After seven hours, the Dacron mesh on the forehead was replaced, and after another seven hours to deplete the reservoir, the rectangular Dacron was replaced with two circular disks of Dacron for a final three-hour collection period, reflecting the sustainable rate of sebum secretion. The lipids were extracted from the bentonite into ethyl ether and ana- lyzed by quantitative thin-layer chromatography. In a variant on this method, the final collection period after depletion of the follicular reservoir was extended to nine hours, and the extracted sebum was quantitated gravimetrically.
More recently, Sebutape (Cuderm Corporation, Dallas, Texas, U.S.A.) has been introduced for assessment of pore patterns and sebum secretion (22,23). This adsor- bant polymeric tape is white but turns transparent at the points where sebum is adsorbed. This tape is used after clearing sebum from the surface, but not depletion of the follicular reservoir, although there is no fundamental reason why this could not be done. Typically, the Sebutape is removed from the forehead after three hours, although it has been suggested that one hour would be adequate (24), and placed on a black background. The pore pattern then appears as black dots on a white back- ground, and the total black area determined by image analysis is proportional to the amount of sebum secreted (25). One can qualitatively assign the pore pattern to one of five categories, referring to images provided by the manufacturer: prepubertal, pubertal, acne, mature, or senescent (26). It is also possible to extract lipids from the Sebutape and to analyze them by thin-layer chromatography (23).
In 1970, Schaefer and Kuhn-Bussius (27) demonstrated that sebum secretion could be measured by collecting it on a frosted glass plate and measuring the transpar- ency. As sebum is adsorbed on the rough surface, it spreads and fills the micro- scopic pockets within the glass. This smooths the surface and will result in less light scattering when the plate is illuminated with a beam of light. The relationship between the amount of adsorbed lipid and light transmission was quantitated and shown to be nonlinear. Subsequently, several instruments based on this principle have become commercially available. The first of this is the Lipometre introduced by L’Oreal (Aulnay’s Bois, France). More recently, the Sebumeter has been intro- duced by Courage & Khazaka (Koln, Germany). These instruments allow measure- ments of the amount of sebum on the skin surface or the sebum secretion rate, which can be made easily within a few minutes. However, calibration can be diffi- cult, and unless the sebaceous reservoir has been depleted, this will be the least accurate means for measuring sebum secretion rates.
Proposed Hybrid Method
The biggest obstacle to accurate measurement of sebum secretion rates is the need to deplete the sebaceous follicles. This can be overcome by 12 to 14 hours of adsorp- tion onto bentonite prior to measurement. Once the reservoir is depleted, the
Sebutape method is appealing for the actual sebum secretion measurement. The collection time could be shortened to one hour, and a digitized image of the Sebu- tape on its black backing should be captured immediately. Subsequently, this image can be analyzed to estimate the sebum secretion rate from the total black area. In addition, information on the number, the pattern and the activity of individual glands can be obtained. The Sebutape can be removed from the backing and the lipids can be extracted and examined by thin-layer chromatography in conjunction with photodensitometry. In this way, one could obtain two measures of the sebum secretion rate, the overall lipid class composition and information on the pore density and variation in the activity of individual glands.
At the time of birth, sebaceous glands are large and active, probably as a result of androgenic stimulation in utero (28). Shortly after birth, the sebaceous glands undergo atrophy and remain relatively small until the onset of puberty. The rate of sebum secretion in prepubertal children is generally low, but there is a measur- able increase in sebum secretion beginning at about an age of seven (29). This is thought to reflect increasing secretion of dehydroepiandrosterone and its sulfated form from the adrenal glands. At the onset of puberty in males, the testes produce increased levels of testosterone. In the skin, this is metabolized to dihydro- testosterone, which binds to a cytoplasmic receptor protein. This complex trans- locates to the cell nucleus and modulates gene expression (30). In females, the ovaries do produce testosterone, androstenedione, and dehydroepiandrosterone; however, adrenal hormones are the major circulating androgens in women (31). In general, sebum secretion rates reach a maximum in the mid-teen years and slowly decline thereafter as circulating androgen levels decline.
Hormonal control of sebaceous gland activity (Thiboutot) and the etiology of acne (Strauss) are discussed at length in later chapters of this book.
SEBUM SECRETION AND AGE
As noted earlier, sebaceous glands are active in utero under the influence of maternal hormones, and sebaceous lipids are major components of the vernix caseosa (13). Vernix caseosa also contains exfoliated corneocytes. In the later stages of development, lung surfactant is produced and can be detected in the amniotic fluid. This surfactant at least partially dislodges vernix caseosa, and this material is ingested (32). Whether this has significance beyond possible provision of nutrients is unknown.
The mean rate of sebaceous wax ester secretion in six-year old boys and girls was 5 mg/10 cm2/3 hr. This increased to about 25 mg/10 cm2/3 hr in seven-year olds and 50 mg/10 cm2/3 hr in eight-year olds (29). There is a major, age-dependent increase in the mean sebum secretion rate for both genders from ages 10 to 15 (33). After age 10, the mean sebum secretion rate at any given age was always greater for males. At age 15, the mean wax ester secretion rates were 0.75 mg/10 cm2/3 hr for boys and 0.55 mg/10 cm2/3 hr for girls (31,33). Sebum secretion rates appear to decline exponentially thereafter, reaching rates approximately half of secretion rates of 15-year olds at about age 50 (33). In individuals older than about 65 years, sebum secretion rates are in the same range as prepubertal children.
FIGURE 1 Relative proportions of ester-linked fatty acids from acylceramide of comedones compared with acylceramide from the skin surface of normal control subjects. Abbreviation: SC, stratum corneum. Source: From Ref. 37.
It has long been established that elevated rates of sebum secretion are associated with acne, and treatments that lower sebaceous gland activity are therapeutic (34). It should be noted that with the bentonite method for measurement of sebum secretion, the mean sebum secretion rates for nonacne control subjects, mild-to-moderate acne, and severe acne are significantly different with higher secretion rates, occurring with more severe disease (35). The role of sebum in come- dogenesis and initiation of acne and the available therapies are discussed in later chapters of this book. In general, any treatment that significantly reduces sebum secretion is therapeutic for acne.
INTERACTION OF SEBACEOUS LIPIDS WITH THE EPIDERMIS
It has been demonstrated that sebaceous lipids can increase the permeability of the skin (36). This was first demonstrated after application of human sebum to neonatal rat skin in vivo. The follicles have not penetrated to the surface of the neonatal rodent skin, so there is no endogenous sebum. It was found that the application of human sebum to this skin surface resulted in a two-fold increase in permeability. In addition, in accord with the localized essential fatty acid hypothesis, it has been shown that in comedones, sebaceous fatty acids can replace linoleate in the acylcer- amide (37). This is illustrated in Figure 1. More recently, it has been reported that barrier function is impaired in acne patients compared to normal controls (38).