Download The Lipid Layer: The Outer Surface of the Ocular Surface Tear Film

Bioscience Reports, Vol. 21, No. 4, August 2001 ( 2002)
MINI REVIEW
The Lipid Layer: The Outer Surface of the Ocular
Surface Tear Film
James P. McCulley1,2 and Ward E. Shine1
Receiûed August 29, 2000
The outer layer of the tear film—the lipid layer—has numerous functions. It is a composite
monolayer composed of a polar phase with surfactant properties and a nonpolar phase. In
order to achieve an effective lipid layer, the nonpolar phase, which retards water vapor
transmission, is dependent on a properly structured polar phase. Additionally, this composite lipid layer must maintain its integrity during a blink. The phases of the lipid layer
depend on both lipid type as well as fatty acid and alcohol composition for functionality.
Surprisingly, the importance of the composition of the aqueous layer of the tear film in
proper structuring of the lipid layer has not been recognized. Finally, lipid layer abnormalities and their relationship to ocular disease are beginning to be clarified.
KEY WORDS: Ocular; tear film; lipid; polar; surfactant; disease.
INTRODUCTION
The eyelid margin is the source of physiologically important lipid secretion, meibum.
These eyelid meibomian gland secretions form the outer layer of the tear film. Functions which have been attributed to this tear film lipid layer are: (1) a lubricant
facilitating the movement of the eyelids during a blink, (2) a barrier preventing
evaporation of the aqueous tear fluid, and (3) a barrier to the entry of microorganisms and organic matter such as pollen (Tiffany, 1987). In addition, it has been
suggested that defects in the lipid layer itself could be responsible for tear breakup
and subsequent dry spots (Kaercher et al., 1994). Through the research efforts of
numerous investigators, a much better understanding of meibum composition has
been attained. However, an understanding of why component lipids are important
in terms of tear film lipid layer functionality has been more difficult to achieve.
Meibum Composition
Many lipid compositions have been reported for meibum from various animal
species, as well as for humans (Tables 1 and 2). Lipids present in the nonpolar lipids
1
Department of Ophthalmology, The University of Texas Southwestern Medical Center at Dallas, Dallas,
Texas.
2
To whom correspondence should be addressed.
407
0144-8463兾01兾0800-0407兾0  2002 Plenum Publishing Corporation
408
McCulley and Shine
Table 1. Composition of Esterified Fatty Acids and Alcohols of Normal Human Meibomian Gland
Secretions
C12–C18
Lipid type
Ester component
Total
n-hyd
n-sat
n-unsat
isat
ai-sat
acid
acid
acid
acid
alcohol
acid
acid
100
100
95
88
1
2
65
1
0
63
0
0
0
0
0
0
100
37
25
6
1
0
31
1
0
0
55
33
0
0
31
0
0
0
9
16
0
1
1
0
0
0
6
33
0
1
2
0
Phospholipids
Sphingolipids
Triglycerides
Wax esters
Wax esters
Cholesterol esters
Free fatty
Hydrocarbons
include wax and sterol esters and hydrocarbons (Nicolaides et al.; 1981, Mathers
and Lane 1998; Shine and McCulley, 1991). On the other hand, composition of
triglycerides and the polar phospholipids is more closely conserved (Shine and
McCulley 1996; Griener et al., 1996). Variation in the composition of other polar
lipids such as the sugar containing cerebrosides (sphingolipids), and even their
importance as a component of meibum are less obvious (Tables 3 and 4).
The importance of composition is evident from analyses of meibum. For
example all polar lipids analyzed to date contain fatty acid chains of carbon length
Table 2. Composition of Esterified Fatty Acids and Alcohols of Normal Human Meibomian
Gland Secretions
C12–C18
Lipid type
Phospholipids
Sphingolipids
Triglycerides
Wax esters
Wax esters
Cholesterol esters
Free fatty
Hydrocarbons
Ester component
Total
n-sat
n-unsat
isat
ai-sat
acid
acid
acid
acid
alcohol
acid
acid
0
0
5
12
99
98
35
99
0
0
0
6
6
4
5
99
0
0
1
0
9
1
5
0
0
0
3
2
2
57
11
0
0
0
1
4
27
40
14
0
Table 3. Liquid Composition of Human Meibum
Cholesterol esters
Wax esters
Triglycerides
Diesters
Free fatty acids
Free cholesterol
Hydrocarbons
Polar lipids
38% (or less)
47% (or more)
4%
2%
2.5% (or less)
1.50%
3–7%
6–16%
Composite: McCulley and Shine (1997); Mathers
and Lane (1998); Nicolaides et al. (1981).
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409
12–18 (C12–C18). These are all normal (straight chain) saturated fatty acids, except
in sphingolipids where a high proportion contain hydroxy groups. The primary nonpolar lipids present in the lipid layer are wax and cholesterol esters (CE), hydrocarbons (HC) and trigylcerides (TG). Cholesterol esters contain mainly long chain
(HC20) saturated branched chain (iso and anteiso) fatty acids that can also functionally substitute for unsaturated fatty acids. The wax esters (WE) contain primarily the monounsaturated C18 fatty acid, oleic acid, and a C17 anteiso fatty acid.
These fatty acids are esterified to long chain iso, anteiso, and normal saturated and
monosaturated alcohols. Triglycerides contain short chain (less than C20) unsaturated (primarily oleic acid) and saturated fatty acids. The free fatty acids are a
mixture of short and long chain fatty acids. The WE fatty acids, TG and free fatty
acids are distinguished by their high levels of unsaturated short chain fatty acids.
Finally the hydrocarbons are straight, long chain and saturated. A minor lipid group
present is the diglycerides that depend on hydrolytic activity for formation from
lipid precursors.
Here we focus on formation of a structurally functional tear film lipid layer,
the importance of the aqueous layer of the tear film in this process, and variation in
lipid composition that can result in lipid layer instability, loss of functionality, and
finally, disease signs.
INVESTIGATIONS UTILIZING IN VITRO LIPID LAYERS
Why Does Meibum Form Monolayers in the Tear Film?
One puzzling question concerns the formation of the lipid layer from meibum;
a related question, previously answered to some extent is what is the lipid layer’s
function? The importance of phospholipid composition in surface monolayer formation and surfactant properties is becoming more apparent. For example, in ûitro
investigations with phospholipids have shown that a small percentage of negatively
charged lipids are required; if these lipids are absent, multilayered assemblies commonly occur (Schindler, 1989). Similarly, a very small amount of protein also
appears to aid in monolayer formation. Since low concentrations of monovalent
ions (e.g., sodium less than 10 mM) or divalent ions (e.g., calcium less than 1 mM)
impede monolayer formation, it was recommended that 120 mM univalent ions and
0.1 mM divalent ions should be added to the aqueous layer (Schindler, 1989). Taking
these recommendations as a whole, it is indeed remarkable that: (1) meibum contains
anionic phospholipids such as phosphatidylserine (7%), phosphatidylinositol (5%)
and cardiolipin (2%), Greiner et al., 1996), (2) the aqueous layer (tears) contain
150 mM univalent ions (primarily sodium with potassium) and 1 mM divalent ions
(primarily calcium with magnesium), as well as (3) small amounts of protein
(Berman, 1991).
Surface bilayers are known to be or have been suggested to be formed only in
unusual conditions. First, in the unusual condition that nonpolar lipids are present
in very limited amounts or absent, a surface bilayer can form. Thus at a certain
critical temperature, depending on phospholipid composition, a bilayer (two monolayers with fatty acid chains facing each other) forms; a quite unexpected attribute
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McCulley and Shine
of this bilayer was a high resistance to water vapor transmission. Once formed,
this bilayer has some temperature stability above and especially below the critical
temperature of formation (Ginsberg and Gershfeld, 1985; Cevc et al., 1990).
Whether these conditions occur in ûiûo is not known. A second example under which
bilayers readily form in ûitro is when polar lipids are mixed with excess cholesterol
esters containing unsaturated fatty acids (Smaby and Brockman, 1987a). Interestingly, however, this type of ‘‘bilayer’’ is actually more like a composite monolayer
with phosphatidylcholine (PC) and small amounts of CE in the polar phase at the
acqueous interface and the remaining CE in the nonpolar phase. When the CE fatty
acids are saturated and very long (HC20), as in meibum, formation of a polar lipid
‘‘bilayer’’ is inhibited with no CE in the polar PC phase. A similar composite monolayer with TG (triolein) as third component showed that more TG than CE was
present in the polar phase (Smaby and Brockman, 1987b). Finally, it has been suggested that in general bilayers are formed when polar lipid monolayers collapse at
high compression (Marsh, 1996), as could occur in the tear film during a blink.
Lipid Layer Compression–Expansion (Reversibility)
Reversible compression and subsequent expansion, without hysteresis (i.e.,
maintenance of integrity during multiple blinks), is essential for the integrity of the
tear film lipid layer. In ûitro studies of lipid monolayers have determined that
depending on the polar lipid present, either trilayers or bilayers can be reversibly
formed. For example, it has been determined that sphingolipid monolayers can be
reversibly compressed into trilayers, presumably by a folding action (Stoffel et al.,
1974). In order to form trilayers, sphingolipids must be in the liquid expanded
(melted) state; stereo configurations as well as saturation兾unsaturation are also
important. Thus trilayer formation is dependent on specific lipid composition and
temperature parameters as well as compression. In this example, bilayers and multilayers were not formed. Similarly, cerebroside (CB) monolayers composed of cerebrosides (sphingolipids with sugar groups) reversibly form bilayers upon compression
(Johnston and Chapman, 1988). In this study, sphingolipids with short chain or
hydroxylated fatty acids (amide bound) were critical. Thus, the hydroxylated CB
rapidly reform monolayers after compression–expansion without hysteresis while
nonhydroxylated CB respond more slowly with definite hysteresis. However, cerebrosides with unsaturated fatty acids such as oleic acid did not form bilayers. Interestingly, when CB with nonhydroxylated (saturated) fatty acids were mixed with PC,
they formed bilayers more readily than when the monolayer contained the CB alone.
In general, hydroxylated CB readily mixes with PC in all proportions, but this is not
true for nonhydroxylated CB (Bunow and Levin, 1988). In summary, formation of
stable trilayers and bilayers is quite dependent on the types of lipid groups present
and on their individual compositions. However, composition also effects lipid ‘‘melting’’ and as discussed above this is important for formation of monolayers that can
undergo compression–expansion without hysteresis.
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411
Optimizing Lipid Layer Functionality
The concept of superlattice structure and lipid layer stability has recently gained
interest. It has been suggested that in many biological systems lipid layers actually
are highly structured in ‘‘superlattices’’, where the individual lipid types form a
specific pattern (Fig. 1). Depending on the number of lipid types (groupings) present,
this model suggests that there are certain lipid arrangements (ratios) that are preferred (Virtanen et al., 1998). These preferred ratios have been found in ûitro in
cholesterol—CB monolayers (Ali et al., 1994) and cholesterol—PC monolayers (Liu
and Chong, 1999). In the latter study decreased phospholipase A2 activity was
directly related to specific superlattice ratios. Furthermore, based on red blood cell
membrane phospholipid analyses from humans and other animal species it was
determined that the superlattice model closely predicts observed in ûiûo compositions
(Virtanen et al., 1998). Thus it was suggested that in ûiûo certain lipid ratios in polar
lipid phases are both preferred and more stable (Somerharju et al., 1999). What the
superlattice model does not address are broader compositional parameters that are
necessary for the model to function effectively in ûiûo.
Fig. 1. Proposed model of tear film.
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McCulley and Shine
IMPORTANCE OF IONIC COMPOSITION OF THE AQUEOUS PHASE
The importance of aqueous layer ionic composition on formation of monolayers has already been referred too. However, calcium ions (Ca2+) are also important and interact with surface pressure to modulate hydration of PC and
phosphatidylserine (Flach et al., 1993). Calcium can also bridge between sulfate
containing glycoaminoglycans (e.g., mucin) and phospholipids, resulting in surface
tension decreases as well as a decrease in molecular (surface) area (Huster et al.,
1999). The ratio of Ca2+ to Na+ (sodium) is also important (Steffan et al., 1994).
The interaction of various constituents of the polar lipid phase with the adjacent
aqueous layer is also quite important but nonetheless complex. For example it has
been suggested that aqueous layer and lipid layer components interact to affect surface tension (Nagyova and Tiffany, 1999); included in these components are tear
lipocalins (Glasgow et al., 1999). Also, although lipids containing choline groups
(e.g., PC and SM, sphingomyelin) are known to be important in polar phase
hydration (Rand and Parsegian, 1989; McIntosh, 1996), it is believed that the surface
potential (i.e., dipole potential, charge separation) and this hydration are closely
related (Brockman, 1994). Other factors important for the structure of the polar
phase are effects of pH and Ca2+ on PE (phosphatidylethanolamine) binding to PC
or to SM (Seimiya et al., 1978; Seimiya and Ohki, 1973). It has even been proposed
that these lipid association not only result in altered pKa (acid dissociation constants) values for NH3+ (amino and PO−4 (phosphate) groups in situ but that the
effective pH (acidity) of the polar lipid phase is quite different from the bulk aqueous
layer (Seimiya et al., 1978, Prats et al., 1986). Acidity (H + ion availability) and Ca2+
concentration may also affect the association of free fatty acids with either the polar
phase or the nonpolar phase of the lipid layer (Kimizuka et al.; 1967, Duzgunes et
al., 1985), depending on the resulting effective polarity of the fatty acid. These structural variations thus affect not only the polar lipid packing density but also surface
potential and hydration.
IMPORTANCE OF VARIATION IN MEIBUM COMPOSITION
Formation of an Effective Lipid Layer
Three functions of the lipid layer are essential. First, the polar lipid layer must
be an effective surfactant, acting as a bridge between the aqueous layer and the
nonpolar lipid phase. Second, the lipid layer must be capable of compression and
expansion as the eye blinks. Third, the lipid layer must be an effective barrier to
water vapor transmission, thus decreasing the loss of the aqueous component of
tears and aiding in the prevention of tear film break-up.
Specific Monolayer Effects of Polar Lipid Differences
It is generally not recognized that specific monolayer effects result from the
polar lipid differences. What is not adequately understood is that both the types of
polar lipids present, and also their individual fatty acid compositions are important.
Thus monolayer compressibility, surface tension, fluidity, viscosity, integrity and
Tear Lipid Layer
413
ability to reversibly form bilayers or trilayers (lack of hysteresis) are all dependent
on lipid type and fatty acid composition. For example, the presence of only 10%
plasmalogen (a polar lipid similar to PC but with an ether bond and polyunsaturated
fatty acid) reduced polar lipid surface tension by 50% and the monolayer viscosity
by 80% (Tolle et al., 1999). Some evidence for the presence of plasmalogens in
meibum has been reported (Greiner et al., 1996). Furthermore, monolayers with
galactosphingolipids (cerebrosides) containing hydroxy-fatty acids readily form
bilayers but if instead the fatty acid is unsaturated or a very long chain saturated
fatty acid, bilayers are only formed with difficulty and after compression may not
expand effectively again (Johnston and Chapman, 1988). Surprisingly both the
monolayer surface potential and compressibility are greatly affected by the saturation or hydroxylation of the galactosphingolipid (Oldani et al., 1975). Other factors
such as temperature and aqueous layer composition interact with lipid composition
to produce an overall effect.
Polar lipid composition can also affect monolayer dipole potential (surface
potential) and hydration, as well as structure. The lipid type not the fatty acid length
has the most effect on this potential. However a remarkable change in potential with
galactosphingolipid fatty acid hydroxylation has been reported (Oldani et al., 1975).
We find high levels of sphingolipid fatty acid hydroxylation in human meibum
(Table 1). Additionally the polar lipid head group itself is quite important. Thus the
neutral (zwitterionic) phospholipids PC and PE have greater dipole potentials than
the anionic PS (phosphatidylserine) and phosphatidic acid (Brockman, 1994). Furthermore, in the melted (liquid-crystalline) state PC binds more water molecules
than PE (23 vs. 9, respectively). On the other hand, PE can form intermolecular
ionic bonds, either alone or with associated hydrogen bonding, involving the NH3+
and PO−4 groups, and possibly water molecules (McIntosh, 1996). This type of intermolecular bonding is sensitive to the aqueous layer pH and is greatly diminished as
the pH approaches pH 8 (the NH4+ group becomes deprotonated and neutral). Concurrently Ca2+ binding to the now free PO+4 groups can occur; the monolayer surface
potential also decays at this point (Seimiya and Ohki, 1973). Finally, the anionic
phospholipids also have an important monolayer function in that their presence in
a monolayer at a level of 10% that of PE inhibits the close approach and fusion
with similar phospholipid monolayers (McIntosh, 1996). Thus a balance between
anionic phospholipids and PE, as well as aqueous layer pH, are critical for monolayer stability.
The importance of the polar lipid fatty acid differences must also be addressed
in terms of environmental temperature and its effect on lipid physical properties.
One must remember that the tear film lipid layer functions at a temperature just
below 36°C (Mori et al., 1997). Furthermore, for a given polar lipid type the melting
temperature (changes in physical structure to a more fluid state) is determined primarily by its fatty acid composition. However, surface pressure also affects lipid
melting (transition) temperature. Thus under medium pressure, PC with C14 acids
melts at 20°C while PC with C16 acids melts at 39°C; the very low surface pressure
encountered with open eyes results in about a 10°C lower melting temperature
(Blume, 1979). On the other hand the melting point for PE with C14 acids is similar
to PC with C14 acids. In contrast to these results, if the phospholipid fatty acids are
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McCulley and Shine
unsaturated the melting point is much lower and with PE the monolayers will have
a tendency to form a different type of structure that is not a monolayer or bilayer
(McIntosh, 1996). Thus fatty acid chain length and unsaturation can profoundly
affect lipid properties depending polar lipid head group, temperature and surface
pressure.
Other more esoteric interactions in monolayers may result from altered polar
lipid phases. Thus it has been suggested that the presence of excess SM in polar lipid
phases can result in formation of SM enriched domains or ‘‘island’’ inclusions (Koiv
et al., 1993). Also, presence of an altered dipole potential, a high surface charge
(e.g., negative or even positive) or excessive amounts of PE (but surprisingly, not
other zwitterionic phospholipids) can result in binding of enzymes or other biomolecules (Pieroni and Verger, 1979; Speelmans et al., 1997), or altered enzymatic activity
(Maggio, 1999). In fact it has been observed that enzymatic action in the polar lipid
phase can result in formation of small domains containing primarily lipid degradation products (Maloney and Grainger, 1993). Some of these degradation products
result in altered surface potential, which in turn promotes formation of these
domains (Maloney et al., 1995). Finally, it has been suggested that polar lipid fatty
acids differences and the influence of aqueous layer ionic composition can result in
domain formation (Hinderliter et al., 1994). These examples of in ûitro polar lipid
phase alteration could be relevant to the tear film in ûiûo and result in a less functional lipid layer.
These examples in fact can easily be related to meibum and the tear film lipid
layer. For example one can now appreciate that it is not by chance that in normal
meibum all of the phospholipids contain C12–C18 saturated normal fatty acids with
the exception that the sphingolipid fatty acids are also highly hydroxylated (Table
1). As we have discussed, proper matching of fatty acid chain lengths and lipid
types affects melting temperature and mixing, hydration, monolayer density (area兾
molecule) and stability, and even domain formation.
In light of the above discussion we also suggest that the nonpolar phase of the
tear film lipid layer has an additional function that has not been generally considered: that of aiding in preserving the integrity of the polar lipid phase. We suggest
that the nonpolar phase CE (that always contains saturated fatty acids) intercalates
into the polar phase times of polar phase stress in order to promote retention of
functionality of the tear film lipid layer (McCulley and Shine, 1997). Triglyceride
(TG) may have similar although mechanistically different function: it is important
to remember that the TG overwhelmingly contains short chain (<C19) fatty acids
that closely match the length of fatty acids in the polar lipids. A related important
point is that the forces binding the lipids together are of numerous types. Much of
the above discussion dealt with fatty acids with similar carbon chain lengths where
bonding dependent on van der Waals forces are most important. This type of bonding is also important in the nonpolar phase where the presence of long chain hydrocarbons are expected to strengthen bonding between other long chain nonpolar
lipids such as those present in CE and WE. We believe that the end result of this
condensing effect is that the rate of water vapor transmission through the nonpolar
lipid phase is greatly reduced.
Tear Lipid Layer
415
An effective tear film polar lipid phase is quite complex. As discussed previously
small amounts of anionic polar lipids seem quite important for proper structuring
of the polar lipid layer. In meibum, PS, as well as diphosphatidylglycerol (cardiolipin) and phosphatidylinositol serve this purpose. Furthermore, as we have discussed (McCulley and Shine, 1997) PE and SM are important structure forming
lipids. Their effectiveness is likely due both to hydrogen and to ionic bonding with
other polar lipids. Analysis of patient meibum suggests that these two lipids are
important determinants for development of some types of dry eye signs (Shine and
McCulley, 1998). Finally, the presence of PC (and SM) has been reported as important for proper lipid layer hydration (McIntosh, 1996) through the structuring of
water molecules.
A SUGGESTED MODEL FOR THE TEAR FILM LIPID LAYER
Based on the known composition of meibum and in ûitro lipid studies a rational
suggestion for the tear film lipid layer has been presented (Fig. 2, McCulley and
Shine, 1997). As discussed, there is scant reason to believe that the tear film lipid
layer is normally a bilayer. We have presented aqueous layer ionic concentrations
and related monolayer parameters, lipid composition and temperature data, all of
which argue against a bilayer. Furthermore there is evidence for reversible formation
of either a bilayer or trilayer; many clinical observations note the fact, however, that
after a blink the tear film is re-established such that it appears almost identical to
that before the blink. This would be mechanistically much more likely to occur with
a folded trilayer than a bilayer. Additionally, the trilayer folding action may aid in
cleansing actions, thus removing undesirable material.
Ultimately functionality depends on composition. For example, increased evaporation from the aqueous layer has been reported in some groups of dry eye patients
(Mathers and Daley, 1996). Other reports have discussed low levels of PE and SM
in meibum from blepharitis patients with dry eye signs (Shine and McCulley, 1998).
Of interest is the in ûitro observation that SM also inhibits peroxidation of unsaturated fatty acids in PC monolayers (Subbaiah et al., 1999). Chronic blepharitis
patients’ meibum phospholipids also have unsaturated fatty acids, especially in those
patients with meibomianitis (McCulley and Shine, 1997); these would tend to destabilize the lipid layer.
Unusual nonpolar lipid compositions have been observed in some normal
patients. Thus in normals and patients with chronic blepharitis, all meibum samples
contained cholesterol esters and unsaturated cholesterol and wax ester fatty acids
and alcohols (Shine and McCulley, 1991). In contrast other normals’ meibum had
no cholesterol esters and no wax ester unsaturated fatty acids of alcohols (Shine and
McCulley, 1993); this lipid pattern was never observed in meibum from chronic
blepharitis patients. Thus there is an association between the presence of cholesterol
esters in meibum and the presence of unsaturated fatty acids and alcohols. Furthermore, when the chronic blepharitis disease state is present, unsaturation in polar
lipid fatty acids is also present. The destabilizing effect of these unsaturated fatty
acids has already been discussed. Subsequent disruption of the integrity of the polar
lipid phase (e.g., superlattice) could increase susceptibility to enzymatic and ROS
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McCulley and Shine
Table 4. Composition of Polar Lipids
from Normal Individuals’ Meibum
Phospholipids (70%)
Phosphatidylcholine (38%)
Phosphatidylethanolamine (18%)
Sphingomyelin (7%)
Unknowns (39%)
Sphingolipids (30%)
Ceramides (20%)
Cerebrosides (80%)
(reactive oxygen species) activity. In fact lipid degradations as well as the initial
polar lipid fatty acid unsaturation could result in formation of domains that would
be expected to further decrease the integrity and functionality of the lipid layer.
Finally, how these changes affect the tear film lipid layer collapse pressure, its
relationship to a blink and lipid layer hysteresis is important but has to be determined. Perhaps the most stable human meibum, however, is infant meibum as illustrated by a lack of hysteresis, as determined in ûitro (Kaercher et al., 1994), and the
fact that infant blink rates are quite long compared to an adults’ rate.
Many factors have been discussed in terms of model systems that are also relevant to a presumed similar functionality in the tear film lipid layer. For example
the importance of the hydrocarbon content in the nonpolar phase is not known.
Similarly the importance of the nonpolar phase, as well as the polar phase, in trapping and eliminating foreign entities, especially in terms of the folded tirlayer model,
is unclear. Finally, the importance of aqueous layer ionic composition and pH, the
significance of sulfated mucin in the aqueous layer (Ellingham et al., 1999), and CB
in the layer (Table 4), in promoting formation and effective structuring of the tear
film lipid monolayer is not well understood. If the composition of infant meibum
were known perhaps many of these uncertainties would be challenged with obvious
answers. What is known is significant however, and obviously becoming more relevant to treatment of ocular diseases.
ACKNOWLEDGMENT
Supported in part by an unrestricted research grant from Research to Prevent
Blindness, Inc., New York, New York.
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