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Influence of Aging on the Polar and Neutral Lipid Profiles in Human Meibomian Gland Secretions
Benjamin D. Sullivan, MS;
James E. Evans;
M. Reza Dana, MD, MSc, MPH;
David A. Sullivan, PhD
Arch Ophthalmol. 2006;124:1286-1292.
ABSTRACT
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Objective To determine whether aging is associated with significant alterations in the polar and neutral lipid profiles in human meibomian gland secretions.
Methods Meibomian gland secretions were collected from both eyes of younger and older men and women. Samples were processed for high-performance liquid chromatography or mass spectrometry and for the analysis of associated spectra of fragment ions. Subjects also underwent slitlamp evaluations of the eyelid.
Results Aging is associated with numerous significant alterations in the lipid profiles of human meibomian gland secretions. Analysis of polar and neutral lipid patterns identified ions that were significantly different in secretions of younger vs older men and women, as well as ions that varied significantly only between men and women. Correlation coefficients within, but not between, groups were high. Aging was accompanied by increased opacity of meibomian gland secretions and by eyelid and eyelid margin changes.
Conclusion Aging is associated with significant sex-related alterations in the polar and neutral lipid profiles of human meibomian gland secretions.
Clinical Relevance The observed changes may contribute to the age-related increase in the prevalence of dry eye syndromes.
INTRODUCTION
During aging, sebaceous glands undergo different structural and functional alterations. These include reduced cellular turnover, epithelial hyperplasia, acinar atrophy, and decreased secretion.1-5 In addition, qualitative changes occur in the neutral lipid composition of sebaceous gland secretions.6-7 These combined alterations, which have been attributed to a decline in endogenous androgen production,2-3 are believed to contribute to the development of dry skin in older persons.7-8
Similarly, the meibomian gland, which is a large sebaceous gland, undergoes anatomical and physiological modifications during aging. These changes include acinar cell atrophy and loss, abnormal features, hyperkeratinization of ductal epithelium, and reduced and viscous secretion.9-17 These age-related alterations, which have also been linked in part to androgen deficiency,18-19 are associated with the development of meibomian gland dysfunction.9, 16, 20 This condition, in turn, promotes hyperosmolarity and instability of the tear film and the condition of evaporative dry eye.10, 21-23
Investigators have speculated that the decreased volume and quality of meibomian gland secretions during aging may be due to alterations in the chemical composition of these secretions.15 Indeed, such compositional changes in meibomian gland secretions may also play a role in the cause of meibomian gland dysfunction and the associated tear film instability.24-25 However, whether significant changes in the lipid profile of human meibomian gland secretions actually occur during aging is unknown.
Given the findings in other sebaceous glands, we hypothesized that the polar and neutral lipid patterns of meibomian gland secretions undergo significant changes during aging. The objective of this investigation was to test our hypothesis.
METHODS
HUMAN SUBJECTS AND CLINICAL EVALUATION
Younger and older men and women (age range, 27-83 years) were recruited from the Boston, Mass, area for these studies. Subjects were excluded if they had active ocular infection, were not in good general health (eg, uncontrolled diabetes mellitus), or could not understand the study objective or procedure.
After giving informed written consent, subjects were asked to answer questions related to their medical histories and current medications and then underwent a slitlamp examination of the lower eyelid and eyelid margin by a corneal external disease specialist (M.R.D.) (Table 1). This examination was based on standard protocols for the identification of erythema, telangiectasia, collarettes, sleeve and scurf, keratinization, irregular posterior margins, and number of expressible meibomian glands.26-29 Eyelids were also evaluated for the presence of meibomian gland orifice metaplasia, a condition defined as an atypical growth and keratinization of duct epithelium,30 as well as for the quality of meibomian gland secretions, according to a reported classification system.27 Clinical data were compared using unpaired 2-tailed t and  2 tests.
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Table 1. Clinical Evaluation of the Eyelid and Eyelid Margin
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Meibomian gland secretions were then obtained from each eye by gently applying digital pressure against the lower eyelid and by collecting the expelled secretions with a chalazion curette. These secretions were obtained while directly visualizing the meibomian gland orifices with a biomicroscope, thereby reducing any possibility of contamination with other eyelid margin lipids. Samples were placed in glass tubes containing a 2:1 mixture of chloroform-methanol, and tubes were capped and stored at –70°C until experimental analysis.
These studies were approved by the human studies committees of the Brigham and Women's Hospital, Boston, and the Schepens Eye Research Institute. Investigations were conducted in accord with guidelines established by the Declaration of Helsinki.
BIOCHEMICAL AND ANALYTICAL PROCEDURES
Meibomian gland secretions were analyzed for molecular species composition in neutral lipids using high-performance liquid chromatography (HPLC) (Spectra-Physics Model 8700; Thermo Electron Corporation, San Jose, Calif) coupled with mass spectrometry (MS) (Finnigan 4500; Thermo Electron Corporation), as previously reported.31 In brief, samples were suspended by sonication in n-heptane for injection and then separated on a 10 cm x 2 mm silica column (Inertsil; Keystone Scientific Inc, Bellefonte, Pa) with a complex multistep gradient. The gradient combined mobile phases of isooctane-tetrahydrofuran, isopropanol-chloroform, and isopropanol-water and had a linear flow velocity of 0.4 mL/min. The vaporizer temperature in the moving-belt interface of the HPLC–mass spectrometer was set at 310°C. Mass spectrometry was conducted in positive-ion, chemical-ionization mode with ammonia reagent gas, and data were acquired using a Teknivent Vector/Two data system (Teknivent Corporation, Maryland Heights, Mo).
Polar lipids in meibomian gland secretions were analyzed according to reported methods.31 Samples were dissolved in 100 µL of methanol, and a 10-µL aliquot was injected into a stream of methanol flowing at 20 µL/min directly into the electrospray ion source of an LCQ quadrupole ion trap MS system (Thermo Finnigan LLC). The heated capillary was maintained at 200°C, and the source voltage was kept at 4.5 kV. Full positive-ion spectra were acquired from mass-charge (m/z) ratios 150 to 950 as the sum of 3 microscans, with the automatic gain control target at 5x107. Spectra were averaged over the elution profile, and background was subtracted using baseline spectra before and after the elution of each sample peak. In these studies, all solvents were Optima grade (Fisher Scientific International, Medford, Mass).
To evaluate and compare HPLC-MS and MS data, dynamic computer programs and algorithms written in Matlab 4.2c and 5.0 (The Mathworks Inc, Natick, Mass) were used, as previously described.31 Briefly, these programs permitted the exportation and translation of the entire mass spectra (ie, including approximately 15 000 data points per sample) of neutral (Teknivent Vector/Two machine format; m/z ratios, 100-900) and polar (ASCII text format; m/z ratios, 150-950) lipid data, as well as the conversion of continuously distributed m/z ratio data into discrete integer-valued m/z ratio units. Statistical analysis of the sum-normalized data (ie, the percentage composition) was performed using the unpaired 2-tailed t test. The HPLC-MS data were integrated from 0 to 16 minutes (ie, the interval of the complete HPLC elution) before comparison. At every m/z ratio, the ratio of the mean difference between groups and the P value from the t test ( /P) was calculated using a custom database structure in Matlab 5.0. Values greater than 2.0 (approximately equal to >0.02 and P<.01) were considered very significant.
Correlation coefficients of m/z ratio distributions within and between groups were also calculated using Matlab programs.31 Iterative calculations within groups produced [n(n–1)/2]–1 values for each internal comparison (n being the number of samples per group) and n x m values for comparisons between groups (n being the number of samples in the first group and m being the number of samples in the second group). The mean correlation coefficients are reported for each comparison.
RESULTS
To determine whether aging is associated with alterations in the polar and neutral lipid profiles of human meibomian gland secretions, secretions were obtained from the left and right lower eyelids of younger (mean ± SE age, 37.3 ± 1.7 years) and older (70.1 ± 1.1 years) men (9-12 men per group) and of younger (36.4 ± 2.4 years) and older (70.6 ± 3.1 years) women (7 women per group). Samples were then processed for the comparative analyses of HPLC-MS and MS mass spectra.
Our results demonstrate that aging is associated with numerous and significant alterations in the lipid profiles of human meibomian gland secretions (Table 2 and Table 3). Indeed, many of these changes appeared to be almost "all" or "none," indicating almost the complete presence or absence of specific ions in different age groups.
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Table 2. Influence of Age on the Polar Lipid Profile in Human Meibomian Gland Secretions*
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Table 3. Influence of Age on the Neutral Lipid Profile in Human Meibomian Gland Secretions*
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Analysis of polar lipid patterns identified major ions that were significantly greater in secretions of younger vs older men (eg, m/z ratio 873) and women (eg, m/z ratio 784) (Figure 1), as well as ions with /P values greater than 2.0 that were significantly different only between men (eg, m/z ratio 306) and women (eg, m/z ratio 761) (Figure 2). Similarly, significant differences existed in neutral lipid profiles between younger and older men and women (eg, m/z ratios 369 and 138) and predominantly between younger and older men (eg, m/z ratio 447) and younger and older women (eg, m/z ratio 111) (Figure 3). No significant differences were found using HPLC-MS between the neutral lipid profiles of younger men and women. Correlation coefficients within, but not between, groups were typically high (Table 4). Aging in men and women was also accompanied by a significant increase in eyelid erythema, telangiectasia, sleeve and scurf, keratinization, irregular posterior margins, meibomian gland orifice metaplasia, and meibomian gland secretion opacity (Table 5).
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Figure 1. Influence of age on the expression of polar lipids in human meibomian gland secretions. Secretions were obtained from the left and right lower eyelids of younger and older men (9-12 men per group) and women (7 women per group). The mass-charge (m/z) ratio is on the x-axis, and the significant difference in the percentage of polar lipid fatty acid fragmentation products is on the y-axis. The graphs focus on ion peaks at m/z ratios 873 and 784.
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Figure 2. Influence of age and sex on the levels of specific polar lipids in human meibomian gland secretions. Samples were obtained and analyzed as described in the legend to Figure 1. The graphs show ions with /P (the ratio of the mean difference between groups and the P value from the t test) values greater than 2.0 that were significantly different only between men (younger > older, mass-charge [m/z] ratios 306 and 366; older > younger, m/z ratios 359 and 425) and women (younger > older, m/z ratio 761).
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Figure 3. Influence of age on the expression of neutral lipids in human meibomian gland secretions. Samples were obtained and analyzed as described in the legend to Figure 1. The graphs show ions that were significantly different between younger and older men and women (mass-charge [m/z] ratios 369 and 138) and between younger and older men (m/z ratio 447) or younger and older women (m/z ratio 111).
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Table 4. Mean Correlation Coefficients (MCC) Within and Between the Lipid Mass Spectra of Meibomian Gland Secretions From Younger and Older Men and Women*
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Table 5. Influence of Age on the Human Eyelid and Meibomian Gland*
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COMMENT
Our results support our hypothesis that aging is associated with significant alterations in the lipid profiles of human meibomian gland secretions. Analysis of polar and neutral lipid patterns identified ions that were expressed at significantly different levels in secretions of younger vs older men and women, as well as ions that varied significantly only between men and women. Aging was also accompanied by an increase in the opacity of meibomian gland secretions and by several eyelid and eyelid margin changes. These meibomian gland findings are analogous to those of other sebaceous glands, which show significant alterations in structure, function, and secretion composition during aging.1-7
This influence of aging on the meibomian gland may be due, at least in part, to androgen deficiency. The meibomian gland, like other sebaceous glands, is an androgen target organ.32-34 Androgens regulate meibomian gland function, enhance the quality or quantity of lipids produced by this tissue, and promote the formation of the lipid layer of the tear film.32, 35-36 These hormone effects seem to be mediated through local androgen synthesis,37 androgen receptor activity within epithelial cell nuclei,38 and control of gene expression.39-40 Conversely, androgen deficiency, such as occurs in menopause, autoimmune disease, antiandrogen treatment, and complete androgen insensitivity syndrome,41-43 is associated with meibomian gland dysfunction, reduced quality of glandular secretions, and significant alterations in the neutral and polar lipid profiles of these secretions.31, 33-34,44-45 Our present findings were obtained by comparing persons aged 37 years and 70 years, and this period between the fourth and eighth decades of life coincides with a dramatic decline in the total androgen pool and with the development of androgen deficiency in both sexes.41
Androgen influence may also have contributed to the sex-related differences observed in the lipid profiles of meibomian gland secretions. Similar sex-associated differences were shown to exist in the m/z ratios of some neutral, but especially polar (eg, m/z ratios 873 and 874), lipids in human meibomian gland secretions, and these were linked to the presence of functional androgen receptors.31 The fact that sex influences meibomian gland function is not surprising. Sex-related differences are known to exist in the anatomy, physiology, gene expression, and secretion of the meibomian gland,18, 31, 44-47 and several of these variations may be associated with sex steroid, particularly androgen, action.18, 47
Our study showed that aging in men and women is accompanied by a significant increase in lower eyelid erythema, telangiectasia, keratinization, irregular posterior margins, orifice metaplasia, and opaque secretions. Other researchers have reported an increase in eyelid margin vascularity, keratinization, telangiectasia, and opacity of meibomian gland secretions during aging.13 These changes, which also occur during androgen deficiency,34, 45 seem to contribute to the generation of tear film instability and evaporative dry eye.10, 23-24
Our analyses identified a significant increase in the levels of a major neutral lipid ion (ie, m/z ratio 369) in the meibomian gland secretions of older men and women compared with younger individuals. The m/z ratio 369 ion is an HPLC-MS peak indicative of cholesterol esters,44 and the presence of these esters is more prevalent in patients with chronic blepharitis.48 This condition, in turn, is commonly associated with meibomian gland dysfunction,49-50 which seemed to be a characteristic of the older populations in this study. For comparison, the expression of the m/z ratio 369 ion is also apparently dependent on the polar lipid pattern of meibomian gland secretions,44 and these profiles changed significantly as a function of age.
During the course of these studies, we found that the expression of a number of ions appeared to be almost "all or none" between comparative samples. These differences could be random, but the probability is extraordinarily small. For example, if given 10 younger and 10 older samples, the chance of having just 3 complete separations over an MS profile is less than 1 in 1 million. The fact that we discovered multiple "all or none" peaks in a single group argues for a physiological basis and not a random event.
To test our hypotheses, we developed HPLC-MS and MS analytical techniques and wrote dynamic computer programs and algorithms to permit us to (1) export and translate the entire mass spectra (ie, including approximately 15 000 data points per sample) of neutral and polar lipid data; (2) convert continuously distributed m/z ratio data into discrete integer-valued m/z ratio units; and (3) perform statistical analyses on all sum-normalized data. However, a limitation in our analytical approach was that our evaluations focused on the m/z ratios of neutral and polar lipids but did not address their specific identities. We hope to apply MS/MS technology in the future, which might facilitate translation of these m/z ratio data into the identification of specific lipid species.
In summary, we discovered that aging is associated with significant alterations in the polar and neutral lipid profiles of human meibomian gland secretions. These changes, which may be due to different factors, may contribute to the age-related increase in the prevalence of tear film hyperosmolarity and dry eye syndromes.10, 51
AUTHOR INFORMATION
Correspondence: David A. Sullivan, PhD, Schepens Eye Research Institute, Harvard Medical School, 20 Staniford St, Boston, MA 02114 (sullivan{at}vision.eri.harvard.edu).
Submitted for Publication: September 13, 2005; final revision received February 4, 2006; accepted March 5, 2006.
Author Contributions: Dr Sullivan had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analyses.
Financial Disclosure: Drs Dana and Sullivan were consultants to Allergan Inc during this study.
Funding/Support: This study was supported by an unrestricted grant from Allergan, Inc, and by grant EY05612 from the National Institutes of Health.
Acknowledgment: We thank Rose M. Sullivan, RN, Jennifer M. Cermak, PhD, and Barbara Evans for their administrative, clinical, and technical assistance.
Author Affiliations: Schepens Eye Research Institute (Mr Sullivan and Drs Dana and Sullivan) and Department of Ophthalmology (Drs Dana and Sullivan), Harvard Medical School, Boston, Mass, and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester (Mr Evans).
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