Spectroscopic evaluation of human tear lipids

A

httwppea©

K

1

fimmlcit(e

f

0

Chemistry and Physics of Lipids 147 (2007) 87–102

Spectroscopic evaluation of human tear lipids

Douglas Borchman a,∗, Gary N. Foulks a, Marta C. Yappert b,Daxin Tang a, Donghai V. Ho a

a Department of Ophthalmology and Visual Science, University of Louisville, 301 E. Muhammad Ali Blvd., Louisville, KY 40202, USAb Department of Chemistry, University of Louisville, Louisville, KY 40292, USA

Received 20 October 2006; received in revised form 30 March 2007; accepted 1 April 2007Available online 5 April 2007

bstract

Infrared and fluorescence spectroscopies were applied to characterize the molecular conformational/structure and dynamics ofuman meibum (ML) and tear lipids (SSL). ML lipids contained more C C and CH3 moieties than SSL. SSL contained OH groupshat were not apparent in the spectra of ML. The C O stretching band observed in the infrared spectra of SSL and ML revealed thathe C O groups are not involved in hydrogen bonds. Bands due to the polar moieties C O and PO2

− did not change significantlyith increasing temperature, suggesting that they may not play an appreciable thermodynamic role in the lipid hydrocarbon chain
hase transition. Components in tears bind to SSL and exclude water at the water–lipid boundary where the polar headgroups ofhospholipids are located. If similar interactions occur in vivo at the tear film lipid–aqueous interface, they would reduce the rate ofvaporation. The results provide a foundation for future studies to assess possible differences with age and sex in tears from normalnd dry eye subjects.
2007 Elsevier Ireland Ltd. All rights reserved.

Meibum
eywords: Fluorescence spectroscopy; Infrared spectroscopy; Lipid;
. Introduction

Lipids intimately associated with tears are found inve regions: the tear-lipid surface-layer, the pool ofeibomian lipid on the lid margins, the lipids in theeibomian glands, the lacrimal lipids (many bound to

ipocalin) and the lipids on the anterior surface of theorneal epithelium that are released into the tears orn the apical membrane. It is generally believed that
ear-film lipids are produced by the meibomian glandsMilder, 1987) and that they may be released by the forcexerted on the eyelid during blinking (Chew et al., 1993;
∗ Corresponding author. Tel.: +1 502 852 7435;ax: +1 502 852 7450.

E-mail address: [email protected] (D. Borchman).

009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.chemphyslip.2007.04.001

; Molecular structure; Tear film

Korb et al., 1994). Meibomian gland expression results inincreased lipid layer thickness and tear stability (Craiget al., 1995). Lipids and mucins stabilize the tear film(Haberich, 1981; Van Haeringen, 1981; Wolff, 1946) andmay inhibit evaporation 4–20-fold (Craig et al., 1995;Craig and Tomlinson, 1997; Iwata et al., 1969; Mathers,1993; Mathers and Daley, 1996; Mishima and Maurice,1961).

Qualitative, chromatographic data demonstrate thatthe lipids from human tear fluid are very different thanthose in the human meibum lipid (Nagyova and Tiffany,1999). Only one published study (Wollensak et al., 1990)and a preliminary report (Stuchell et al., 1984a) mea-
sured the complete lipid composition of tear fluid. Waxand cholesterol esters compose 45% of the tear lipid,similar to the meibum. Non-esterified cholesterol andphospholipid compose 15% each of the lipid in tears,
ed.

mailto:[email protected]

dx.doi.org/10.1016/j.chemphyslip.2007.04.001

nd Phys
88 D. Borchman et al. / Chemistry a
much higher than in the meibum which contains only0–7% of these species (Wollensak et al., 1990).

Quantitative comparison of compositional differ-ences between lipids excreted from the meibomian gland(ML) and lipids in tear fluid is difficult because only threecomprehensive human meibum lipid compositional stud-ies have been published that encompass the percentageof all species of wax esters, cholesterol and choles-terol esters, phospholipids and diglycerides (Cory et al.,1973; Nicolaides et al., 1981; Tiffany, 1978). Numer-ous studies to quantify individual classes of lipid, suchas phospholipids (Greiner et al., 1996) have been pub-lished. The accuracy and precision of measurements ofthe lipid composition of meibum is complicated by thelarge variation in composition from person to person(Tiffany, 1978), the paucity of sample and the complexityof lipid species (Nicolaides et al., 1981). It is estimatedthat there are over 30,000 molecular species of lipids inhuman meibum (Nicolaides and Santos, 1985). There isloose agreement on the composition of meibum lipid;for instance, esters compose between 20 and 80% ofthe meibum and alkanes compose 0–36% of the meibumwhich is very unusual for animal lipids (Cory et al., 1973;Nicolaides et al., 1981; Tiffany, 1978).

Molecular structure defines how molecules arearranged in space. Specific lipid molecular interactions atthe hydrocarbon chain interface and head group regionsof the lipid tear layer affect the rate of evaporation. Forinstance, if the hydrocarbon chains are all arranged ina trans conformation (conformation is how the atomsare arranged across a bond), the hydrocarbon chainswill be extended and the van der Waals interactionswill be maximal and the distance between the hydro-carbon chains minimal. To evaporate, water must breakthe lipid–lipid interactions in passing through the lipid-tear-film layer. If the lipid–lipid interactions are strong,the rate of evaporation will be low. ML structure is alsoimportant because it contributes to the viscosity (or flu-idity) and thus the ability of the lipid to flow out ofthe meibomian gland orifice onto the edge of the eye-lid at the temperature of the eyelid (Nagymihalyi etal., 2004). The molecular structure of ML has not beeninvestigated.

In this study, we evaluated the wealth of informationavailable from infrared and fluorescence spectroscopiesas a foundation for future studies designed to define tearlipid composition, structure function relationships withage, sex and dry eye symptoms. In addition, four general
questions were answered:
1. Are tear-fluid lipids different from meibum lipids andcan these differences be discerned spectroscopically?

ics of Lipids 147 (2007) 87–102

2. Is it possible for tear lipids to enter into the aqueoustear-fluid, and do tear proteins have the capacity tobind to them?

3. Could lipid on Schirmer strips that are usuallydiscarded, serve as a source for measurement of tear-fluid lipid?

4. What is the molecular conformation of tear lipidand how much do lipid hydrophobic and hydrophilicgroups contribute to it?

2. Experimental procedures

2.1. Materials

Unmarked Schirmer strips without ink ruling werepurchased from Alcon Laboratories (Fort Worth, TX).All solvents were purchased from Fisher Scien-tific, Pittsburgh PA. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadeconoyl-sn-3-phosphoethanolamine, tri-ethyl ammonium salt (NBD-PE) was purchased fromMolecular Probes (Eugene, OR, USA). Lipid standardswere purchased from Sigma Chemical, St Louis, MO.

2.2. Collection of tear lipids

ML was obtained from living subjects as describedin Kilp et al. (1986), except that meibomian gland exc-reta was collected with a platinum spatula. About 1 mgof lipid were collected per individual for direct spectro-scopic study.

Schirmer test strips were used to obtain tear-fluidlipids (SSL). The leading 5 mm of the Schirmer stripwas bent at a 90◦ angle and subsequently placed over theedge of the lower eyelid for 3 min. Tear film is wickedup the strip. To collect polar tear-fluid lipids (PSSL) thatare soluble in methanol, the lower portion of the stripswas cut off and placed in a vial filled with methanol thathad been bubbled with argon to remove oxygen. The vialwas filled with argon before sealing to prevent oxidation.Care was taken not to contaminate the lower end of thetest strip with finger lipids. Donors with contact lensesor eye liner were excluded.

2.3. Lipid extraction and sample preparation

To extract polar lipids from Schirmer Strips,a monophasic lipid extraction protocol was used(Byrdwell et al., 2002). Waxes and long-chain hydro-
carbons are not extracted using this method. Lipids wereextracted from 20 to 140 Schirmer strips in a glass vialusing 30 ml of methanol which had been bubbled withargon for 5 min. The strips and methanol were sonicated

nd Phys

iswtwladrDtltaottwtst

SobvBtfspdsatadr

ofoTtuppthlaA

D. Borchman et al. / Chemistry a

n an ultrasonic bath (Branson 1510, Branson Ultra-onics Co., Danbury, CT) for 5 min and the methanolas decanted and centrifuged at 10,000 rpm for 15 min

o remove methanol-insoluble impurities. The methanolas decanted again with care not to disturb the pel-

et, and evaporated under a stream of argon. Hexanend isopropanol (2:1, v:v; 10 ml) were added to thery lipid film and sonicated with a Sonifier® cell dis-upter microprobe for 15 s (Branson Ultrasonics Co.,anbury, CT). The solution was transferred to a cen-

rifuge tube and centrifuged at 5,000 rpm for 1 h. Theipid-containing supernatant was decanted into anotherube, taking care not to disturb the pellet, and the hexanend isopropanol were evaporated directly under a flowf nitrogen. Methanol (150 �l) was added to solubilizehe lipid. The extracted lipid was added, 5 �l at a time,o the center of a 2-mm thick, 13-mm diameter AgClindow while drying with a gentle stream of argon until

he entire sample was deposited onto the window. Theample window was lyophilized at least 4 h to removehe methanol.

To extract all lipids from the Schirmer strips, 25chirmer strips were placed in a glass vial with 30 mlf tetrahydrofuran and methanol (3:1, v:v) which hadeen bubbled with argon for 5 min. The strips and sol-ent were sonicated in an ultrasonic bath (Branson 1510,ranson Ultrasonics Co., Danbury, CT) for 15 min and

he solvent was decanted and centrifuged at 10,000 rpmor 15 min to remove solvent-insoluble impurities. Theolvent was decanted again with care not to disturb theellet, and evaporated under a stream of argon. Tetrahy-rofuran and methanol (150 �l, 3:1, v:v) was added toolubilize the lipid. The extracted lipid was added, 5 �lt a time, to the center of a 2-mm thick 13-mm diame-er AgCl window while drying with a gentle stream ofrgon until the entire sample was deposited onto the win-ow. The sample window was lyophilized at least 4 h toemove the solvent.

Reflex human tears (RHT) were obtained by exposingne 52-year-old male Caucasian to the lachrymatory-actor (Imai et al., 2002) in the vapor of freshly cutnions for about 3 min at a time (Frey et al., 1981;iffany and Marsden, 1986). There is no difference in

he viscosity/shear rate between stimulated and unstim-lated tears (Pandit et al., 1999). Pooled RHT wasrocessed to remove mucins, lipids, debris and otherroteins from 0.5 ml of RHT by centrifugation and fil-ering through a 5000 kDa cut-off filter. The resulting
uman tear aqueous (HTA) was used to hydrate tearipids. Anhydrous tear lipid was hydrated by addingbout 10 �l of HTA to the anhydrous lipid on thegCl infrared window prepared as described above. A
ics of Lipids 147 (2007) 87–102 89

0.10 mm Teflon spacer and a second AgCl window wereadded to complete the sample holder. Anhydrous tearlipid was hydrated by adding about 10 �l of HTA to theanhydrous lipid on the AgCl infrared window preparedas described above. A 0.10 mm Teflon spacer and a sec-ond AgCl window were added to complete the sampleholder.

2.4. Fourier transform infrared spectroscopy

Infrared spectra were measured using a Nicolet 5000Magna Series Fourier transform infrared spectrometer(Thermo Fisher Scientific, Inc., Waltham, MA). Theprocedure was similar to that developed in this labora-tory (Borchman et al., 1991, 1999, 2004). Anhydrouslipid samples on the AgCl window were placed in atemperature-controlled infrared cell. The cell was jack-eted by an insulated water coil connected to a NeslabR-134A (NESLAB Instruments, Newton, NH) circulat-ing water bath. The sample temperature was measuredand controlled by a thermistor touching the AgCl sam-ple cell window. The water bath unit was programmed tomeasure the temperature at the thermistor and to adjustthe bath temperature so the sample temperature was atthe desired set point. The rate of heating or cooling atthe sample was also adjusted by the water bath. Tem-peratures were maintained within ±0.1 ◦C. Exactly 300interferograms were recorded. Spectral resolution wasset to 1.0 cm−1.

Infrared data analysis was performed withGRAMS/386 software (Galactic Industries, Salem,NH). In this study we used the symmetric stretching fre-quency of the CH2 band near 2850 cm−1 to estimate thecontent of trans and gauche rotomers in the hydrocarbonchains. The ratio of trans to gauche rotomers provides ameasure of hydrocarbon chain conformational order, asdescribed below. The CH2 symmetric stretching bandfrequency was calculated by first base line levelingthe OH–CH stretching region. The center of mass ofthe CH2 symmetric stretching band was calculated byintegrating the top 10% of the intensity of the band. Thebaseline for integrating the top 10% of the intensity ofthe band was parallel to the OH–CH region baseline.Lipid order was calculated from the CH2 symmetricstretching band frequencies by Eq. (1) from Borchmanet al. (1991).

2.5. Measurement of fluorescent probe dynamics
and interactions
The fluorescent probe NBD-PE, that partitions nearthe water–lipid interface region, was mixed with lipids

nd Phys
extracted from Schirmer strips (SSL) at a weight ratioof 0.005–1 (probe to lipid). Phase transitions were mea-sured as described for the hydrated SSL samples aboveexcept that 1 ml of HTA was used. Probe anisotropy andfluorescence intensity measurements were performedas reported previously (Borchman and Tang, 1996a;Cenedella et al., 2004; Grami et al., 2005; Tang andBorchman, 1998; Tang et al., 1998a,b, 2003) on an ISSPC1 photon counting spectrofluorometer (Champagne,IL) with a polarization accessory unit. The excitationand emission wavelengths used were 460 and 540 nm,respectively. The fluorescence signal was measured asthe ratio of the signals from the sample and referencedetectors.

To measure if SSL–tear protein interactions occur,HRT tears were used for comparison with the resultsobtained using HTA processed to remove material largerthan 5 kDa as described above.

2.6. Potential differences between the lipids at thetip and non-tip segments of Schirmer strips

To determine if meibum lipid from the lid mar-gin, potentially present at the tips of the Schirmerstrips, influence the spectral characteristics of tear-fluid lipids extracted from the non-tip segments, lipidswere extracted from 26 Schirmer strips that were sep-arated and pooled into tip (front 5 mm segment) andnon-tip segments. Lipids were extracted with tetrahy-drofuran/methanol as described in Section 2.3.

2.7. Quantification of lipids excluded from a 5 kDacut-off filter

To measure if lipids are free or bound, Gouveia andTiffany (2005), used a 5 kDa cut-off centrifugal filter toseparate bound lipids from free lipids. We tested waxstandards, meibum lipid and Schirmer strip lipids thatwere not tested in a previous qualitative study (Gouveiaand Tiffany, 2005). We quantified the amount of lipidretained by the filter using UV spectroscopy.

Eighteen Schirmer strips collected from a 53-year-old male Caucasian donor were used. ML was alsocollected from the same donor. SSL, ML, dimyris-toylphosphatidylcholine (DMPC), palmityloleate wax(W) and DMPC/W (1:1, w:w) were each mixed at1 mg/ml with balanced salt phosphate buffer (BSPB),without calcium and magnesium. The mixtures were
sonicated in an ultrasonic bath (Branson 1510, Bran-son Ultrasonics Co., Danbury, CT) for 15 min andmixed vigorously with a vortex Genie-2 mixer (Ther-moFisher Scientific, Waltham, MA). One milliliter of
ics of Lipids 147 (2007) 87–102

lipid emulsion was placed into the upper chamberof an Amicon, Ultrafree-CL, 5 kDa cut-off, cellulosemembrane centrifugal filter assembly (Millipore Cor-poration, Billerica, MA). One-hundred microliters ofthe lipid emulsion was removed for spectral analysisprior to filtration. Lipid-filter assemblies were cen-trifuged at 7000 rpm, 23 ◦C, in a Beckman, ModelTJ-R Centrifuge (Beckman Coulter, Inc., Fullerton,CA).

To quantify the lipid, samples were diluted 2–10-fold with BSPB and absorbance spectra were measuredfrom 200 to 450 nm using a DU® 640 spectrophotome-ter equipped with a 50 �l quartz micro cell accessory(Beckman Coulter, Inc., Fullerton, CA). Light scatteringwas corrected by extrapolating the region of the spectradue entirely to light scattering, 350–450 nm from theremainder of the spectra, 200–350 nm. As described inthe manual provided with the spectrometer, the equa-tion: As = aλb was used for the extrapolation, were As isthe absorbance due to scatter at a particular wavelength,and a and b are constants. To quantify lipid prior to andafter filtration, the absorbance at 205 nm and the inte-grated area under the peaks from 205 to 450 nm weremeasured using GRAMS/386 software (Galactic Indus-tries, Salem, NH). The absorbance at 205 nm is not themaximum absorbance, but was chosen because it is dis-tant enough from the 201 nm wavelength cut-off of thedetector.

2.8. Statistics

Data are presented as the average ± the standard errorof the mean. Significance was determined using theStudent’s t-test. Values of p < 0.01 were considered sta-tistically significant.

3. Results

3.1. Human material

Human donors of SSL were recruited from themembers and associates of Dr. Borchman’s labora-tory. Written informed consent was obtained from alldonors and protocols and procedures were reviewed bythe University of Louisville Institutional Review Boardand procedures were in accord with the Declaration ofHelsinki. For the infrared spectroscopic study, about20–140 Schirmer strips were pooled for lipid extraction.
The median age of the donors ranged from 20 to 53years. The male to female distribution was 1.7–3 malesper female except for two pools which were collectedfrom 1 male, 48 years of age. None of the donors had

nd Phys

ems2o

sa2

p

3S

ficifdfsalarlt1aa‘

F(sfNDt

The intensity of bands due to OH/NH stretchingvibrations, 3100–3400 cm−1 (Fig. 1 bands 1–4), were


ver experienced dry eye symptoms. Females with eyeake up were excluded. ML for the infrared spectro-

copic study were collected from three male donors: a1-year-old Asian; a 17-year-old Caucasian; a 61-year-ld Caucasian.

ML and SSL lipid for the filtration UV spectroscopictudy were collected from a 53-year-old Caucasian. Themount of lipid extracted from 18 Schirmer strips was.3 mg.

Samples were not meant to be representative of anyopulation.

.2. Meibum and tear-fluid lipid distribution onchirmer strips

The average infrared spectra of tear lipids extractedrom Schirmer strips and lipids from meibum are shownn Fig. 1. The spectrum of meibum (Fig. 1, ML), does notontain any OH stretching (3500–3100 cm−1) or bend-ng (1650 cm−1) bands in contrast to tear-fluid lipidsrom Schirmer strips (Fig. 2, top three spectra). Theseifferences were used to determine if meibum lipid trans-erred from the lid margin to the tip of the Schirmertrips. For this determination, Schirmer strips were sep-rated into the 5 mm tip that contacts the meibum on theid margin when the strip is placed on the lower lid tobsorb the tear fluid. The non-tip segment composes theemainder of the Schirmer strip. The infrared spectrum ofipid extracted from the tip of the Schirmer strip (Fig. 2,ip SSL) was similar to that of lipid extracted from the
5 mm section of the Schirmer strip that contained thequeous tear-fluid (Fig. 2, no tip SSL). Based on the rel-tive intensity of the OH stretching and bending bands intip SSL and ‘no tip SSL’, we estimate that ML composes
ig. 1. Infrared CH and OH stretching regions: (A) human meibum;B) palmitoyloleate; (C) ‘total’ tear lipids extracted from Schirmertrips with tetrahydrofuran and methanol; (D) polar tear lipids extractedrom Schirmer strips with methanol; (E) palmitoyl sphingomyelin.umbers correspond with tentative band assignments in Table 1.ifferences in spectra indicate dramatic conformational and composi-

ional differences in samples.

ics of Lipids 147 (2007) 87–102 91

less than 5% of the ‘tip SSL’ lipid. Because the spectrumof ‘tip SSL’ and ‘no tip SSL’ were similar, they wereaveraged and named SSL in subsequent figures. Basedon the intensity of the infrared bands between 2500 and3500 cm−1, we estimate that there was twice as muchlipid in the 5 mm tip region of the Schirmer strips, com-pared with the 15 mm section of the Schirmer strip thatcontained the aqueous tear-fluid. The relative intensity ofthe OH stretching and bending bands was much higherin the spectrum of polar Schirmer strip lipids (Fig. 2,PSSL) compared with total Schirmer strip lipids (Fig. 2,middle two spectrum) and ML.

3.3. The CH and OH infrared stretching region,3500–2800 cm−1

The CH stretching regions of the infrared spectra cor-responding to SSL, PSSL and ML are shown in Fig. 1.The CH stretching region is composed of four majorbands (Table 1).

The intensity of bands assigned to the C C doublebond CH stretch (Fig. 1, band 5) at 3010 cm−1, theCH3 terminal methyl stretch at 2953 cm−1 (Fig. 1, band6) and CH2 symmetric stretch at 2850 cm−1 (Fig. 1,band 8) were lower for SSL (C) and PSSL (D) com-pared to ML (A). The frequencies of the CH2 stretchingbands for SSL, PSSL and ML at 32.4 ◦C, are given inTable 2.

very low or absent in the ML spectra compared to SSLand PSSL (Fig. 1). Three bands were resolved in the SSL

Fig. 2. Complete infrared spectra of tear lipids. (PSSL) Polar tear lipidsextracted from Schirmer strips with methanol. (Tip SSL) Tear lipidsextracted from the tip of a 5 mm Schirmer strip that contacts the lowerlid margin. Lipid was extracted with tetrahydrofuran and methanol.(No tip SSL) Tear lipids extracted from Schirmer strips in which the5 mm tip of the strip that contacts the lower lid margin was removed.Lipid was extracted with tetrahydrofuran and methanol. (ML) Meibumlipid. No differences were detected between tip SSL and no tip SSLindicating that there was minimal ML associated with the Schirmerstrip tips.

92 D. Borchman et al. / Chemistry and Physics of Lipids 147 (2007) 87–102

Table 1Tentative infrared band assignments

Peak # in figures Meibomian lipid band (cm−1)a Schirmer lipid band (cm−1)a Tentative band assignmentb

1–3 3450, 3347, 3200 OH stretch4 3283 OH stretch, NH stretch (3195 shoulder)5 3010 3010 C C CH stretch6 2953 2953 CH3 terminal methyl stretch7 2920 2920 CH2 asymmetric stretch8 2850 2850 CH2 symmetric stretch9 1739 1737 C O, ester linkage

10 1650 1668 Conjugated dienes11 1623 Not assigned12 1650 Amide I, aldehydes13 1550 Amide II14 1469 1461 C–H in-plane bend, CH2 groups15 1408 Symmetric stretch, COO− groups16 1366, 1378 1349, 1370 CH2 wag, C–H bend CH3 groups17 1272 Asymmetric stretch, PO2

− groups18 1245 C–O19 1172 1171 C–O20 1067 Symmetric stretch, PO2

− groups21 1043 C–O–P–O–C, diester stretch22 960 965 C–N–C23 918 903 OH carboxylic acid24 738 CH2, rock25 723 718 CH2, rock

5

me banur tear l

bands at 1667 and 1622 cm were observed in allSSL spectra (C) and in some spectra of PSSL (D) thatwere not obscured due to incomplete removal of watervapor.

26 69

a Frequencies are approximate they are temperature dependent in sob Band assignments are tentative because the lipid composition of o

spectra of the OH stretching region. Curve fitting showsthese bands were centered at 3450, 3347 and 3200 cm−1.

3.4. The infrared finger print region

3.4.1. Comparison of the infrared finger printregion of ML, PSSL and SSL lipid spectra

Tentative band assignments for the ‘finger print
region’ 1800–600 cm−1 (Figs. 3–5) are listed in Table 1.The frequency of the C O stretching band averaged1738 cm−1 for PSSL and SSL and 1736 cm−1 for MLwith a standard deviation of ±0.6.
Table 2

Sample CH2 symmetricstretching band, λ

(wavenumbers)

% transrotomers

Number ofsamples

PSSL 2851.53 ± 0.08 53.9 ± 1.5 10PSSL + HTA 2850.88 ± 0.04 65.8 ± 0.7 3SSL 2851.0 ± 0.3 55 ± 6 2ML 2851.50 ± 0.05 53.9 ± 1.5 3

PSSL, polar lipids extracted from Schirmer strips with methanol; HTA,human tear fluid aqueous filtered to remove molecules greater than5 kDa; SSL, ‘total’ lipids extracted from Schirmer strips with tetrahy-drofuran and methanol; ML, human meibum.

CH2, rock

ds.ipid samples has not been determined.

Bands in the 1600–1700 cm−1 region are evidentin the infrared spectra of SSL (Fig. 3C), and PSSL(D) but are not obvious in the ML spectrum (A). Two

−1

Fig. 3. Infrared finger print regions: (A) human meibum; (B) palmi-toyloleate; (C) tear lipids extracted from Schirmer strips withtetrahydrofuran and methanol; (D) polar tear lipids extracted fromSchirmer strips with methanol; (E) palmitoyl sphingomyelin. Numberscorrespond with tentative band assignments in Table 1. Differencesin spectra indicate dramatic conformational and compositional differ-ences in samples.

D. Borchman et al. / Chemistry and Phys

Fig. 4. Infrared phosphate and C–O regions of: (A) human meibum;(B) palmitoyloleate; (C) human tear lipids extracted from Schirmerstrips; (D) polar human tear lipid extracted from Schirmer stripswith methanol. (E) Polar human tear lipid extracted from Schirmerstrips with methanol then hydrated with human tear fluid aqueous. (F)Palmityl sphingomyelin. Spectra were averaged from all samples withat an average temperature of 35 ◦C. Numbers correspond with tentativebc

adatthpcaafe

Fepp

and assignments in Table 1. Differences in spectra indicate dramaticonformational and compositional differences in samples.

The band near 1465 cm−1 (Fig. 3, band #14) isssigned to the CH2 bending mode. In half of the anhy-rous PSSL samples, water vapor bands did not interferend it was possible to measure the average center of masso be 1461.7 ± 0.7 cm−1, n = 5. The frequency at the cen-er of mass of the CH2 bending band was significantlyigher in the spectra of ML, 1467.4 ± 0.2 cm−1, n = 3,= 0.0007. The bandwidth at half peak height was alsoalculated by curve fitting the CH2 bending band regionnd calculated to be 21.3 ± 2.4 and 15.0 ± 3.6 cm−1 for
nhydrous PSSL and ML, respectively. The bandwidthsor the CH2 bending band were not significantly differ-nt, p > 0.05.
ig. 5. Infrared C–C rocking bands: (A) human meibum; (B) tear lipidsxtracted from Schirmer strips with tetrahydrofuran and methanol; (C)olar tear lipids extracted from Schirmer strips with methanol; (D)almitoyl sphingomyelin.

ics of Lipids 147 (2007) 87–102 93

The intensity of the bands #17 and 18 near 1250 cm−1

was relatively weak in the ML spectrum (Fig. 4A) com-pared with that seen in the trace for anhydrous SSL(C) and PSSL (D). The bands are in the frequencyregion of the PO2

− asymmetric stretching band, butmay also be due to C–O stretching modes (see Sec-tion 4). For PSSL, the center of mass of the PO2

−asymmetric stretching band labeled #17 (Fig. 4D),appeared at an average of 1266.5 ± 1.6 cm−1, n = 9and was significantly (p < 0.001) higher (20 cm−1) thanthe average center of mass for the ML band labeled#18 (Fig. 4A) at 1246.1 ± 0.1 cm−1, n = 3. The MLband was significantly (p = 0.001) broader, 22 ± 1 cm−1,n = 3 compared to the PO2

− asymmetric stretchingband for anhydrous SSL, with an average width of15.9 ± 0.8 cm−1, n = 9. No bands are present between1200 and 1400 cm−1 in the infrared spectrum of anhy-drous phosphorylcholine (data not shown), so bands17 and 18 in Fig. 4 are likely to arise from phospho-lipids. Two intense bands appear at 1079 and 1147 cm−1

in the infrared spectra of phosphorylcholine (data notshown).

The band(s) between 721 and 685 cm−1 (Fig. 5) areassigned to the methylene rocking modes. In the spec-trum of anhydrous PSSL (Fig. 5C), three CH2 rockingbands could be resolved by curve fitting with a centerof mass and bandwidth at half peak height in parenthe-sis at: 738.6 ± 3.4 (38.7 ± 8.7) cm−1, n = 9; 718.5 ± 1.6(14.2 ± 1.0) cm−1, n = 6; 695.9 ± 4.7 (19.5 ± 2.5) cm−1,n = 8. The CH2 stretching bands were near the lowerlimit of the frequency range for the infrared detector andbecause of difficulties determining the background inthis region, only some of the spectra provided reliablesignals. The value of n reflects the number of reliablemeasurements. One major CH2 rocking band was appar-ent in the spectra of ML (Fig. 5A) and SSL (B) centered at723.1 ± 0.8 cm−1, with a bandwidth at half peak heightof 16.2 = 0.6 cm−1, n = 3. A broad shoulder at 739 cm−1

is visible in the spectra of ML and SSL (A and B).In contrast, only one band at 720 cm−1 is present forsphingomyelin (D).

3.4.2. Influence of hydration of anhydrous PSSL onthe finger print region spectra

The CH2 rocking band region in the spectra ofhydrated PSSL was not significantly different from thatobserved for anhydrous PSSL spectra and displayed
bands with a center of mass and bandwidth at half peakheight in parenthesis at: 741 ± 10 (13.0 ± 4.5) cm−1,n = 3; 715.8 ± 6.4 (18.8 ± 9.1) cm−1, n = 3; 688.7 ± 1.8(31.6 ± 9.9) cm−1, n = 3.

nd Phys
The center of mass of the CH2 bending band wassignificantly higher in the spectra of hydrated PSSL,1466.6 ± 1.0 cm−1, n = 3, p = 0.004, in comparison toanhydrous PSSL, 1461.7 ± 0.7 cm−1, n = 5. The band-width at half peak height of the CH2 bending band wascalculated by curve fitting and calculated to be 21.3 ± 2.4and 14.2 ± 1.2 cm−1 for anhydrous and hydrated PSSL,respectively.

Curve fitting showed that upon hydration of PSSLwith HTA, the center of mass of the PO2

− asymmetricstretching band decreased significantly (p = 0.004) from1266.5 ± 1.6 (Fig. 4D, band #17) to 1260.5 ± 0.6 cm−1,n = 3 (E). The bandwidth at half peak height did notchange with hydration and averaged 14.6 ± 0.2 cm−1,n = 3. With hydration, the center of mass of thePO2

− symmetric stretching band shifted significantly,p = 0.006, from 1078.1 ± 1.1 cm−1, n = 9 in the anhy-drous PSSL (D, band #20) to 1087.2 ± 1.8 cm−1, n = 3(E). The band also broadened significantly, p = 0.004,with hydration from a peak width at half peak height of15.4 ± 2.3 cm−1, n = 9 in the anhydrous PSSL spectra to35.7 ± 12.9 cm−1, n = 3.

3.4.3. Temperature and the infrared C O andPO2

− regionsThe frequency of the C O stretching band

in the infrared spectra of PSSL changed from1737.6 ± 0.3 cm−1 at 15 ◦C to 1739.2 ± 0.1 cm−1

(±S.D. of the mean, p = 0.001) at 60 ◦C. In addition,a small but significant (p = 0.005) increase in the band-width from 13.2 ± 0.4 at 15 ◦C to 15.7 ± 0.9 at 60 ◦C wasobserved (Fig. 6). The PO2

− bands near 1270 (asymmet-

Fig. 6. Typical infrared C O stretching region spectra of polar tearlipids extracted from Schirmer strips with methanol. This region wasused to measure lipid composition and structure. (—) 60 ◦C; (- - -)35 ◦C; (—) 10 ◦C. Spectra show that carbonyl hydrogen bonding doesnot occur and that changes in carbonyl hydrogen bonding do not takeplace with the temperature-induced gel to liquid crystalline phase tran-sition of the hydrocarbon chains.

ics of Lipids 147 (2007) 87–102

ric stretch) and 1080 cm−1 did not change between thetemperature range of 15–60 ◦C (data not shown).

3.5. The influence of temperature, hydration andtear-fluid proteins on the dynamics of PSSL inferredfrom fluorescent probes

The fluorescent probe, NBD-PE, partitions into thelipid–water interface region. The motion (1/anisotropy)and environment (fluorescence intensity) of the fluo-rescent probe NBD-PE was measured to estimate themotion of lipids at the lipid–water interface regions. InPSSL hydrated with tears processed to remove mate-rial above 5 kDa, HTA, the fluorescence intensity andanisotropy of NBD-PE, decreased linearly with temper-ature and no sharp transition was evident (Fig. 7A and B(�)). A similar change with temperature was observedwith HTA except the fluorescence intensity was abouttwice as high (Fig. 2B (�)). At 32 ◦C, the magnitude ofthe increase in NBD-PE anisotropy (Fig. 7C, p < 0.001,n = 5) with HTA used as a hydrating medium comparedto RHT was less than that for the magnitude of the fluo-rescence intensity increase (Fig. 7D, p = 0.001, n = 5).

The anisotropy and fluorescence intensity of theprobe DPH increase due to its immobility and theexclusion of water, respectively. DPH partitions in thehydrophobic core region of lipids. Thus variations inthe anisotropy and fluorescence intensity of DPH sug-gest changes in lipid mobility and environment at thehydrophobic region. In hydrated PSSL, the decreasein the anisotropy of the DPH probe with increasingtemperature (Fig. 7E) could be fit to a four-parametersigmoidal function (Fig. 7F, Borchman et al., 2004),with c = 0.025 ± 0.001; b = −6.8 ± 0.6; T50 = 35.3 ± 0.5;Yo = 0.276 ± 0.001. The definition of the parameters are:the phase-transition temperature, Tm; magnitude of thetransition, a; the relative lipid cooperativity, c; minimumlipid order, Yo. Cooperativity is the ability of one lipidto influence the melting of an adjacent lipid. Highercooperativity results in a sharper phase transition curve.No change in the fluorescence intensity of DPH withtemperature was detected.

3.6. Comparison of infrared ML and PSSL lipidwith lipid standards

As stated earlier, the infrared spectrum of ML(Fig. 8B) closely corresponds to that of a wax (A).
Infrared bands at 1378, 1368, 840 and 799 cm−1 inthe spectrum of ML (B) corresponded to bands in theinfrared spectrum of cholesterol (C) suggesting thatwaxes and cholesterol predominate in ML. Numerous

D. Borchman et al. / Chemistry and Physics of Lipids 147 (2007) 87–102 95

Fig. 7. (A) Anisotropy (1/wobble) and (B) fluorescence intensity of the probe NBD-PE in Schirmer strip human tear lipids (�) hydrated with humantear aqueous without material above 5 kDa, (�) hydrated with human tears. NBD-PE partitions near the water–lipid interface. Lower fluorescenceintensity indicates exclusion of water. Lines are the best fit of a linear correlation. Data indicate that the dynamics of the temperature induced order todisorder phase transition for the hydrocarbon chain region does not influence the dynamics of the water–lipid interface region and that a componentin tears binds to the lipid excluding water at the lipid surface. Difference in (C) anisotropy and (D) fluorescence intensity of the probe NBD-PE inSchirmer strip human tear lipids (filled bar) hydrated with human tear aqueous without material above 5 kDa, or (open bar) hydrated with humantears. Data are the average plus the standard deviation of five experiments on the same sample at 32 ◦C. (E) Anisotropy of the DPH fluorescent probeDPH in hydrated polar lipids extracted from Schirmer strips with methanol. A higher anisotropy indicates lower mobility. Open and closed circlesrepresent two independent experiments of the same sample. Data were fit to a five-parameter sigmoidal function shown in (F). DPH partitions intothe hydrophobic domain of lipid hydrocarbon chains. (F) Plot of the four-parameter Hill curve-fitting equation used to fit the temperature–% lipidd of theY ipid to ii

itgS

3c

tSer

isorder data in (E). The phase transition temperature, Tc; magnitude

o, were calculated for data in (E). Cooperativity is the ability of one ln a sharper phase transition curve.

nfrared bands in the spectrum of SSL (Fig. 9C) matchhose corresponding to phosphatidylcholine (A), sphin-omyelin (B), cholesterol (E) and wax (D) suggestingSL may contain these lipids.

.7. Quantification of lipids excluded from a 5 kDaentrifugal cut-off filter

UV spectra from 200 to 450 nm were used to quan-
ify lipid. Light scattering (Fig. 10A) from emulsions ofSL (a) and ML (b) were more intense than that for themulsions of lipid standards (c–e) as evident from theelatively steep increase of the background from 350 to
transition, a; the relative lipid cooperativity, c; minimum lipid order,nfluence the melting of an adjacent lipid. Higher cooperativity results

450 nm. After correcting for light scattering, the inte-grated area of UV absorbance from 205 to 450 nm wasused to quantify lipid (e). The integrated area was linearwith concentration (B). The correlation coefficients forstandard curves using dimyristoylphosphatidylcholine(DMPC), palmitoyloleate (W), a mixture of W andDMPC, 1:1, mole:mole (W&PC), ML and SSL were0.9954, 0.9943, 0.9924, 0.9984, and 0.9959, respec-tively. Standard curves using the absorbance at 205 nm,
after correcting the spectra for light scattering, were evenmore linear than the standard curves prepared from thearea. Standard curves using DMPC, W, W&PC, ML andSSL gave correlation coefficients of: 0.9997, 0.9982,

96 D. Borchman et al. / Chemistry and Physics of Lipids 147 (2007) 87–102

Fig. 8. Infrared spectra of (A) palmityloleate; (B) spectra averagedfrom all meibum samples; (C) cholesterol; (D) bovine brain sphin-gomyelin; (E) dipalmitoylphosphatidylcholine; (F) palmitic acid. All

Fig. 9. Infrared spectra of: (A) dipalmitoylphosphatidylcholine; (B)bovine brain sphingomyelin; (C) spectra averaged from all Schirmer

mate tear-fluid volume but with questionable reliability

samples were placed onto a silver chloride window and dried in vacuo.Spectra were measured at 25 ◦C, except for (B) which was measuredwith an average temperature of 35 ◦C.

0.9979, 0.9988, and 0.9992, respectively. About 80% ofDMPC passed through the filter (C). The filter retained40% of ML and SSL. The most lipid retained by the filter,60%, was for the W and W&PC samples (C).

The wax, arachidylsterate and a mixture of
DMPC:arachidylsterate (1:1, w/w) would not form anemulsion. All other emulsions were studied immediatelybut, remained suspended for at least weeks.
Fig. 10. (A) UV absorbance spectra of lipids in methanol: (a) ML, SSL, Smeibum; (c) DMPC, dimyristoylphosphatidylcholine; (d) Wax, dimyristoylintegrated area of under the curve was used quantify the total amount of lipidlipid. Shaded bars, concentration of lipid in filtrate after passing through a 5equal amount by weight of DMPC and W.

strip lipid samples; (D) palmityloleate; (E) cholesterol; (F) palmiticacid. All samples were placed onto a silver chloride window and driedin vacu. Spectra were measured at 25 ◦C, except for (B) which wasmeasured at an average temperature of 35 ◦C.

4. Discussion

4.1. Schirmer strips as a source of tear-fluid lipid

Schirmer strips are routinely used clinically to esti-

(Cho and Yap, 1993a,b). The technique involves bend-ing the leading 5 mm of the Schirmer strip at a 90◦angle and subsequent placement over the edge of the

chirmer strip lipid extracted with tetrahydrofuran and methanol; (b)oleate; (e) spectrum of SSL after correcting for light scattering. The(B) SSL standard curve. (C) Solid bars, normalized concentration of

kDa cut-off centrifugal filter. Labels are defined in (A). W&PC is an

nd Phys

lSatiptptodelap

ilsssftoi

4cs

a32Tdhetc

tMwrach3iap


ower eyelid for 5 min. Tear film is wicked up the strip.chirmer strips from patients are readily available andre a potential source of tear lipid, however, composi-ional analysis should be interpreted cautiously sincenflammation induced by the strips cause changes inrotein composition (Stuchell et al., 1984b). It has yeto be determined if specific Schirmer strip lipids couldotentially serve as markers of specific dry eye classifica-ions (Foulks and Bron, 2003). We used SSL as a sourcef tear-fluid lipids. SSL were extracted with tetrahy-rofuran/methanol. Polar tear-fluid lipids (PSSL), werextracted with methanol, since these lipids are moreikely to be in an equilibrium state bound to proteinsnd are more likely to be associated with the aqueoushase.

The −5 mm portion of the tip of the Schirmer strip isn direct contact with the lid margin. We tested if meibumipid present on the lid margin, was detectable in infraredpectra of lipid extracted from the tip of the Schirmertrip. Based on the relative intensity of the infrared OHtretching and bending bands in ‘tip SSL and ‘no tip SSL’ractions (Fig. 1), we estimate that ML composes lesshan 5% of the ‘tip SSL’ lipid (Section 3.2). This amountf ML did not contribute to any major differences in thenfrared spectra.

.2. Are tear-fluid lipids different from meibum andan these differences be discerned by infraredpectroscopy?

At physiological temperature, the intensity of bandsssigned to the C C double bond CH stretch at010 cm−1, and the CH3 terminal methyl stretch at953 cm−1 were lower for SSL compared to ML (Fig. 1).hese differences suggest that the SSL contain fewerouble bonds and less hydrocarbons or perhaps longerydrocarbon chains compared to ML. Resistance tovaporation is dependent on the packing geometry ofhe hydrocarbon chains and is exponentially related tohain length (Quicken and Barnes, 1978).

The intensity of bands due to OH stretching vibra-ions, 3100–3400 cm−1, were very low or absent in the

L spectra compared to SSL (Fig. 2). Three bandsere resolved in the SSL spectra of the OH stretching

egion. Curve fitting showed these bands to be centeredt 3450, 3347 and 3200 cm−1. All of these peaks areharacteristic of hydrogen bonded OH moieties as non-ydrogen bonded OH bands appear between 3700 and
500 cm−1 (Dugan et al., 1949). Lipid hydroperoxidesn olive oil exhibit a band at 3447 cm−1 (van de Voort etl., 1994), fatty acids, such as oleic acid in olive oil dis-lay bands at 3310 and 3200 cm−1 (Ismail et al., 1993),
ics of Lipids 147 (2007) 87–102 97

however no bands were detectable in our spectra ofpalmitic acid (data not shown). The infrared spectra foranhydrous cholesterol, a potential tear lipid, showed OHstretchs at 3360 and 3440 cm−1 (data not shown). TheOH stretching band in the infrared spectra of anhydroussphingomyelin, another potential tear lipid, appears asa shoulder at 3360 cm−1 on the larger N–H stretchingbands centered at 3283 cm−1 (Lamba et al., 1991) whichalso has a shoulder at 3195 cm−1 (Fig. 1E).

The CH2 bending band is narrower and at a higherfrequency in the infrared spectra of ML (Fig. 3A) com-pared to those observed for SSL (C) and PSSL (D).In addition, the CH2 rocking band is also narrower inthe infrared spectrum of ML (Fig. 5A) compared to theinfrared spectra of SSL which exhibits three broad com-ponents (C). Splitting of CH2 bending bands may bedue to correlation field splitting and suggests that theorientation of the hydrocarbon chains is in an orthorhom-bic packing geometry (Zhang et al., 1997). Although asimilar splitting pattern was observed in the PSSL spec-tra, the greater broadening and lower frequencies of theCH2 bending bands could be the result of a less tightlypacked network of hydrocarbon chains. The basis forthis observation may become clearer pending analysisof the sample composition. We observe that the numberof gauche rotomers in the hydrocarbon chains is simi-lar for PSSL and ML (Table 2). Therefore, kinks in thehydrocarbon chains due to rotomers may not be responsi-ble for packing differences. It is reasonable to speculatethat the OH groups uniquely found in PSSL and SSL,if positioned near the hydrophobic region of the lipids,could disrupt lipid packing.

Bands in the 1600–1700 cm−1 region are evident inthe infrared CH2 spectra of PSSL (Fig. 3D) that are notobvious in the ML spectra (A). The 1600–1700 cm−1

region may contain bands from aldehydes, ketones, transdouble bonds (Ismail et al., 1993; van de Voort et al.,1994) and the amide I bands from sphingolipids (Lambaet al., 1991). In some spectra of PSSL, two bands at 1667and 1622 cm−1 were resolved.

Bands in the infrared spectral region of phosphatemoieties were much less intense in ML (Fig. 4A) spec-tra compared to PSSL spectra (D). This may be becauseML contain less phospholipids, 0–7% (Cory et al., 1973,Nicolaides et al., 1981; Tiffany, 1978), compared to SSLwhich may be representative of tear lipids that contain16% phospholipids (Wollensak et al., 1990). Based onthe close correspondence of the infrared spectrum of ML
(Fig. 4A) to that of a wax (B), it is probable that the bandsat 1245 and 1090 cm−1 are due to wax moieties ratherthan phosphate stretching bands. The center of mass ofthese bands were significantly higher and narrower in the

nd Phys

mation; van der Waals interactions and bilayer packing


spectra of SSL lipids compared to ML. Infrared bandsat 1378, 1368, 840 and 799 cm−1 in the spectrum of ML(Fig. 8B) corresponded to bands in the infrared spectrumof cholesterol (C) suggesting that waxes and cholesterolpredominate in ML. Numerous infrared bands in thespectrum of PSSL (Fig. 9C) match those correspondingto phosphatidylcholine (A), sphingomyelin (B), choles-terol (E) and wax (D) suggesting PSSL may contain theselipids.

The differences observed between the spectra ofPSSL and ML, indicate major compositional differencesbetween the lipids in tears and meibum. This is in agree-ment with chromatographic data (Nagyova and Tiffany,1999) and also consistent with the compositional dif-ferences reported in the only comprehensive studies
published that encompass the percentage of all speciesof wax esters, cholesterol and cholesterol esters, phos-pholipids and diglycerides for ML (Cory et al., 1973;
Fig. 11. Hypothetical diagram of Schirmer strip lipids as they could appear ingel phase and (bottom) disordered between the ordered (top) and disordered l

ics of Lipids 147 (2007) 87–102

Nicolaides et al., 1981; Tiffany, 1978) and tear-fluidlipids (Wollensak et al., 1990). Studies are underwayto quantify the differences between ML and SSL usingmatrix-assisted time-of-flight mass spectroscopy.

4.3. What are the molecular conformation anddynamics of tear lipids and how much do lipidhydrophilic and hydrophobic groups contribute tothem?

4.3.1. Temperature-induced changes in PSSLhydrocarbon chain dynamics and structure

When lipids are in an ordered state (gel phase), theCH2 in the hydrocarbon tails are in an all-trans confor-

are maximized. In the disordered liquid-crystallinephase, with the introduction of gauche rotomers, van derWaals interactions and bilayer packing are diminished

an infrared spectrometer with (top) ordered hydrocarbon chains in aiquid crystalline phase (bottom).

nd Phys

(fia

igiut1bAtbm(motscidtwt

2emTa(

oaDatdhwttg2

4r

rw


Fig. 11). When lipid is heated, or pressure on a lipidlm is diminished, lipids may undergo a transition fromn ordered state to a disordered one.

The CH2 stretching bands are predominant in thenfrared spectra of lipids due to the large number of CH2roups in their hydrocarbon tails. The symmetric stretch-ng frequency of the CH2 band near 2850 cm−1 may besed to estimate the gauche to trans to rotomer con-ent of the hydrocarbon chains (Borchman et al., 1991,999, 2004; Kota et al., 1999). The frequency of thisand increases with the number of gauche rotomers.t 32 ◦C, about 55% of the CH2 rotomers were in the

rans conformation which indicates that the hydrocar-on chains are about half ordered (Table 2). Because theobility of lipid could affect its delivery, the motion

1/anisotropy) of DPH was measured to estimate theotion of lipids in the hydrophobic hydrocarbon region

f hydrated PSSL. The anisotropy of the DPH probehat partitions into the hydrophobic region exhibited aharp transition with increasing temperature which indi-ates that the motion of the hydrocarbon chain regionncreases with an increase in the hydrocarbon structuralisorder (Fig. 7E). The order to disorder phase transitionemperature of PSSL was centered at 35 ◦C, in agreementith the infrared spectroscopic results that indicate that

he lipids are 55% ordered (Table 2).Lipid hydrocarbon chain order did not change below

5 ◦C or above 40 ◦C so when physically warming theye lid margin as a therapy to increase ML delivery to theargin (Harrison and Lawlor, 1998; Mori et al., 1999;hygeson, 1946), the temperature must be above 25 ◦Cnd there is little value in heating the eye lid above 40 ◦CFig. 7E).

The temperature range at which the phase transitionccurs (20–40 ◦C, see Fig. 7E) matches that visu-lly assessed and reported earlier for ML (Brown andervichian, 1969; Linton, 1961; Nicolaides, 1965; Ong

nd Larke, 1990; Tiffany and Marsden, 1986). As theemperature increased from 25 to 45 ◦C, a significantecrease in the refractive index has been reported foruman ML (Tiffany, 1986). In line with these trends,e found that lipid hydrocarbon motion increased when

he temperature was raised from 25 to 40 ◦C (Fig. 7E),he same temperature range lipid delivery to the mar-ins were observed to increase (Nagymihalyi et al.,004).

.3.2. Temperature-induced changes in PSSL polar
egion dynamics and structure
The motion (1/anisotropy) and environment (fluo-escence intensity) of the fluorescent probe NBD-PEas measured to estimate the motion of PSSL at the

ics of Lipids 147 (2007) 87–102 99

lipid–water interface regions. The fluorescence inten-sity and anisotropy of NBD-PE, that partitions intothe lipid–water interface region, decreased linearly withtemperature and no sharp transition was evident (Fig. 7Aand B). The decrease in fluorescence intensity withtemperature indicates that water penetrates deeper intothe lipid interface region with increasing temperature.Motion at the interface region (A and B) did not fol-low the sharp change observed in the conformation andmotion of the hydrocarbon chain region (E). The gradualchange (A and B) may be the result of increased motiondue to increased thermal energy rather than a relativelysharper change in conformation.

The infrared C O and PO2− bands, presumed to

be in the interfacial and headgroup region, did notchange significantly with increasing temperature, sug-gesting that these moieties may not play an appreciablethermodynamic role in the phase transition. This isin agreement with the dynamic changes (Fig. 7A andB). The frequency of the infrared C O band is rela-tively high (1738 cm−1), and suggests that the carbonylsare not involved in strong hydrogen bonds. The C Ostretching frequency and band width increased by only2 cm−1 between 15 and 60 ◦C (Fig. 6). In compar-ison, the difference between hydrogen bonded andnon-hydrogen bonded carbonyls could be 10 timeslarger (Mushayakarara et al., 1986). The lack of sig-nificant changes in these regions is possibly due totheir dehydrated state or unique lipid composition. Inother systems, such as membranes prepared from sphin-gomyelin in an aqueous medium, significant temperatureinduced changes in the infrared bands corresponding tothe head group and interface regions have been reportedand attributed to alterations in hydrogen bonding inter-actions (Rujoi et al., 2002).

4.3.3. Hydration-induced changes in SSLHydration of PSSL with tears processed to remove

material greater than 5 kDa caused significant changesin molecular structure. Notably, lipid hydrocarbon chainorder at 32.3 ◦C increased from 54 to 66% (Table 2).It is clear that interactions between PSSL and tearaqueous take place. Since water does not interact withthe hydrophobic hydrocarbons studied in this project,it must affect hydrophilic moieties in the head groupor interfacial region. Interactions between tear lipidand components of tear aqueous phase are believed tobe responsible for the surface tension of human tears
(Nagyova and Tiffany, 1999) and viscosity (Gouveia andTiffany, 2005). Spectroscopic studies are in progress inour laboratory to assess the molecular nature of theseinteractions.

nd Phys
Upon hydration, the asymmetric PO2− band observed

for the PSSL shifted to lower frequencies (Fig. 4) indi-cating more hydrogen bonding in the head group regionwith hydration. A similar trend was observed for thehydration of other model phospholipid systems (Goniand Arrondo, 1986; Hadzi et al., 1992; Pohle et al.,2004; Rujoi et al., 2002). The asymmetric PO2

− bandstretching frequency for anhydrous PSSL at 1266 cm−1

(Fig. 4D) was comparable to that for anhydrous phos-phatidylcholine. Upon hydration of phosphatidylcholine(Pohle et al., 2004) and sphingomyelin (Lamba et al.,1991; Rujoi et al., 2002) the frequency of the PO2

−asymmetric stretching infrared band shifts to 1235 cm−1,25 cm−1 lower than that for hydrated SSL indicatingthat phosphate group hydrogen bonding is not as strongin hydrated SSL compared to model systems. In con-trast to the PO2

− asymmetric band, the PO2− symmetric

stretching band center of mass shifted significantly tohigher frequencies with and broadened with hydration.This change is consistent with conformational changesof the phosphate group upon hydration or changes inthe overlapping C–O–P–O–C stretching band. In conclu-sion, although polar moieties do not influence tear lipidhydrocarbon chain order in the anhydrous state, they maybe important in tear-fluid interactions and interactionswith proteins, as discussed in the following section.

4.4. Is it possible for tear lipids to partition into theaqueous tear-fluid and do tear proteins have thecapacity to bind to them?

Lipocalin, present in tears is capable of binding tolipid. Lipocalin sequesters 99.5% of fatty acids in the tearfilm (Gasymov et al., 1999). Lipocalin also sequesterscholesterol, fatty acids, glycolipids and glycerophos-pholipids (Glasgow et al., 1995). Lipocalin may besecreted from the lacrimal gland completely saturatedwith lacrimal lipids (Nagyova and Tiffany, 1999). Con-formational changes in lipocalin are evident when lipidbinds (Gasymov et al., 1998). It has been proposedthat lipocalin scavenges lipid from the corneal surface(Glasgow et al., 1999) and may enhance the transport andequilibration of lipid in the lipid surface layer. Gouveiaand Tiffany (2005) report that there are no free lipids intear fluid.

In this study we tested if it is possible for free lipids toenter the tear fluid and if tear proteins could bind to theselipids, even though they are saturated with lacrimal lipids
(Nagyova and Tiffany, 1999). We found that severallipids (SSL, ML, DMPC, W, W and DMPC), poten-tially free in tears, could form emulsions that were stablefor weeks. The wax emulsions were very viscous. One
ics of Lipids 147 (2007) 87–102

approach to measuring lipid–protein binding is to sepa-rate free from bound lipids. We did this by centrifugationto quantify �-crystallin binding to lens lipids (Gramiet al., 2005; Tang et al., 1998b). Gouveia and Tiffany(2005) used a centrifugal filter to separate bound fromfree lipids. They measured lipids qualitatively, and foundthat all of the free phospholipids passed through the filterand that lipids bound to protein did not. We found that80% of DMPC passed through the filter, in agreementwith their findings (Fig. 10C). Unfortunately, waxes arethe predominant lipid in tears (Wollensak et al., 1990)and only 40% of waxes, free from protein, pass thor-ough the filter. When DMPC is mixed with wax, it too isretained by the filter. About 40% of free SSL and ML areretained by the filter. In our hands the use of a centrifugalfilter to quantify the amount of free and protein-boundlipid was not practical.

Another approach, using a fluorescent probe to char-acterize the binding of �-crystallin and �2GP1 to lipidswas successful (Borchman and Tang, 1996a; Borchmanet al., 1996b; Tang et al., 1998a,b). We used the sametechnique to measure binding of PSSL to tear-fluid pro-teins in this study. The over two-fold increase in NBD-PEfluorescence intensity when PSSL was hydrated withRHT compared with hydration with HTA (Fig. 7B–D)is because components in RHT that are absent in HTAbind to the surface of PSSL and exclude water from theprobe environment, a process that results in an increasein NBD-PE fluorescence intensity. If similar interactionsoccur in vivo at the tear-film lipid–aqueous interface,they would reduce the rate of evaporation. Mucins,lysozyme, lipocalin and other proteins present in RHTand not in HTA could potentially bind to PSSL. Tear-fluid proteins could potentially bind to the lipids at theinterface between the lipid-tear-film and aqueous inter-face or to lipids present in tears that are free for shortor long periods of time. Although the presence of freelipids in tears is unlikely, this possibility has yet to bedetermined. We are currently applying this technique tomeasure the binding constants of a variety of lipids withthe major tear-fluid proteins.

This work highlights the power of infrared andfluorescence spectroscopes to characterize molecularstructure/conformation, dynamics and packing of humanML and SSL. These spectroscopic techniques andothers, including mass spectrometry and nuclear mag-netic resonance will be applied next to study tear-filmlipid composition–structure–function relationships and
lipid–protein interactions in relation to age, sex and dryeye symptoms. These future studies could provide a basisof knowledge necessary to develop therapies to abrogatedry eye symptoms.

nd Phys

A

ELRp

R

B

B

B

B

B

B

B

C

C

C

C

C

C

C

D

F

F

G


cknowledgements

Supported by Public Health Service research grantY017094-01 (Bethesda, MD, USA) the Kentuckyions Eye Foundation, an unrestricted grant fromesearch to Prevent Blindness Inc. and The Jewish Hos-ital Foundation of Louisville.

eferences

orchman, D., Tang, D., 1996a. Binding capacity of alpha-crystallinto bovine lens lipids. Exp. Eye Res. 63, 407–410.

orchman, D., Cenedella, R.I., Lamba, O.P., 1996b. Role of cholesterolin the structural order of lens lipids. Exp. Eye Res. 62m, 191–197.

orchman, D., Tang, D., Yappert, M.C., 1999. Lipid composition,membrane structure relationships in lens and muscle sarcoplasmicreticulum membranes. Biospectroscopy 5, 151–167.

orchman, D., Yappert, M.C., Afzal, M., 2004. Lens lipids and maxi-mum lifespan. Exp. Eye Res. 79, 761–768.

orchman, D., Yappert, M.C., Herrell, P., 1991. Structural character-ization of human lens membrane lipid by infrared spectroscopy.Invest. Ophthalmol. Vis. Sci. 32, 2404–2416.

rown, S.I., Dervichian, D.G., 1969. The oils of the meibomianglands. Physical and surface characteristics. Arch. Ophthalmol.82, 537–540.

yrdwell, W.C., Sato, H., Schwarz, A.K., Borchman, D., Yappert,M.C., Tang, D., 2002. 31P NMR quantification and monophasic sol-vent purification of human and bovine lens phospholipids. Lipids37, 1087–1092.

enedella, R.J., Jacob, R., Borchman, D., Tang, D., Neely, A.R.,Samadi, A., Mason, R.P., Sexton, P., 2004. Direct perturbation oflens membrane structure may contribute to cataracts caused byU18666A, an oxidosqualene cyclase inhibitor. J. Lipid Res. 45,1232–1241.

hew, C.K., Jansweijer, C., Tiffany, J.M., Dikstein, S., Bron, A.J.,1993. An instrument for quantifying meibomian lipid on the lidmargin: the meibometer. Curr. Eye Res. 12, 247–254.

ho, P., Yap, M., 1993a. Schirmer test. I. A review. Opt. Vis. Sci. 70,152–156.

ho, P., Yap, M., 1993b. Schirmer test. II. A clinical study of itsrepeatability. Opt. Vis. Sci. 70, 157–159.

ory, C.C., Hinks, W., Burton, J.L., Shuster, S., 1973. Meibomiangland secretion in the red eyes of rosacea. Br. J. Dermatol. 89,25–27.

raig, J.P., Blades, K., Patel, S., 1995. Tear lipid layer structure andstability following expression of the meibomian glands. Ophthal.Physiol. Opt. 15, 569–574.

raig, J.P., Tomlinson, A., 1997. Importance of the lipid layer in humantear film stability and evaporation. Optom. Vis. Sci. 74, 8–13.

ugan, L.R., Beadle, B.W., Henick, A.S., 1949. An infrared absorptionstudy of autoxidized methyl linoleate. J. Am. Oil Chem. Soc. 26,681–685.

oulks, G.N., Bron, A.J., 2003. Meibomian gland dysfunction: a clin-ical scheme for description, diagnosis, classification, and grading.Ocul. Surf. 1, 107–126.

rey, W.H., DeSota-Johnson, D., Hoffman, C., McCall, J.T., 1981.Effect of stimulus on the chemical composition of human tears.Am. J. Ophthalmol. 92, 559–567.

asymov, O.K., Abduragimov, A.R., Yusifov, T.N., Glasgow, B.J.,1999. Binding studies of tear lipocalin: the role of the conserved

ics of Lipids 147 (2007) 87–102 101

tryptophan in maintaining structure, stability and ligand affinity.Biochim. Biophys. Acta 1433, 307–320.

Gasymov, O.K., Abduragimov, A.R., Yusifov, T.N., Glasgow, B.J.,1998. Structural changes in human tear lipocalins associated withlipid binding. Biochim. Biophys. Acta 1386, 145–156.

Glasgow, B.J., et al., 1999. Tear lipocalins: potential lipid scav-engers for the corneal surface. Invest. Ophthalmol. Vis. Sci. 40,3100–3107.

Glasgow, B.J., Abduragimov, A.R., Farahbakhsh, Z.T., Faull, K.F.,Hubbell, W.L., 1995. Tear lipocalins bind a broad array of lipidligands. Curr. Eye Res. 14, 363–372.

Goni, F.M., Arrondo, J.L., 1986. A study of phospholipid phosphategroups in model membranes by Fourier transform infrared spec-troscopy. Faraday Discuss. Chem. Soc. 81, 117–126.

Gouveia, S.M., Tiffany, J.M., 2005. Human tear viscosity: an inter-active role for proteins and lipids. Biochim. Biophys. Acta 1753,155–163.

Grami, V., Marrero, Y., Huang, L., Tang, D., Yappert, M.C., Borch-man, D., 2005. alpha-Crystallin binding in vitro to lipids from clearhuman lenses. Exp. Eye Res. 81, 138–146.

Greiner, J.V., Glonek, T., Korb, D.R., Leahy, C.D., 1996. Meibomiangland phospholipids. Curr. Eye Res. 15, 371–375.

Haberich, F.J., 1981. Der praekorneale tranenfilm (PKTF). Dtsch. Opt.Ztg., 24–33.

Hadzi, D., Hodoscek, J., Grdadolnik, J., Avbelj, F., 1992. Inter-molecular effects on phosphate frequencies in phospholipids:infrared study and ab initio model calculation. J. Mol. Struct. 266,9–19.

Harrison, D.A., Lawlor, D., 1998. Experiences in treating patients forblepharitis. Arch. Ophthalmol. 116, 1133–1134.

Imai, S., Tsuge, N., Tomotake, M., Nagatome, Y., Sawada, H., Nagata,T., Kumagaiet, H., 2002. Plant biochemistry: an onion enzyme thatmakes the eyes water. Nature 419, 685.

Ismail, A.A., van de Voort, F.R., Emo, G., Sedman, J., 1993. Rapidquantitative determination of free fatty acids in fats and oils byFourier transform infrared spectroscopy. J. Am. Oil Chem. Soc.70, 335–341.

Iwata, S., Lemp, M.A., Holly, F.J., Dohlman, C.H., 1969. Evaporationrate of water from the precorneal tear film and cornea in the rabbit.Invest. Ophthalmol. 8, 613–619.

Kilp, H., Schmid, E., Kirchner, L., Zipf-Pohl, A., 1986. In: Holly, F.J.(Ed.), The preocular tear film in health, disease and contact lenswear. The Dry Eye Institute, Lubbock, TX, p. 564.

Korb, D.R., Baron, D.F., Herman, J.P., Finnemore, V.M., Exford, J.M.,Hermosa, J.L., Leahy, C.D., Glonek, T., Greiner, J.V., 1994. Tearfilm lipid layer thickness as a function of blinking. Cornea 13,354–359.

Kota, Z., Debreczeny, M., Szalontai, B., 1999. Separable contributionsof ordered and disordered lipid fatty acyl chain segments to �CH2

bands in model and biological membranes: a Fourier transforminfrared spectroscopic study. Biospectroscopy 5, 169–178.

Lamba, O.P., Borchman, D., Sinha, S.K., Lal, S., Yappert, M.C., Lou,M.F., 1991. Structure and molecular conformation of anhydrousand of aqueous sphingomyelin bilayers determined by infrared andRaman Spectroscopies. J. Mol. Struct. 248, 1–24.

Linton, R.G., 1961. The meibomian glands: an investigation into thesecretion and some aspects of the physiology. Br. J. Ophthalmol.
45, 718–723.
Mathers, W.D., 1993. Ocular evaporation in meibomian gland dys-function and dry eye. Ophthalmology 100, 347–351.

Mathers, W.D., Daley, T.E., 1996. Tear flow and evaporation in patientswith and without dry eye. Ophthalmology 103, 664–669.

nd Phys
Milder, B., 1987. Clinical applications. In: Mossa, F., Hart, W.M.(Eds.), Adler’s Physiology of the Eye. Mosby Co., St Louis, pp.15–35.

Mishima, S., Maurice, D.M., 1961. The oily layer of the tear film andevaporation from the corneal surface. Exp. Eye Res. 1, 39–45.

Mori, A., Oguchi, Y., Goto, E., Nakamori, K., Ohtsuki, T., Egami, F.,Shimazaki, J., Tsubota, K., 1999. Efficacy and safety of infraredwarming of the eyelids. Cornea 18, 188–193.

Mushayakarara, E.C., Wong, P.T., Mantsch, H.H., 1986. Detection byhigh pressure infrared spectrometry of hydrogen-bonding betweenwater and triacetyl glycerol. Biochem. Biophys. Res. Commun.134, 140–145.

Nagymihalyi, A., Dikstein, S., Tiffany, J.M., 2004. The influence ofeyelid temperature on the delivery of meibomian oil. Exp. Eye Res.78, 367–370.

Nagyova, B., Tiffany, J.M., 1999. Components responsible for thesurface tension of human tears. Curr. Eye Res. 19, 4–11.

Nicolaides, N., 1965. Skin lipids II. Lipid class composition of samplesfrom various species and anatomical sites. J. Am. Oil Chem. Soc.42, 691–702.

Nicolaides, N., Kaitaranta, J.K., Rawdah, T.N., Macy, J.I., Boswell III,F.M., Smith, R.E., 1981. Meibomian gland studies: comparisonof steer and human lipids. Invest. Ophthalmol. Vis. Sci. 20, 522–536.

Nicolaides, N., Santos, E.C., 1985. The di- and triesters of the lipidsof steer and human meibomian glands. Lipids 20, 454–467.

Ong, B.L., Larke, J.R., 1990. Meibomian gland dysfunction: some clin-ical, biochemical and physical observations. Ophthalm. Physiol.Opt. 10, 144–148.

Pandit, J.C., Nagyova, B., Bron, A.J., Tiffany, J.M., 1999. Physicalproperties of stimulated and unstimulated tears. Exp. Eye Res. 68,247–253.

Pohle, W., Gauger, D.R., Bohl, M., Mrazkova, E., Hobza, P., 2004.Lipid hydration: headgroup CH moieties are involved in waterbinding. Biopolymers 74, 27–31.

Quicken, T.I., Barnes, G.T., 1978. Evaporation through monolayers-theoretical treatment of effect of chain-length. J. Colloid Interf.Sci. 67, 415–422.

Rujoi, M., Borchman, D., DuPre, D.B., Yappert, M.C., 2002. Inter-actions of Ca2+ with sphingomyelin and dihydrosphingomyelin.Biophys. J. 82, 3096–3104.

Stuchell, R.N., Slomian, B.L., Jowiale, Z., Marty, V.L.N., Siomi-any, A., Farris, R.L., 1984a. Lipid composition of human tears.

ics of Lipids 147 (2007) 87–102

Meeting Abstract. Invest. Ophthalmol. Vis. Sci. 25 (Suppl.),320.

Stuchell, R.N., Feldman, J.J., Farric, R.L., Mandel, I.D., 1984b. Theeffect of collection technique on tear composition. Invest. Ophthal-mol. Vis. Sci. 25, 374–377.

Tang, D., Borchman, D., 1998. Temperature induced structural changesof beta-crystallin and sphingomyelin binding. Exp. Eye Res. 67,113–118.

Tang, D., Borchman, D., Harris, N., Pierangeli, S., 1998a. Lipid inter-actions with human antiphospholipid antibody, beta 2-glycoprotein1, and normal human IgG using the fluorescent probes NBD-PEand DPH. Biochim. Biophys. Acta 1372, 45–54.

Tang, D., Borchman, D., Yappert, M.C., Cenedella, R.J., 1998b. Influ-ence of cholesterol on the interaction of alpha-crystallin withphospholipids. Exp. Eye Res. 66, 559–567.

Tang, D., Borchman, D., Yappert, M.C., Vrensen, G.F., Rasi, V., 2003.Influence of age, diabetes, and cataract on calcium, lipid–calcium,and protein–calcium relationships in human lenses. Invest. Oph-thalmol. Vis. Sci. 44, 2059–2066.

Thygeson, P., 1946. The etiology and treatment of blepharitis: a studyin military personnel. Milit. Surg. 98, 191–200.

Tiffany, J.M., Marsden, R.G., 1986. The influence of composition onphysical properties of meibomian secretion. In: Holly, F.J. (Ed.),The Preocular Tear Film in Health, Disease and Contact Lens Wear.Dry Eye Institute, Lubbock, TX, pp. 597–608.

Tiffany, J.M., 1978. Individual variations in human meibomian lipidcomposition. Exp. Eye Res. 27, 289–300.

Tiffany, J.M., 1986. Refractive index of meibomian and other lipids.Curr. Eye Res. 5, 887–889.

van de Voort, F.R., Ismail, A.A., Sedman, J., Emo, G., 1994. Moni-toring the oxidation of edible oils by Fourier transform infraredspectroscopy. J. Am. Oil Chem. Soc. 71, 242–253.

Van Haeringen, N.J., 1981. Clinical biochemistry of tears. Surv. Oph-thalmol. 26, 84–96.

Wolff, E., 1946. Muco-cutaneous function of the lid margin and the dis-tribution of the tear fluid. Trans. Opthtalmol. Soc. UK 66, 291–308.

Wollensak, G., Mur, E., Mayr, A., Baier, G., Gottinger, W., Stoffler, G.,1990. Effective methods for the investigation of human tear film
proteins and lipids. Graefes Arch. Clin. Exp. Ophthalmol. 228,78–82.
Zhang, Y.P., Lewis, R.N., McElhaney, R.N., 1997. Calorimetric andspectroscopic studies of the thermotropic phase behavior of the n-saturated 1,2-diacylphophatidylglycerols. Biophys. J. 72, 779–793.

Documents

Spectroscopic evaluation of human tear lipids