Introduction
Cerumen is a complex mixture of products secreted by the sebaceous and ceruminal glands, together with squames of epithelium, dust and other foreign debris in the external auditory canal.Reference Hanger and Mulley1, Reference Okuda, Bingham, Stoney and Hawke2 Two types of cerumen are present – ‘wet’ and ‘dry’. These differ in their lipid composition and appearance. In the present study, only the ‘wet’ type of cerumen was examined.Reference Hawke3 The lipid portion of cerumen consists of squalene, cholesteryl esters, wax esters, triacylglycerols, fatty acids, cholesterol, ceramides and cholesterol sulphate.Reference Inaba, Chung, Kim, Choi and Kim4, Reference Bortz, Wertz and Downing5
Ceruminal lipids are of great importance, since they are known to execute various functions, including antimicrobial activity.Reference Chai and Chai6 Despite these potentially important functions, only limited data are available regarding changes in the ceruminal lipid profile with age, sex and menstrual stage.
Endocrine mechanisms are known to play a major role in sebaceous gland control, and the levels of various hormones vary with age, sex and menstrual stage.
The objective of the present study was to correlate quantitative changes in the lipid composition of cerumen with changes in age, sex and menstrual cycle stage, so that alterations in cerumen properties could be defined. This may be of importance in determining predisposing factors to disorders of the auditory canal, e.g. otitis. Cerumen samples were collected from subjects of varying age, sex and menstrual stage, and then analysed by high performance thin layer chromatography. The lipid composition of these cerumen samples was found to vary with age, sex and menstrual stage.
Materials and methods
Subjects
One hundred and fifty-two subjects (75 males and 77 females) aged four to 60 years were investigated. A total of 212 cerumen samples was analysed. None of the subjects was receiving any systemic or topical therapy at the time of the study.
The following age groups were investigated: group I, one to 10 years (15 males (mean age 7.0 years) and 18 females (mean age 4.61 years)); group II, 11–18 years (15 males (mean age 14.8 years) and 15 females (mean age 15.2 years)); group III, 19–40 years (30 males (mean age 30.1 years) and 90 females (mean age 29.2 years)); and group IV, 40 years and over (15 males (mean age 49.5 years) and 14 females (mean age 50.0 years)).
Cerumen samples were collected from women aged 19–40 years with established menstrual cycles. On the first day of menstruation, the ear canal was cleaned. The average time between germinative cell division in the sebaceous gland and excretion of oily material to the skin surface (i.e. cell transition time) is in the order of 13–14 days.Reference Downing and Strauss7 The transition time was taken into account when determining the cerumen sampling day. Cerumen samples were taken on day seven or eight of menstruation, representing the luteal phase of the preceding menstrual cycle. The ear canal was sampled 17–18 days and 27–28 days after cessation of menstruation, representing the menstrual and follicular phases of the preceeding menstrual cycle, respectively.
Collection of cerumen
The study was approved by the local ethics committee. Informed consent was obtained from each subject.
A sterile cotton bud was used to collect the samples. Cerumen was sampled from the ear canal and extracted using 1 ml of hexane/diethylether/acetic acid (80/20/7 by volume). All the cerumen samples taken were ‘wet’, with a yellowish, sticky appearance.
Separation and identification of cerumen lipids
High performance thin layer chromatography plates were used to separate and identify the cerumen lipids. A standard lipid mixture was used as a control, comprising l-phosphatidylcholine, l-α-phosphatidylethanolamine, cholesterol-3-sulphate, galactocerebrosides (type I and type II), N-palmitoyl-d-sphingosine, cholesterol, palmitic acid, triolein, cholesteryl oleate, beeswax and squalene. We applied 3–6 µg of the control standard and 10–60 µl of ceruminal extract to high performance thin layer chromatography plates.
Thin layer chromatography was conducted as described elsewhere. After separation of all lipids, the plates were sprayed with an aqueous solution of 10 per cent copper (II) sulphate in 8 per cent phosphoric acid and charred at 180°C.Reference Menlik, Hollmann, Erler, Verhoeven and Plewig8, Reference Röpke, Augustin and Gollnick9
Cerumen lipids were separated into the following classes: squalene, wax esters plus cholesteryl esters, triacylglycerol, and free fatty acids.
Quantification of lipids
All high performance thin layer chromatography plates were scanned using a Desaga CD 60 densitometer (Desaga, Heidelberg, Germany). We used the absorbance mode with a wavelength of 600 nm. Desaga CD 60 software was employed for quantitation. The percentage of each lipid class was calculated, measuring the peak height for each spot.
Statistical analysis
Statistical analysis was conducted using the Statistical Package for the Social Sciences for Windows version 11.0 software. All data were reported as means ± standard deviation (SD). Normality for continuous variables in groups was determined by the Shapiro–Wilks test. The variables did not show normal distribution (p < 0.05); therefore, the Mann–Whitney U test was used for comparison of variables between groups. A value of p < 0.05 was considered significant.
Results and analysis
Ceruminal lipid composition in male subjects
Ceruminal lipids were sampled from the ear canal, recovered by extraction into hexane/diethylether/acetic acid (80/20/7 by volume) and analysed. Figure 1 shows a typical densitometric scanning pattern for the charred high performance thin layer chromatogram of the standard lipid mixture. Figure 2 shows a densitometry chromatogram for the ceruminal extract. Table I shows the percentage of each lipid class detected in subjects' cerumen samples.
Fig. 1 Densitometric scanning pattern for high performance thin layer chromatogram of standard lipid mixture. Solvent development and visualisation procedures are described in the text. Peaks are as follows: 1 = l-phosphatidylcholine; 2 = l-α-phosphatidylethanolamine; 3 = cholesterol-3-sulphate; 4 & 5 = galactocerebrosides (types I & II, respectively); 6 = N-palmitoyl-d-sphingosine; 7 = cholesterol; 8 = palmitic acid; 9 = triolein; 10 = cholesteryl oleate + beeswax; 11 = squalene.
Fig. 2 Densitometric scanning pattern for high performance thin layer chromatogram of cerumen lipids. Peaks are as follows: 1 = phospholipids; 2 = cholesterol; 3 = free fatty acids; 4 = triacylglycerol; 5 = wax esters + cholesterol esters; 6 = squalene.
Table I Cerumen lipid composition by age and sex
Data are given as mean ± standard deviation. *p < 0.05, compared with 1–10 year group; †p < 0.05, compared with preceding age range; ‡p < 0.05, compared with same age range for males. Yrs = years; SQ = squalene; WE + CE = wax esters + cholesterol esters; TG = triacylglycerol; FFA = free fatty acids; C = cholesterol
Group I samples showed the lowest percentages of squalene and triacylglycerol and the highest percentages of wax esters plus cholesteryl esters, compared with other age groups. The percentage of squalene was highest in group II subjects, and declined slightly in older age groups. Groups III and IV showed the highest levels of free fatty acids. The ceruminal content of wax esters plus cholesteryl esters diminished with age; the most prominent decline was seen between groups I and II. The cholesterol content was observed to peak in group I, reduce in group II, and then level off in the older age groups. The squalene and triacylglycerol levels peaked at puberty, while levels of cholesterol and wax esters plus cholesteryl esters decreased with age.
Ceruminal lipid composition in female subjects
Age-related variations in ceruminal lipid composition exhibited a similar pattern in female subjects to that in males. Levels of squalene, triacylglycerol and free fatty acids were lowest in the youngest age group. Squalene and triacylglycerol levels peaked in groups II and III, respectively, then both levelled out in the older age groups. Levels of free fatty acids were highest in group III. In contrast, levels of wax esters plus cholesteryl esters peaked in group I, dropped significantly in group II, then levelled off in groups III and IV.
Comparison of ceruminal lipids by sex and age
Table I compares the ceruminal lipid composition of both sexes for the different age groups. In group III, squalene levels were lower in female samples compared with male subjects. In the same group, cholesterol levels were higher in female samples compared with male samples.
Effect of menstrual stage on ceruminal lipids
Menstrual stage-related variations in ceruminal lipid composition are shown in Table II. In cerumen specimens taken during the follicular stage (during which the influence of oestrogens is prominent), squalene and cholesterol levels were higher and free fatty acids levels were lower, compared with the other menstrual stages. In the luteal phase, free fatty acid levels rose significantly; however, cholesterol levels declined, compared with the follicular stage.
Table II Cerumen lipid composition by menstrual stage
Data are given as mean ± standard deviation. n = 30. *p < 0.05, compared with menstrual stage; †p < 0.05, compared with follicular stage. SQ = squalene; WE + CE = wax esters + cholesterol esters; TG = triacylglycerol; FFA = free fatty acids; C = cholesterol
We found no difference in ceruminal lipid composition, comparing the menstrual and luteal stages.
Discussion
The lipid composition of cerumen is important because of its effect on bactericidal activity and protective function within the external ear canal.Reference Hanger and Mulley1
The lipid fraction of cerumen has been reported to mainly contain squalene (6.4–20 per cent), triacylglycerols and free fatty acids (25.7–59.5 per cent), cholesterol (5.1–20.9 per cent), cholesterol esters (9.6 per cent), ceramides (18.6 per cent), cholesterol sulphate (2.0 per cent), and wax esters (9.3 per cent).Reference Okuda, Bingham, Stoney and Hawke2, Reference Inaba, Chung, Kim, Choi and Kim4, Reference Bortz, Wertz and Downing5, Reference Harvey10 These proportions generally agree with our results, except regarding wax esters plus cholesteryl esters. The evaluation of wax esters data is difficult, since the chromatography technique employed did not satisfactorily separate wax and sterol esters. Therefore, these lipid fractions were considered together (i.e. wax esters plus cholesteryl esters). Since the contribution of cholesterol ester to sebum lipids is very limited, peaks for wax esters plus cholesteryl esters could represent mostly wax esters.
In our study, the ceruminal content of wax esters plus cholesteryl esters ranged from 32 to 45 per cent, whereas Inaba et al. Reference Inaba, Chung, Kim, Choi and Kim4 reported it to be approximately 19 per cent. This discrepancy may be accounted for by differences in the type of cerumen analysed (dry or wet) and in the extraction and analysis methods employed.
The origins of the various surface lipid fractions differ. Squalene and wax esters are predominantly sebaceous in origin, whilst triglyceride is derived from both epidermis and sebaceous glands. Free fatty acids are derived from triacylglycerol by lipolysis, either by skin micro-organisms or by esterases present in the sebaceous ducts. Cholesterol esters and cholesterol are predominantly epidermal in origin.Reference Nicolaides11, Reference Stewart, Downing, Pochi and Strauss12
Changes in the composition of skin surface lipids have been used as an index of sebaceous gland activity. It has been suggested that differences in the wax esters content and the wax ester to sterol + sterol esters ratio of sebum may reflect differences in sebaceous gland activity. During periods of increased activity, the sebaceous glands would be expected to synthesise more wax esters.Reference Thody and Shuster13 Studies of age-related differences in sebaceous lipids have revealed that the wax esters percentage rises significantly as puberty commences.Reference Yamamato, Serzawa, Ito and Sato14–Reference Sansone-Bazzano, Cummings, Seeler and Reisner18 To our surprise, we failed to detect any significant increase in cerumen wax ester content, relative to other lipids, comparing prepubertal with pubertal subjects. It appears that the sebaceous glands in the ear canal respond differently to pubertal stimuli, compared with those in other regions. This may be attributed to regional variation in sebaceous lipid synthesis. At puberty, hormonal stimuli prompt synthesis of squalene, and to a lesser extent triacylglycerol, in cerumen. In contrast, other authors have indicated that puberty prompts a significant increase in sebum wax esters content, compared with triacylglycerol and squalene. It appears that the sebaceous glands of the ear canal respond to pubertal stimuli differently to those of other regions. Further work will be necessary to elucidate the reason for this discrepancy.
It is generally accepted that endocrine mechanisms play a major role in sebaceous gland control, and that there is evidence that hormones affect both proliferation and lipogenic activity of sebaceous cells. Androgens are the best known stimulators of the sebaceous glands. Oestrogens tend to inhibit sebaceous gland activity in humans.Reference Thody and Shuster13, Reference Shuster and Thody19 Pubertal alteration in the lipid composition of cerumen can largely be explained by concomitant changes in androgen levels; however, the possible role of other hormones, including growth hormone and thyroid hormone, cannot be ruled out.Reference Pochi, Strauss and Downing16
In group III, we found that the cerumen samples from female subjects contained less squalene and more cholesterol, compared with those from male subjects. On the other hand, Cipriani et al. Reference Cipriani, Taborelli, Gaddia, Melagrana and Rebora20 reported no sex differences in the lipid composition of cerumen, in contrast with the sex differences seen in sebum composition. However, these authors analysed only the cholesterol and triacylglycerol content of cerumen, and compared these two parameters for females and males aged between 25 and 42 years. They used colorimetric, enzymic methods to analyse triacylglycerol and cholesterol content, not high performance thin layer chromatography. Their method of cerumen extraction also differed from ours.
Chiang et al. Reference Chiang, Lowry and Senturia21 found that, among adults, there was little age-related variation in the lipid or protein content of cerumen, and no significant sex-related difference, in either children or adults. However, their study lacked detailed lipid analysis, since only total lipid and total protein fractions were analysed. On the other hand, Cotterill et al. found that the squalene content of skin surface lipids was maximal in men aged 26–40 years;Reference Cotterill, Cunliffe, Williamson and Bulusu22 this agrees with our findings.
In cerumen samples from both male and female subjects, the free fatty acids content peaked in the 19–40 year age range, at an average age of 30 years. It is well established that free fatty acids are not present in sebaceous glands but are formed from triacylglycerol by the lipolytic enzymes of skin bacteria.Reference Nicolaides11, Reference Stewart, Downing, Pochi and Strauss12 Leyden et al. Reference Leyden, McGinley, Mills and Kligman23 reported, that after puberty, microorganism counts on the human face start to rise, and peak after 20 years, young adults having significantly higher values than older adolescents. This agrees well with our data.
• Cerumen is a complex mixture of products secreted by the sebaceous and ceruminal glands, together with epithelial squames, dust and other foreign debris in the external auditory canal
• Age, sex and menstrual stage all affect the lipid composition of cerumen
• Ceruminal lipids are important as they act as a physical barrier, excluding water, harmful materials and micro-organisms
• Alterations in the cerumen lipid profile due to age, sex and menstrual stage may affect the predisposing factors of ear canal disorders
From our results, it can be seen that cerumen samples taken during the follicular phase of the menstrual cycle contained more squalene and cholesterol and less free fatty acids, compared with those taken during the menstrual phase. Since the major hormone in the follicular phase is oestrogen, this finding can be accounted for by the effect of oestrogens on cerumen composition. Oestrogens have been shown to suppress sebaceous gland activity in humans.Reference Thody and Shuster13 The inhibitory action of oestrogens on the sebaceous glands is thought to be at the level of lipid synthesis. However, Alves et al. Reference Alves, Thody, Fisher and Shuster24 found that ovariectomy decreased preputial sebaceous gland cell lipogenesis and also altered the pattern of lipid synthesis, producing a relative decrease in the proportion of polar lipids and an increase in the proportion of triacylglycerol. They concluded that oestrogen was able to stimulate preputial gland cell lipogenesis in female rats.
In contrast, no significant differences in cerumen lipid composition was detected between the luteal and menstrual phases. The major hormone of the luteal phase is progesterone, although oestrogens are also present to some extent. However, in the luteal phase, the effect of oestrogen on cerumen composition appears to lessen, or be neutralised by high progesterone levels. In the cerumen samples, the observed increase in squalene and cholesterol and decrease in free fatty acid levels in follicular phase samples, compared with menstrual phase samples, were almost reversed in the luteal phase, although the squalene level decrease was not statistically different. The effect of progesterone on the sebaceous glands, however, is controversial. Response by sebaleous glands to progesterone has been reported to be dependent on changes in the early endocrine environment.Reference Thody and Shuster13
Conclusion
It appears that age, sex and menstrual stage all affect the lipid composition of cerumen. Endocrine mechanisms appear to play an important role in determining the cerumen lipid profile. Other authors have reported that the increase in sebaceous gland activity at puberty is accompanied by a concomitant increase in the wax esters content of sebum, compared with other lipids. However, this is not the case for cerumen. In pubertal subjects, cerumen levels of squalene and triglyceride increased while those of wax esters plus cholesteryl esters and cholesterol decreased, compared with prepubertal subjects. The importance of ceruminal lipids lies in their ability to act as a physical barrier, excluding water, harmful materials and micro-organisms.Reference Huang, Fixter and Little25, Reference Pata, Ozturk, Akbas, Gorur, Unal and Ozcan26 The viscosity and hydrophobic properties of cerumen result mainly from its lipid composition. Since squalene is less polar than wax esters, it may afford better protection against wetting and micro-organisms and, as a result, against external auditory canal pathology. Alterations in cerumen lipid profile due to age, sex and menstrual stage may have an important effect on the predisposing factors of ear canal disorders.
Acknowledgements
This research was supported by the Scientific Research Projects Unit of İnönü University (Malatya, Turkey) (Project 2004 Güz-3).
