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Figure 1.
Association of chronic blepharitis (CB), with and without keratoconjunctivitis sicca signs (CB-KCS), with levels of phosphatidylethanolamine and sphingomyelin polar lipids in meibomian secretions.

Association of chronic blepharitis (CB), with and without keratoconjunctivitis sicca signs (CB-KCS), with levels of phosphatidylethanolamine and sphingomyelin polar lipids in meibomian secretions.

Figure 2.
Association of chronic blepharitis (CB) with and without keratoconjunctivitis sicca signs (CB-KCS), with levels of phosphatidylethanolamine plus sphingomyelin polar lipids in meibomian secretions.

Association of chronic blepharitis (CB) with and without keratoconjunctivitis sicca signs (CB-KCS), with levels of phosphatidylethanolamine plus sphingomyelin polar lipids in meibomian secretions.

Association of Phosphatidylethanolamine (PE) and Sphingomyelin (SM) Levels With Chronic Blepharitis (CB) and Keratoconjunctivitis Sicca (KCS)*
Association of Phosphatidylethanolamine (PE) and Sphingomyelin (SM) Levels With Chronic Blepharitis (CB) and Keratoconjunctivitis Sicca (KCS)*
1.
McCulley  JPSciallis  GF Meibomian keratoconjunctivitis. Am J Ophthalmol. 1977;84788- 793
2.
Dougherty  JMMcCulley  JP Tear lysozyme measurements in chronic blepharitis. Ann Ophthalmol. 1985;1753- 57
3.
Bron  AJ Duke-Elder Lecture: prospects for the dry eye. Trans Ophthalmol Soc UK. 1985;104801- 826
4.
Fraunfelder  FTWright  PTripathi  RC Corneal mucus plaques. Am J Ophthalmol. 1977;83181- 197
5.
Tabbara  KFOstler  HBDaniels  TE  et al.  Sjögren's syndrome: a correlation between ocular findings and labial salivary gland history. Trans Am Acad Ophthalmol Otolaryngol. 1974;78OP467- OP478
6.
Gudmundsen  KJO'Donnell  BFPowell  FC Schirmer testing for dry eyes in patients with rosacea. J Am Acad Dermatol. 1992;26211- 214Article
7.
Mathers  WD Ocular evaporation in meibomian gland dysfunction and dry eye. Ophthalmology. 1993;100347- 351Article
8.
Mathers  WDDaley  TE Tear flow and evaporation in patients with and without dry eye. Ophthalmology. 1996;103664- 669Article
9.
McCulley  JPDougherty  JMDeneau  DG Classification of chronic blepharitis. Ophthalmology. 1982;891173- 1180Article
10.
Dougherty  JMMcCulley  JP Analysis of the free fatty acid component of meibomian secretions in chronic blepharitis. Invest Ophthalmol Vis Sci. 1986;2752- 56
11.
Dougherty  JMOsgood  JKMcCulley  JP The role of wax and sterol ester fatty acids in chronic blepharitis. Invest Ophthalmol Vis Sci. 1991;321932- 1937
12.
Shine  WEMcCulley  JP Role of wax ester fatty alcohols in chronic blepharitis. Invest Ophthalmol Vis Sci. 1993;343515- 3521
13.
Shine  WEMcCulley  JP Meibomian gland triglyceride fatty acid differences in chronic blepharitis patients. Cornea. 1996;15340- 346Article
14.
Dougherty  JMMcCulley  JPSilvany  REMeyer  DR The role of tetracycline in chronic blepharitis: inhibition of lipase production in staphylococci. Invest Ophthalmol Vis Sci. 1991;322970- 2975
15.
Bernhard  WLinck  MCreutzburg  H  et al.  High-performance liquid chromatographic analysis of phospholipids from different sources with combined fluorescence and ultraviolet detection. Anal Biochem. 1994;220172- 180Article
16.
Greiner  JVGlonek  TKorb  DRBooth  RLeahy  CD Phospholipids in meibomian gland secretion. Ophthalmic Res. 1996;2844- 49Article
17.
Nicolaides  NSantos  ECSmith  REJester  JV Meibomian gland dysfunction, III: meibomian gland lipids. Invest Ophthalmol Vis Sci. 1989;30946- 951
18.
Shine  WEMcCulley  JP The role of cholesterol in chronic blepharitis. Invest Ophthalmol Vis Sci. 1991;322272- 2280
19.
McCulley  JPShine  WE A compositional based model for the tear film lipid layer. Trans Am Ophthalmol Soc. 1997;9579- 93
20.
Holly  FJ Formation and rupture of the tear film. Exp Eye Res. 1973;15515- 525Article
21.
Wang  ZHall  SBNotter  RH Dynamic surface activity of films of lung surfactant phospholipids, hydrophobic proteins, and neutral lipids. J Lipid Res. 1995;361283- 1293
22.
Greiner  JVGlonek  TKorb  DRLeahy  CD Meibomian gland phospholipids. Curr Eye Res. 1996;15371- 375Article
23.
Dougherty  JMMcCulley  JMeyer  DR Keratoconjunctivitis sicca and tear lysozyme in chronic blepharitis. Holly  FJed.The Preocular Tear Film Lubbock, Tex Dry Eye Institute1984;176- 181
24.
Dougherty  JMMcCulley  JP Tear lysozyme measurements in chronic blepharitis. Ann Ophthalmol. 1985;1753- 57
25.
Dougherty  JMMcCulley  JP Bacterial lipases and chronic blepharitis. Invest Ophthalmol Vis Sci. 1986;27486- 491
26.
Bowman  RWDougherty  JMMcCulley  JP Chronic blepharitis and dry eyes. Int Ophthalmol Clin. 1987;2727- 35Article
27.
Fennema  OKester  JJ Resistance of lipid films to transmission of water vapor and oxygen. Adv Exp Med Biol. 1991;302703- 719
28.
Martin-Polo  MVoilley  ABlond  GColas  BMesnier  MFloquet  N Hydrophobic films and their efficiency against moisture transfer, 2: influence of the physical state. J Agric Food Chem. 1992;40413- 418Article
29.
Lee  Y-CZheng  YoTaraschi  TFJanes  N Hydrophobic alkyl headgroups strongly promote membrane curvature and violate the headgroup volume correlation due to "headgroup" insertion. Biochemistry. 1996;353677- 3684Article
30.
Seimiya  TAshida  MHayashi  MMuramatsu  THara  I Hydrogen bond among the ionic groups of ampholytic phospholipid. Chem Phys Lipids. 1978;2169- 76Article
31.
Seimiya  TOhki  S Ionic structure of phospholipid membranes, and binding of calcium ions. Biochim Biophys Acta. 1973;298546- 561Article
32.
Boggs  JM Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function. Biochim Biophys Acta. 1987;906353- 404Article
33.
McCulley  JPDougherty  JM Blepharitis associated with acne rosacea and seborrheic dermatitis. Int Ophthalmol Clin. 1985;25159- 172Article
34.
Shine  WEMcCulley  JP Significant differences in polar lipids associated with chronic blepharitis. Invest Ophthalmol Vis Sci. 1997;385156ARVO Abstract.
35.
Zengin  NTol  HGunduz  KOkudan  SBalevi  SEndogru  H Meibomian gland dysfunction and tear film abnormalities in rosacea. Cornea. 1995;14144- 146Article
36.
Asherson  RAFei  H-MStaub  HLKhamashta  MAHughes  GRVFox  RI Antiphospholipid antibodies and HLA associations in primary Sjögren's syndrome. Ann Rheum Dis. 1992;51495- 498Article
37.
Fullard  RJTucker  DL Changes in human tear protein levels with progressively increasing stimulus. Invest Ophthalmol Vis Sci. 1991;322290- 2301
38.
Keizenga  AStolwijk  TRvan Agtmaal  EJvan Haeringen  NJKijlstra  A Detection of secretory IgM in tears of IgA deficient individuals. Curr Eye Res. 1990;9997- 1005Article
39.
Harmer  IJLoizou  SThompson  KMSo  AKLWalport  MJMackworth-Young  C A human monoclonal antiphospholipid antibody that is representative of serum antibodies and is germline encoded. Arthritis Rheum. 1995;381068- 1076Article
Clinical Sciences
July 1998

Keratoconjunctivitis Sicca Associated With Meibomian Secretion Polar Lipid Abnormality

Author Affiliations

From the Department of Ophthalmology, The University of Texas Southwestern Medical Center at Dallas.

Arch Ophthalmol. 1998;116(7):849-852. doi:10.1001/archopht.116.7.849
Abstract

Objective  To determine whether an association between keratoconjunctivitis sicca (KCS) and meibomian gland lipids exists in patients with chronic blepharitis.

Methods  Meibomian gland lipids were collected from normal patients and those with chronic blepharitis. Some of the chronic blepharitis patients had an ocular surface abnormality with apparent aqueous deficiency similar to KCS. Lipids were separated by thin-layer chromatography and polar lipids were further separated by high-pressure liquid chromatography with detection by UV absorbance. Lipids were identified by retention time with comparison with standards and by gas chromatography and mass spectroscopy.

Results  A strong association between specific lipids and KCS signs was observed only with the polar lipids. Low levels of 2 phospholipids, identified as phosphatidylethanolamine and sphingomyelin, were significantly (P<.05) associated with ocular surface abnormalities that were consistent with KCS.

Conclusions  Evaporative KCS syndrome (rather than tear insufficiency) in many individuals may be the result of polar lipid abnormalities. We believe that the 2 associated phospholipids identified in the patients with chronic blepharitis act as important structural components in the polar phase of the tear film lipid layer. We suggest that a deficiency in these lipids results in a poorly structured polar phase that in turn affects the nonpolar phase. Ultimately water transmission through the tear film lipid layer increases, thus resulting in evaporative KCS. These results should aid in development of tear film substitutes directed toward specific abnormalities.

CHRONIC BLEPHARITIS (CB) is an ocular disorder commonly seen by ophthalmologists. Among the many ocular symptoms described by patients are foreign body sensation, itching, and dryness. We have observed that CB often has an associated ocular surface abnormality similar to that seen in keratoconjunctivitis sicca (KCS), but an understanding of how it was related to abnormal meibomian gland secretions (meibum) was lacking.1 In earlier studies we observed that 45% of patients with CB (n=21), evenly distributed as to sex, had an associated KCS2; the patients with KCS were also distinguished by significantly (P<.01) lower tear lysozyme levels than other patients with CB. In a classic sense, KCS is defined by aqueous insufficiency as determined by the Schirmer test, as well as other tests.3 However, sicca syndromes have been associated not only with CB,4 but also with Sjögren's syndrome5 and ocular rosacea.6 More recently it has been determined that signs of KCS associated with CB can result not only from low tear production but also from insufficient secretion from the meibomian gland.7 Subsequent research determined that high rates of tear film evaporation were correlated with this KCS condition.8 Thus, KCS conditions can result not only from insufficient production of tears but also from high rates of tear film evaporation.

Our investigations of CB have included numerous microbial and lipid assays. Microbial analysis included extraocular and meibomian gland secretions9; lipid assays included the nonpolar lipids in meibomian gland secretions1013 and the effects of tetracycline on ocular flora and lipids.14 In these investigations, we have observed associations with a picture of KCS often without a clear mechanism being apparent. Herein we report an association of these KCS-like ocular surface abnormalities with polar lipid changes in meibum that may represent the mechanism behind this association. These findings also provide insight into the fact that the lipid layer is most likely highly structured with the thin polar lipid layer serving as a surfactant similar to that of the glycocalyx at the epithelial aqueous-mucin tear interface but with a reverse polarity, ie, converting a hydrophilic environment to one of hydrophobicity.

PATIENTS AND METHODS

Informed consent was obtained from all patients and the study was conducted according to the tenets of the Declaration of Helsinki. Patients who had any form of CB in our classification system (thus all types were included) for at least 6 months were carefully evaluated.1,9 The patient population was 56% male (n=61); of this male population, CB-KCS was present in 39% (n=24) and CB-KCS was present in 50% (n=23) of the female population. If the patients with a staphylococcal infection were omitted, the occurrence of CB-KCS in the female population was 38% (n=11) and 41% (n=23) in the male population. In a previous study, we published2 this patient population's association with decreased levels of production of tear lysozyme in the CB-KCS patients as compared with the CB patients. Of these patients in the present study(n=19), 7 had a clinical picture consistent with KCS that persisted after the CB had been controlled and treatment with ocular medications discontinued, ie, interpalpebral epithelial erosions with staining of conjunctiva and cornea with a vital stain (rose bengal), and decreased tear meniscus on slitlamp examination. None of these patients had a general meibum deficiency. Relatedly, it has been observed that lacrimal gland function is decreased in patients with KCS and increased evaporation.8

Meibomian gland secretions were collected as follows. After instillation of 1 drop of lidocaine onto the ocular surface, a lid conformer was placed into either the inferior or superior cul-de-sac. A sterile cotton swab was then passed over the lid margin to remove excessive debris and tears. The tarsal plate containing the meibomian glands was then squeezed between the swab and conformer. The secretions were harvested with a sterile platinum spatula. Samples were washed off the spatula with chloroform, collected in glass tubes, and evaporated to dryness with nitrogen. Tubes were then sealed (under nitrogen) and stored at −70°C.

Lipid classes were first separated by thin-layer chromatography as previously described.10 Samples were dissolved in chloroform and spotted on silica gel H plates, previously activated at 120°C. The plates were developed in hexane–diethyl ether–acetic acid (75:25:1), the region of origin containing the polar lipids was scraped off, and then it was placed in nitrogen-flushed screw-capped tubes.

The polar lipids were further separated by high-pressure liquid chromatography. The high-pressure liquid chromatography system (Waters Corporation, Milford, Mass)) consisted of a universal liquid chromatograph injector (model U6K), automated gradient controller (model 680), high-pressure liquid chromatography pump (model 510), tunable absorbance detector (No. 484) set at a wavelength of 220 nm, and data module (model 740) to collect data. The mobile phase was a modification of a previously reported method15 and consisted of 73% acetonitrile, 25% methanol, 1.5% water, and 2.25×10-4% methylphosphonic acid (Aldrich, St Louis, Mo); pH was adjusted to 6.3 with ammonium hydroxide. The sample was dissolved in chloroform, evaporated to 4 to 6 µL, injected into the Waters system, and the polar lipids were separated on an aminopropyl silica column (Nucleosil-NH2, 5 µm, 250×4.6 mm (Sigma-Aldrich, St Louis) with an aminopropyl guard column. Column fractions were collected in screw-capped vials and evaporated under nitrogen at room temperature to reduce the volume.

Column fractions were transmethylated under nitrogen as follows. After transferring the samples to small screw-capped reaction vials (Pierce Reacti-Vial, Rockford, Ill) and adding sodium methoxide in methanol (Supelco, Bellefonte, Pa), they were heated in a heating block at 85°C for 15 minutes to transesterify the glycerolipids. The methyl esters were removed with heptane. After adding more sodium methoxide in methanol, the samples were heated for 18 hours at 85°C to transmethylate the sphingolipids.

Methyl esters were analyzed by a chemical ionization–gas chromatography–mass spectrometry (CI-GC-MS) system. This system (Hewlett Packard, Palo Alto, Calif) consisted of a 5890 series II GC and a 5971 mass selective detector with chemical ionization option. Samples were separated on a column (J & W Scientific 17HT, J & W Scientific, Folsom, Calif) (30-m, 0.25-mm inner diameter, 0.15-µm film thickness) with helium carrier gas and methane as the chemical ionization gas.

Lipids were identified using these 2 methods. Thus individual polar lipids were identified by retention time and comparison with lipid standards (sources: Sigma Chemical Co, St Louis; Avanti Polar Lipids, Alabaster, Ala; and Matreya, Pleasant Gap, Pa). Differential transmethylation and GC-MS identified the fatty acids and type of lipid (glycerolipid or sphingolipid). Data were analyzed statistically (t test) using the Statistica program (StatSoft, Tulsa, Okla).

RESULTS

Meibomian lipids are quite varied and more complex than previously understood.1618 In our investigation of CB, we find that meibum polar lipids of normal individuals were not notably different from those of patients with CB without a KCS-like picture, composition of which is given in the Table 1. The most striking observation was that in patients with CB with a KCS-like picture; phosphatidylethanolamine (PE) and sphingomyelin (SM) were significantly lower (P<.05) than in the patients with CB without associated KCS (Table 1, Figure 1). It was also observed that the significance (P<.02) of low PE was much more important than low SM in male patients (Table 1). When the sum of PE and SM was compared for all patients, the sum for the CB-KCS group was significantly lower (P<.005) than that for the CB group (Figure 2).

COMMENT

We have presented elsewhere19 an analysis of the various lipid classes of the lipid layer of the tear film and proposed that the lipid layer is highly structured. The polar lipid layer may be only 1 to 3 molecules thick20 but serves a very critical function, ie, that of a surfactant creating a transition from a hydrophilic aqueous-mucin phase to a hydrophobic nonpolar lipid phase. Phosphatidylethanolamine and SM are most likely to be the key molecules in establishing the integrity and function of the polar layer. These 2 phospholipids are prominent in lung surfactant phospholipids and are much more effective than phosphatidylcholine in maintaining in vitro lipid layer integrity and in lowering surface tension (surfactant properties).21 These polar lipids have also been observed to be dominant in rabbit meibomian glands.22

Although evidence has accumulated suggesting that meibomian disease and KCS syndrome may be related,1,6,9,2326 this is the first evidence that specific lipids could be involved. Low levels of both PE and SM are significantly related to an ocular surface abnormality similar to KCS (Table 1), but PE appears to be more important, at least in men. Although we believe that it is the nonpolar lipid phase of the tear film lipid layer that ultimately determines the tear film evaporation rate, the importance of the supporting structure of the polar lipid phase should not be ignored; as suggested by model systems27,28 both are important. We believe that it is both the structural and water transmission (activity) characteristics of the polar lipid phase that are compromised by low levels of PE and SM. The results presented in Figure 2 suggest that when PE and SM are at low levels, KCS is much more likely to occur.

The functionality of PE and SM in the polar lipid phase can be altered by associated conditions. The uniqueness of PE and its role in promoting a functional polar lipid phase can be understood in terms of the structure of PE. Thus, because of the molecular shape of the PE molecule, it has been proposed that the presence of PE reduces curvature stress,29 as would occur at the tear film surface. Also because of its molecular structure, and unlike phosphatidylcholine and SM, the amino (NH3+) group of PE which lacks the trimethylation (N[CH3]3+) of the choline group in phosphatidylcholine and SM is not stearically hindered. Therefore, PE is the only lipid present in meibum that contains an NH3+ group and thus can form ionic bonds and multiple hydrogen bonds (eg, to other lipids in the polar lipid phase).30 Finally, the amino group of PE (with a dissociation constant of about a pH of 7.5 in a membrane environment) can readily lose hydrogen-bonding ability at higher pHs, but especially when calcium levels are high.30,31 These characteristics of PE enhance both its structure-forming and surfactant properties, but under abnormal conditions these properties can be diminished. Finally, the hydroxyl groups present in SM but not phosphatidylcholine also increase its hydrogen-bonding ability and therefore its structure-forming properties.32 On the basis of these reports and our results, we believe that the functional polar lipid layer of the tear film can be compromised by abnormal conditions such as low levels of key polar lipids such as PE and SM.

Our results in no way suggest that all KCS conditions, whether due to high rates of aqueous evaporation, aqueous hyposecretion, or other lipid or mucin abnormalities, are the result of low levels of these 2 phospholipids. For example, we have reported an association of one type of CB, ie, rosacea, with meibomian keratoconjunctivitis33 and apparent nonpolar and polar lipid abnormalities34; more recently, there has been another report of an abnormal tear film associated with ocular rosacea.35 As a point of future interest, we would also like to point out that in Sjögren's syndrome, with a sparsity of aqueous production, the presence of serum antiphospholipid antibodies (primarily IgA) are a commonly associated condition.36 In general high levels of IgA and lower levels of IgM are found in human tears37; in individuals with apparent IgA deficiency, IgM levels may be elevated.38 A recent study has determined that serum IgM but not IgG binds significantly to both PE and SM.39 Further studies will clarify the relevance of these points.

We, therefore, propose that in some KCS conditions, hyperevaporation of aqueous tears may result from low levels of PE and SM in meibum (as in the present investigation) and/or loss of PE and SM from the tear film because of antiphospholipid antibodies, lipases, or even reactive aldehydes. The resulting clinical picture would be that of a sicca syndrome (KCS) and would be caused by increased rates of aqueous evaporation. With these insights it may be possible to devise a tear replacement to correct the defect in many patients with aqueous deficiency and especially those with an evaporative KCS condition.

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Article Information

Accepted for publication May 1, 1998.

Supported in part by an unrestricted grant from Research to Prevent Blindness, Inc, New York, NY, and by grant EYO3650 from the National Institutes of Health, Bethesda, Md.

Corresponding author: Ward Shine, PhD, Department of Ophthalmology, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75235-9057.

References
1.
McCulley  JPSciallis  GF Meibomian keratoconjunctivitis. Am J Ophthalmol. 1977;84788- 793
2.
Dougherty  JMMcCulley  JP Tear lysozyme measurements in chronic blepharitis. Ann Ophthalmol. 1985;1753- 57
3.
Bron  AJ Duke-Elder Lecture: prospects for the dry eye. Trans Ophthalmol Soc UK. 1985;104801- 826
4.
Fraunfelder  FTWright  PTripathi  RC Corneal mucus plaques. Am J Ophthalmol. 1977;83181- 197
5.
Tabbara  KFOstler  HBDaniels  TE  et al.  Sjögren's syndrome: a correlation between ocular findings and labial salivary gland history. Trans Am Acad Ophthalmol Otolaryngol. 1974;78OP467- OP478
6.
Gudmundsen  KJO'Donnell  BFPowell  FC Schirmer testing for dry eyes in patients with rosacea. J Am Acad Dermatol. 1992;26211- 214Article
7.
Mathers  WD Ocular evaporation in meibomian gland dysfunction and dry eye. Ophthalmology. 1993;100347- 351Article
8.
Mathers  WDDaley  TE Tear flow and evaporation in patients with and without dry eye. Ophthalmology. 1996;103664- 669Article
9.
McCulley  JPDougherty  JMDeneau  DG Classification of chronic blepharitis. Ophthalmology. 1982;891173- 1180Article
10.
Dougherty  JMMcCulley  JP Analysis of the free fatty acid component of meibomian secretions in chronic blepharitis. Invest Ophthalmol Vis Sci. 1986;2752- 56
11.
Dougherty  JMOsgood  JKMcCulley  JP The role of wax and sterol ester fatty acids in chronic blepharitis. Invest Ophthalmol Vis Sci. 1991;321932- 1937
12.
Shine  WEMcCulley  JP Role of wax ester fatty alcohols in chronic blepharitis. Invest Ophthalmol Vis Sci. 1993;343515- 3521
13.
Shine  WEMcCulley  JP Meibomian gland triglyceride fatty acid differences in chronic blepharitis patients. Cornea. 1996;15340- 346Article
14.
Dougherty  JMMcCulley  JPSilvany  REMeyer  DR The role of tetracycline in chronic blepharitis: inhibition of lipase production in staphylococci. Invest Ophthalmol Vis Sci. 1991;322970- 2975
15.
Bernhard  WLinck  MCreutzburg  H  et al.  High-performance liquid chromatographic analysis of phospholipids from different sources with combined fluorescence and ultraviolet detection. Anal Biochem. 1994;220172- 180Article
16.
Greiner  JVGlonek  TKorb  DRBooth  RLeahy  CD Phospholipids in meibomian gland secretion. Ophthalmic Res. 1996;2844- 49Article
17.
Nicolaides  NSantos  ECSmith  REJester  JV Meibomian gland dysfunction, III: meibomian gland lipids. Invest Ophthalmol Vis Sci. 1989;30946- 951
18.
Shine  WEMcCulley  JP The role of cholesterol in chronic blepharitis. Invest Ophthalmol Vis Sci. 1991;322272- 2280
19.
McCulley  JPShine  WE A compositional based model for the tear film lipid layer. Trans Am Ophthalmol Soc. 1997;9579- 93
20.
Holly  FJ Formation and rupture of the tear film. Exp Eye Res. 1973;15515- 525Article
21.
Wang  ZHall  SBNotter  RH Dynamic surface activity of films of lung surfactant phospholipids, hydrophobic proteins, and neutral lipids. J Lipid Res. 1995;361283- 1293
22.
Greiner  JVGlonek  TKorb  DRLeahy  CD Meibomian gland phospholipids. Curr Eye Res. 1996;15371- 375Article
23.
Dougherty  JMMcCulley  JMeyer  DR Keratoconjunctivitis sicca and tear lysozyme in chronic blepharitis. Holly  FJed.The Preocular Tear Film Lubbock, Tex Dry Eye Institute1984;176- 181
24.
Dougherty  JMMcCulley  JP Tear lysozyme measurements in chronic blepharitis. Ann Ophthalmol. 1985;1753- 57
25.
Dougherty  JMMcCulley  JP Bacterial lipases and chronic blepharitis. Invest Ophthalmol Vis Sci. 1986;27486- 491
26.
Bowman  RWDougherty  JMMcCulley  JP Chronic blepharitis and dry eyes. Int Ophthalmol Clin. 1987;2727- 35Article
27.
Fennema  OKester  JJ Resistance of lipid films to transmission of water vapor and oxygen. Adv Exp Med Biol. 1991;302703- 719
28.
Martin-Polo  MVoilley  ABlond  GColas  BMesnier  MFloquet  N Hydrophobic films and their efficiency against moisture transfer, 2: influence of the physical state. J Agric Food Chem. 1992;40413- 418Article
29.
Lee  Y-CZheng  YoTaraschi  TFJanes  N Hydrophobic alkyl headgroups strongly promote membrane curvature and violate the headgroup volume correlation due to "headgroup" insertion. Biochemistry. 1996;353677- 3684Article
30.
Seimiya  TAshida  MHayashi  MMuramatsu  THara  I Hydrogen bond among the ionic groups of ampholytic phospholipid. Chem Phys Lipids. 1978;2169- 76Article
31.
Seimiya  TOhki  S Ionic structure of phospholipid membranes, and binding of calcium ions. Biochim Biophys Acta. 1973;298546- 561Article
32.
Boggs  JM Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function. Biochim Biophys Acta. 1987;906353- 404Article
33.
McCulley  JPDougherty  JM Blepharitis associated with acne rosacea and seborrheic dermatitis. Int Ophthalmol Clin. 1985;25159- 172Article
34.
Shine  WEMcCulley  JP Significant differences in polar lipids associated with chronic blepharitis. Invest Ophthalmol Vis Sci. 1997;385156ARVO Abstract.
35.
Zengin  NTol  HGunduz  KOkudan  SBalevi  SEndogru  H Meibomian gland dysfunction and tear film abnormalities in rosacea. Cornea. 1995;14144- 146Article
36.
Asherson  RAFei  H-MStaub  HLKhamashta  MAHughes  GRVFox  RI Antiphospholipid antibodies and HLA associations in primary Sjögren's syndrome. Ann Rheum Dis. 1992;51495- 498Article
37.
Fullard  RJTucker  DL Changes in human tear protein levels with progressively increasing stimulus. Invest Ophthalmol Vis Sci. 1991;322290- 2301
38.
Keizenga  AStolwijk  TRvan Agtmaal  EJvan Haeringen  NJKijlstra  A Detection of secretory IgM in tears of IgA deficient individuals. Curr Eye Res. 1990;9997- 1005Article
39.
Harmer  IJLoizou  SThompson  KMSo  AKLWalport  MJMackworth-Young  C A human monoclonal antiphospholipid antibody that is representative of serum antibodies and is germline encoded. Arthritis Rheum. 1995;381068- 1076Article
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