we obtained cdcl3 , sq , tetramethylsilane ( tms ) , and reagents from sigma - aldrich corp .
meibomian glands were expressed by compressing the eyelid between cotton - tipped applicators with strict attention to avoid touching the eyelid margin during expression .
all four eyelids were expressed , and approximately 0.5 mg meibum ( ml ) was collected per individual for direct spectroscopic study .
the ml was collected with a platinum spatula and dissolved in a vial of chloroform .
the subjects for nmr spectroscopic analysis were recruited from the kentucky lion 's eye center ( louisville , ky , usa ) .
normal status was assigned when the subject 's meibomian gland orifices showed no evidence of keratinization or plugging with turbid or thickened secretions , and no dilated blood vessels were observed on the eyelid margin . lipid absorbent tape ( sebutape ; cuderm corp . , dallas , tx , usa ; fig .
2a ) is a microporous film that was designed to collect sebum from the skin .
the tape ( cuderm corp . ) was pressed for 45 seconds onto the nose to collect sebum .
as the sq content in the nose and eyelid skin sebum is presumably identical , sebum from the skin of the nose was used because it is easier to collect higher sample amounts suitable for repetitive measurements .
sebum was collected from a 59-year - old caucasian donor once in the morning and once at night for a period of a week .
a total of 56 tape ( cuderm corp . ) samples were removed from the cardboard backing and placed directly into a 15-ml glass scintillation vial containing 5 ml chloroform .
the samples were sonicated under an atmosphere of argon gas in an ultrasonic bath ( branson 1510 ; branson ultrasonics , danbury , ct , usa ) for 10 minutes .
the tape ( cuderm corp . ) was removed from the vial and placed into another vial containing 5 ml chloroform .
the tape ( cuderm corp . ) was removed and the chloroform from the two extractions was mixed and the chloroform was evaporated under a stream of nitrogen gas .
the sample was sonicated under an atmosphere of argon gas in an ultrasonic bath ( branson ultrasonics ) for 10 minutes and placed into an nmr tube for spectral measurement .
( a ) sebutape ( cuderm corp . ) is a thin lipid absorbent surface that covers the gray box shown in the figure on a cardboard backing .
sebum lipid absorbed into the tape after pressing the tape onto the forehead is visible as dark dots on the gray surface .
the length of the cardboard backing is 4.5 cm . the tape ( cuderm corp . )
can be removed from the cardboard backing and the amount and composition of lipid can be measured directly using infrared spectroscopy , or extracted and quantified by nmr spectroscopy as in this study or quantified chemically .
( b ) sebum was also collected using a stainless steel blackhead remover ( revlon , inc . ) by rubbing the nose and forehead with the remover .
, new york , ny , usa ) by rubbing the nose and forehead with the blackhead remover ( revlon , inc . ; fig .
sebum was collected from the same 59-year - old caucasian donor from which sebum was collected with tape ( cuderm corp . ) .
the sebum that accumulated in the small holes of the device was dislodged from the remover by placing it into a weighed 15-ml glass scintillation vial containing 2 ml chloroform and sonicated under an atmosphere of argon gas in an ultrasonic bath ( branson ultrasonics ) for 10 minutes .
we added cdcl3 ( 500 l ) to the 11.9 mg of collected sebum lipid .
the sample was sonicated under an atmosphere of argon gas in an ultrasonic bath ( branson ultrasonics ) for 10 minutes and placed into an nmr tube for spectral measurement .
spectral data were acquired using a spectrometer ( varian vnmr 700 mhz nmr ; varian , lexington , ma , usa ) equipped with a 5 mm h{c / n } c  enhanced cold probe ( varian , palo alto , ca , usa ) .
spectra were acquired with a minimum of 250 scans , 45 pulse width , and a relaxation delay of 1.000 seconds .
we performed hsqc using 512 increments with 16 scans per increment , 45 pulse width , and a relaxation delay of 1.000 seconds , mixing time of 0.080 seconds , and a one - bond coupling constant threshold of 140 hz .
spectra were analyzed using chemistry software ( mestrenova , version 7.1.2 - 10008 ; mestrelab research s.l .
, salem , nh , usa ) was used for spectral deconvolution and curve fitting .
the cell was jacketed by an insulated water coil connected to a circulating water bath ( model r-134a ; neslab instruments , newton nh , usa ) .
the sample temperature was measured and controlled by a thermistor touching the sample cell window .
the water bath unit was programmed to measure the temperature at the thermistor and to adjust the bath temperature so that the sample temperature could be set to the desired value .
the rate of heating or cooling ( 1c/15 minutes ) at the sample was also adjusted by the water bath unit .
infrared spectra were measured using a fourier transform infrared spectrometer ( nicolet 5000 magna series ; thermo fisher scientific , inc .
phase transitions were run four times with care taken to obtain data points in the sharp phase - transition temperature region .
the frequency of the ch2 symmetric ch2 stretching band near ( 2850 cm ) was used to estimate the content of trans and gauche rotamers in the hydrocarbon chains .
although the 2954 cm asymmetric ch2 stretching band is useful for measuring phase - transition parameters , we chose to use the 2850 cm band rather than the band near 2954 cm , because measurement of the asymmetric band frequency is complicated by the adjacent ch3 symmetric stretching band near 2955 cm and the ch2 symmetric stretching band near 2852 cm .
the symmetric stretch was calculated by first baseline leveling the oh - ch stretching region between 3500 and 2700 cm . the center of mass of the ch2 symmetric stretching band , sym ,
the baseline for integrating the top 10% of the intensity of the band was parallel to the oh - ch region baseline .
lipid ch2 groups in the hydrocarbon chains are present as gauche rotamers , prevalent in disordered hydrocarbon chains , or trans rotamers , more abundant in ordered hydrocarbon chains .
thus , lipid hydrocarbon chain order may be evaluated in terms of the relative amount of ch2 
 trans rotamers .
the frequency of the ch2 symmetric stretch is dependent on the amount of trans or gauche rotamers and has been used to characterize lipid phase transitions and to measure the trans rotamer content of lipid hydrocarbon chains with changes in temperature . since rotamers are either in trans or gauche conformations , phase transitions can be described by a two - state sigmoidal equation , as described by borchman et al .
lipid order at 33.4c was calculated by extrapolating the sym at 33.4c from the fit of the phase transition and then converting sym to the percentage of trans rotamers , a measure of lipid conformational order .
the data for percentage of trans rotamers were used to calculate the phase - transition enthalpy and entropy from the slopes of arrhenius plots , as described in borchman et al .
surface pressure - area profiles of human meibum and human sebum were recorded using a computer - controlled single barrier langmuir teflon trough ( nima 102 m ; nima technology ltd . ,
the surface pressure was measured by a pressure sensor with a wilhelmy plate ( whatman , chr 1 filter paper ) .
the trough was enclosed in a transparent pmma ( perspex ; lucite international , lancashire , uk ) cabinet to avoid air currents and dust particles .
the trough was filled with an artificial tear ( at ) solution that emulated the salt composition of human tears ( nacl 6.6
g / l ; cacl2.2h2o 0.15 g / l ; nah2po4.h2o 0.1g / l ; mops 4.18
purified water ( milli - q , resistance > 18.2 m ; millipore corp . , billerica , ma , usa ) was used for preparing the at solution .
the at solution surface was cleaned with a vacuum aspirator until a clean surface was achieved ( pressure change
< 0.02 mn / m when the surface area was compressed and expanded completely ) .
the lipid sample in chloroform was spread dropwise on the surface of the at solution using a microsyringe ( hamilton co. , bonaduz , switzerland ) and chloroform was allowed to evaporate for 10 minutes .
the lipid film was compressed and expanded with a barrier speed of 15 cm / minute and changes in pressure (  ) with area ( a ) were recorded as -a profiles .
the sebum -a profiles ( 23.8 mg / ml ) and meibum ( 1 mg / ml ) alone was recorded .
sebum was mixed with meibum ( s : m ) at mole fraction ratios : 0.25:0.75 , 0.70:0.30 , and 0.97:0.03 ( based on estimated molecular weights 490 and 720 g / mole , respectively ) and -a profiles of mixtures were recorded .
the excess area of mixing ( aex ) of sebum and meibum in the mixed films was determined by the following equation : 
 where a1,2 , a1 , and a2 are the molecular areas of the mixture and the pure components , and x1 and x2 are the mole fractions of the two components in the mixture .
a negative value means molecules in the mixture occupy less surface area than expected ( condensation effect ) , while a positive value indicates the opposite , that is , an expansion effect . 
 
we obtained cdcl3 , sq , tetramethylsilane ( tms ) , and reagents from sigma - aldrich corp .
meibomian glands were expressed by compressing the eyelid between cotton - tipped applicators with strict attention to avoid touching the eyelid margin during expression .
all four eyelids were expressed , and approximately 0.5 mg meibum ( ml ) was collected per individual for direct spectroscopic study .
the ml was collected with a platinum spatula and dissolved in a vial of chloroform .
the subjects for nmr spectroscopic analysis were recruited from the kentucky lion 's eye center ( louisville , ky , usa ) .
normal status was assigned when the subject 's meibomian gland orifices showed no evidence of keratinization or plugging with turbid or thickened secretions , and no dilated blood vessels were observed on the eyelid margin .
lipid absorbent tape ( sebutape ; cuderm corp . , dallas , tx , usa ; fig .
2a ) is a microporous film that was designed to collect sebum from the skin .
the tape ( cuderm corp . ) was pressed for 45 seconds onto the nose to collect sebum .
as the sq content in the nose and eyelid skin sebum is presumably identical , sebum from the skin of the nose was used because it is easier to collect higher sample amounts suitable for repetitive measurements .
sebum was collected from a 59-year - old caucasian donor once in the morning and once at night for a period of a week .
a total of 56 tape ( cuderm corp . ) samples were removed from the cardboard backing and placed directly into a 15-ml glass scintillation vial containing 5 ml chloroform .
the samples were sonicated under an atmosphere of argon gas in an ultrasonic bath ( branson 1510 ; branson ultrasonics , danbury , ct , usa ) for 10 minutes .
the tape ( cuderm corp . ) was removed from the vial and placed into another vial containing 5 ml chloroform .
the tape ( cuderm corp . ) was removed and the chloroform from the two extractions was mixed and the chloroform was evaporated under a stream of nitrogen gas .
the sample was sonicated under an atmosphere of argon gas in an ultrasonic bath ( branson ultrasonics ) for 10 minutes and placed into an nmr tube for spectral measurement .
( a ) sebutape ( cuderm corp . ) is a thin lipid absorbent surface that covers the gray box shown in the figure on a cardboard backing .
sebum lipid absorbed into the tape after pressing the tape onto the forehead is visible as dark dots on the gray surface .
the length of the cardboard backing is 4.5 cm . the tape ( cuderm corp . )
can be removed from the cardboard backing and the amount and composition of lipid can be measured directly using infrared spectroscopy , or extracted and quantified by nmr spectroscopy as in this study or quantified chemically . (
b ) sebum was also collected using a stainless steel blackhead remover ( revlon , inc . ) by rubbing the nose and forehead with the remover .
, new york , ny , usa ) by rubbing the nose and forehead with the blackhead remover ( revlon , inc . ; fig .
sebum was collected from the same 59-year - old caucasian donor from which sebum was collected with tape ( cuderm corp . ) .
the sebum that accumulated in the small holes of the device was dislodged from the remover by placing it into a weighed 15-ml glass scintillation vial containing 2 ml chloroform and sonicated under an atmosphere of argon gas in an ultrasonic bath ( branson ultrasonics ) for 10 minutes .
we added cdcl3 ( 500 l ) to the 11.9 mg of collected sebum lipid .
the sample was sonicated under an atmosphere of argon gas in an ultrasonic bath ( branson ultrasonics ) for 10 minutes and placed into an nmr tube for spectral measurement .
spectral data were acquired using a spectrometer ( varian vnmr 700 mhz nmr ; varian , lexington , ma , usa ) equipped with a 5 mm h{c / n } c  enhanced cold probe ( varian , palo alto , ca , usa ) .
spectra were acquired with a minimum of 250 scans , 45 pulse width , and a relaxation delay of 1.000 seconds .
we performed hsqc using 512 increments with 16 scans per increment , 45 pulse width , and a relaxation delay of 1.000 seconds , mixing time of 0.080 seconds , and a one - bond coupling constant threshold of 140 hz .
spectra were analyzed using chemistry software ( mestrenova , version 7.1.2 - 10008 ; mestrelab research s.l .
the cell was jacketed by an insulated water coil connected to a circulating water bath ( model r-134a ; neslab instruments , newton nh , usa ) .
the sample temperature was measured and controlled by a thermistor touching the sample cell window .
the water bath unit was programmed to measure the temperature at the thermistor and to adjust the bath temperature so that the sample temperature could be set to the desired value .
the rate of heating or cooling ( 1c/15 minutes ) at the sample was also adjusted by the water bath unit .
infrared spectra were measured using a fourier transform infrared spectrometer ( nicolet 5000 magna series ; thermo fisher scientific , inc . ,
phase transitions were run four times with care taken to obtain data points in the sharp phase - transition temperature region .
the frequency of the ch2 symmetric ch2 stretching band near ( 2850 cm ) was used to estimate the content of trans and gauche rotamers in the hydrocarbon chains .
although the 2954 cm asymmetric ch2 stretching band is useful for measuring phase - transition parameters , we chose to use the 2850 cm band rather than the band near 2954 cm , because measurement of the asymmetric band frequency is complicated by the adjacent ch3 symmetric stretching band near 2955 cm and the ch2 symmetric stretching band near 2852 cm .
the symmetric stretch was calculated by first baseline leveling the oh - ch stretching region between 3500 and 2700 cm . the center of mass of the ch2 symmetric stretching band , sym ,
the baseline for integrating the top 10% of the intensity of the band was parallel to the oh - ch region baseline .
lipid ch2 groups in the hydrocarbon chains are present as gauche rotamers , prevalent in disordered hydrocarbon chains , or trans rotamers , more abundant in ordered hydrocarbon chains .
thus , lipid hydrocarbon chain order may be evaluated in terms of the relative amount of ch2 
 trans rotamers .
the frequency of the ch2 symmetric stretch is dependent on the amount of trans or gauche rotamers and has been used to characterize lipid phase transitions and to measure the trans rotamer content of lipid hydrocarbon chains with changes in temperature . since rotamers are either in trans or gauche conformations , phase transitions can be described by a two - state sigmoidal equation , as described by borchman et al .
lipid order at 33.4c was calculated by extrapolating the sym at 33.4c from the fit of the phase transition and then converting sym to the percentage of trans rotamers , a measure of lipid conformational order .
the data for percentage of trans rotamers were used to calculate the phase - transition enthalpy and entropy from the slopes of arrhenius plots , as described in borchman et al .
surface pressure - area profiles of human meibum and human sebum were recorded using a computer - controlled single barrier langmuir teflon trough ( nima 102 m ; nima technology ltd . ,
the surface pressure was measured by a pressure sensor with a wilhelmy plate ( whatman , chr 1 filter paper ) .
the trough was enclosed in a transparent pmma ( perspex ; lucite international , lancashire , uk ) cabinet to avoid air currents and dust particles .
the trough was filled with an artificial tear ( at ) solution that emulated the salt composition of human tears ( nacl 6.6
g / l ; cacl2.2h2o 0.15 g / l ; nah2po4.h2o 0.1g / l ; mops 4.18
purified water ( milli - q , resistance > 18.2 m ; millipore corp . , billerica , ma , usa ) was used for preparing the at solution .
the at solution surface was cleaned with a vacuum aspirator until a clean surface was achieved ( pressure change
< 0.02 mn / m when the surface area was compressed and expanded completely ) .
the lipid sample in chloroform was spread dropwise on the surface of the at solution using a microsyringe ( hamilton co. , bonaduz , switzerland ) and chloroform was allowed to evaporate for 10 minutes .
the lipid film was compressed and expanded with a barrier speed of 15 cm / minute and changes in pressure (  ) with area ( a ) were recorded as -a profiles .
the sebum -a profiles ( 23.8 mg / ml ) and meibum ( 1 mg / ml ) alone was recorded .
sebum was mixed with meibum ( s : m ) at mole fraction ratios : 0.25:0.75 , 0.70:0.30 , and 0.97:0.03 ( based on estimated molecular weights 490 and 720 g / mole , respectively ) and -a profiles of mixtures were recorded .
the excess area of mixing ( aex ) of sebum and meibum in the mixed films was determined by the following equation : 
 where a1,2 , a1 , and a2 are the molecular areas of the mixture and the pure components , and x1 and x2 are the mole fractions of the two components in the mixture .
a negative value means molecules in the mixture occupy less surface area than expected ( condensation effect ) , while a positive value indicates the opposite , that is , an expansion effect . 

for the infrared spectroscopy meibum / sebum phase transition study , meibum was pooled from three donors : mc4 , mc4 , fc6 .
meibum from children was used as a first test rather than meibum from adults since the difference between the phase transition temperature of meibum from children and sebum was greater than the difference between the phase transition temperature of meibum from adults and sebum .
if we were to see a change with sebum , our best chance would be to first use meibum from children .
for the infrared spectroscopy meibum / sq phase transition study , meibum was pooled from seven donors : ma21 , fc 21 , fb21 , mc59 , mc22 , fa 42 , and fa19 , where m is male , f is female , a is asian , c is caucasian , b is black , and the number is the age of the donor in years .
this age range reflects the majority of adults without major age- or dry eye  related changes in their infrared phase transition parameters . for langmuir trough studies ,
meibum was pooled from four donors : mc37 , fc56 , mc59 , and mc67 .
the phospholipid composition of this sample was typical of sebum collected from a wide range of donors ( table 1 ) .
the composition of sebum was identical within 1% for samples collected with tape ( cuderm corp . ) or
mole fractions of components of human sebum from a 59-year - old caucasian male and literature lipid phase transition parameters sebum ( 20 l ) spread on an at solution at the maximum area had a very high baseline spreading pressure ( 26 mn / m ; fig .
with further compression , there was a steep increase in pressure that continued until approximately 70 mn / m , after which the film collapsed .
since the baseline and surface pressures for sebum were quite high , lower volumes were also tested .
the baseline pressure for 10 l was also very high ( 13 mn / m ) and a very low volume ( 2 l ) showed a baseline pressure near zero , but the increase in pressure was seen immediately on starting the compression ( fig .
films prepared with lower amounts of sebum showed a continuous increase in pressure upon compression and were highly compressible .
the pressure - area profile of 20 l meibum was typical with a slow and continuous increase in pressure upon compression ( fig .
pressure - area profiles of various volumes of sebum on the artificial tear solution at 35c .
data show compression parts of the profiles for 2 , 10 , and 20 l sebum .
pressure - area profiles of the mixtures of sebum with meibum on the artificial tear solution at 35c .
data show compression parts of the profiles of sebum : meibum mixtures ( s : m in mole fraction ratios 0.25:0.75 , 0.70:0.30 , and 0.97:0.03 ) and meibum ( m ) .
sebum increased the average molecular area in the 0.25:0.75 mixture , did not affect it much in the 0.70:0.30 mixture , and decreased it in the 0.97:0.03 mixture , in comparison with the molecular area of meibum alone .
sebum was mixed with meibum to observe how it influenced the biophysical properties of meibum at the air - liquid interface .
the pressure - area profile of 0.25:0.75 mixture showed first pressure rise upon compression nearly 30 cm earlier than that for meibum alone which shows that sebum caused meibum to expand by around 30 cm ( fig .
sebum also increased the surface pressure of the mixture at the maximum compression by around 10 mn / m .
increasing amounts of sebum caused an increase in the baseline pressures of mixtures 0.70:0.30 and 0.97:0.03 ( fig .
the surface pressure at the maximum compression also increased by 17 mn / m and 38 mn / m , respectively .
the shape of the pressure - area curves for all mixtures showed a continuous increase in pressure with high compressibility , typical of liquid films .
although pressure - area profiles of meibum / sebum mixtures indicated an increase in surface area with increasing amount of sebum , plots of surface pressure versus molecular area show that sebum caused an increase in average molecular area for only 0.25:0.75 mixture with respect to that of meibum ( fig .
4b ) . the average molecular area of 0.70:0.30 mixture did not change much and that of 0.97:0.03 mixture was reduced substantially in comparison with meibum ( fig .
4b ) . the excess area of mixing calculated for mixed films of sebum and meibum at different surface pressures ( fig .
5 ) showed that in 0.25:0.75 mole fraction mixture sebum caused an expansion effect ( as indicated by the positive values of aex ) .
the 0.70:0.30 mole fraction mixture also showed slight expansion but the highest mole fraction of sebum ( 0.97:0.03 ) caused a condensation effect on the lipid mixture at the pressure of 15mn / m ( as indicated by the negative values of aex ) .
the baseline pressure for 0.97:0.03 mixture was 15 mn / m , so aex values below this pressure could not be calculated for this mixture .
similarly , the maximum surface pressure for meibum was 15 mn / m , so aex values above this pressure could not be calculated for mixtures . in spite of these technical difficulties , the data indicated that sebum can cause expansion or condensation of meibum depending on its concentration in the mixture .
excess area of mixing of the mixed films of sebum and meibum at different surface pressures .
the data indicate that sebum exhibits an expansion effect on meibum in 0.25:0.75 mole fraction ratio , a slight expansion effect in 0.70:0.30 mole fraction ratio , but a condensation effect in 0.97:0.03 mole fraction ratio .
ftir was used to measure the biophysical properties of meibum , sebum , sq and meibum mixtures .
the changes in hydrocarbon chain conformation with temperature ( phase transition ) of sebum or sq have never been measured .
6a ) centered at 27c ( table 2 ) . as expected from the melting temperature of sq ( 75c ) , sq was 87% disordered and did not undergo a phase transition between 20 and 80c ( fig . 6b ) .
the slight increase ( 0.4 cm ) in the ch2 symmetric stretching frequency of sq between 20 and 80c may be due to increased bond rotational motion upon heating .
the infrared ch2 symmetric stretching band center of mass ( frequency ) was measured to calculate trans / gauche rotamer ratios as a lipid order parameter .
( a ) ( ) lipid phase transition of sebum from a 61-year - old caucasian male .
( ) lipid phase transition of meibum from children mixed with sebum ( 20% by weight ) .
( b ) no order - disorder transition was detected for squalene through the temperature range measured .
( d ) phase transitions for a pool of human meibum pooled from adults and squalene ( 3:1 , weight : weight ) .
lipid phase transition parameters the lipid phase transition parameters of meibum from children ( table 2 ) were characteristic of those measured previously for children .
meibum from children was much more ordered than meibum from adults ( table 3 ) in agreement with a previous study . at physiological temperature , 33.4c ,
sebum was less ordered than meibum from children ( table 2 ) and more ordered than meibum from adults ( table 3 ) .
meibum from children was mixed with sebum to test if sebum had an effect on the phase transition parameters of meibum .
when meibum from children was mixed with sebum ( 20% sebum by weight ) , the mixture became much less ordered compared with meibum alone and all of the phase transition parameters were significantly different ( fig .
sebum lowered the enthalpy of the phase transition and lowered the phase transition temperature of meibum ( fig .
confirmation of h and c nmr resonance assignments for squalene and human sebum sebum contains 28% sq ( table 4 ) so we tested if sq could contribute to the disordering of meibum by sebum .
squalene has a much lower melting point ( 75c ) than the transition temperature of both sebum ( table 2 ) and meibum ( 2835c ) .
a ratio of sq well above expected physiological levels was used to clearly delineate an effect should one occur .
squalene caused significant ( p < 0.05 ) changes in the phase transition parameters for a pool of human meibum ( figs .
although sq caused a small 5c change in the phase transition temperature of meibum , because the phase transition temperature of adult meibum is close to physiological temperature , sq caused a large ( 21% ) decrease in meibum lipid order from 30% to 23.8% .
the phase transition temperature , enthalpy , entropy and order at 34c all decreased with sq ( table 3 ) .
the = ch resonance region ( figs . 7a , 7ab , 7ba , 7bb ) , and ch2 and ch3 regions ( figs .
7ac , 7ad , 7bc , 7bd ) of the nmr spectra of human sebum extracted from tape ( cuderm corp . ; figs .
7aa , 7ac ) were almost identical to those observed in the spectrum of human sebum collected directly using an extractor tool ( fig .
however , there were slight differences in the resonances at 2.38 and 0.847 ppm that were more intense in the nmr spectrum of human sebum extracted from sebutape compared to sebum extracted directly .
( a ) h nmr spectra of sebum from a 59-year - old caucasian donor .
the = ch resonance regions of ( a ) sebum extracted from tape ( cuderm corp . ) ; ( b ) sebum collected directly .
the ch2 and ch3 resonance regions of ( c ) sebum extracted from tape ( cuderm corp . ) ; ( d ) sebum collected directly .
( b ) h nmr spectra of : ( a , c ) sebum collected directly from a 59-year - old caucasian donor ; ( b , d ) squalene .
the = ch resonance region accounts for 6 of the 50 protons of sq ( fig .
the close correspondence between the nmr resonance intensities ( table 2 ) and chemical shifts ( table 3 ) of human sebum ( figs .
7bb , 7bd ) strongly suggests that the resonances in this region for human sebum are due to terpenoids such as sq .
vicinal coupling constants ( jhh ) , the frequency difference between the split peaks , are larger for anticlinal than for synclinal isomers .
the vicinal coupling constants for the peaks near 5.15 are 7.6  0.7 hz , thus confirming the proposed anticlinal isomer assignment .
the largest resonance in this region was observed at 5.32 ppm with a shoulder at 5.35 ppm ( figs .
these partially overlapped resonances are assigned to protons of the = ch moieties from hydrocarbon chains and to the proton attached to carbon # 6 of cholesterol esters , respectively .
the spectra hsqc confirm the resonance assignments for this region of the h and c spectra of human sebum ( fig .
h nmr spectra of human sebum collected directly from a 59-year - old caucasian donor atop the hsqc spectra .
7bc ) and hsqc spectra confirm the resonance assignments for the h and c spectra of human sebum ( fig .
9b ; table 3 ) for this region . from the intensities of the nmr ester resonances in figure 7ab
, we calculated the mole fractions of sq , cholesteryl , and wax esters and triglycerides in a sample of human sebum ( table 4 ) .
for the infrared spectroscopy meibum / sebum phase transition study , meibum was pooled from three donors : mc4 , mc4 , fc6 .
meibum from children was used as a first test rather than meibum from adults since the difference between the phase transition temperature of meibum from children and sebum was greater than the difference between the phase transition temperature of meibum from adults and sebum .
if we were to see a change with sebum , our best chance would be to first use meibum from children .
for the infrared spectroscopy meibum / sq phase transition study , meibum was pooled from seven donors : ma21 , fc 21 , fb21 , mc59 , mc22 , fa 42 , and fa19 , where m is male , f is female , a is asian , c is caucasian , b is black , and the number is the age of the donor in years .
this age range reflects the majority of adults without major age- or dry eye  related changes in their infrared phase transition parameters . for langmuir trough studies ,
meibum was pooled from four donors : mc37 , fc56 , mc59 , and mc67 .
the phospholipid composition of this sample was typical of sebum collected from a wide range of donors ( table 1 ) .
the composition of sebum was identical within 1% for samples collected with tape ( cuderm corp . ) or
mole fractions of components of human sebum from a 59-year - old caucasian male and literature lipid phase transition parameters
sebum ( 20 l ) spread on an at solution at the maximum area had a very high baseline spreading pressure ( 26 mn / m ; fig .
, there was a continuous increase in pressure until approximately 50 mn / m . with further compression
, there was a steep increase in pressure that continued until approximately 70 mn / m , after which the film collapsed .
since the baseline and surface pressures for sebum were quite high , lower volumes were also tested .
the baseline pressure for 10 l was also very high ( 13 mn / m ) and a very low volume ( 2 l ) showed a baseline pressure near zero , but the increase in pressure was seen immediately on starting the compression ( fig .
films prepared with lower amounts of sebum showed a continuous increase in pressure upon compression and were highly compressible .
the pressure - area profile of 20 l meibum was typical with a slow and continuous increase in pressure upon compression ( fig .
pressure - area profiles of various volumes of sebum on the artificial tear solution at 35c .
data show compression parts of the profiles for 2 , 10 , and 20 l sebum .
pressure - area profiles of the mixtures of sebum with meibum on the artificial tear solution at 35c .
data show compression parts of the profiles of sebum : meibum mixtures ( s : m in mole fraction ratios 0.25:0.75 , 0.70:0.30 , and 0.97:0.03 ) and meibum ( m ) .
sebum increased the average molecular area in the 0.25:0.75 mixture , did not affect it much in the 0.70:0.30 mixture , and decreased it in the 0.97:0.03 mixture , in comparison with the molecular area of meibum alone .
sebum was mixed with meibum to observe how it influenced the biophysical properties of meibum at the air - liquid interface .
the pressure - area profile of 0.25:0.75 mixture showed first pressure rise upon compression nearly 30 cm earlier than that for meibum alone which shows that sebum caused meibum to expand by around 30 cm ( fig .
sebum also increased the surface pressure of the mixture at the maximum compression by around 10 mn / m .
increasing amounts of sebum caused an increase in the baseline pressures of mixtures 0.70:0.30 and 0.97:0.03 ( fig .
the surface pressure at the maximum compression also increased by 17 mn / m and 38 mn / m , respectively .
the shape of the pressure - area curves for all mixtures showed a continuous increase in pressure with high compressibility , typical of liquid films .
although pressure - area profiles of meibum / sebum mixtures indicated an increase in surface area with increasing amount of sebum , plots of surface pressure versus molecular area show that sebum caused an increase in average molecular area for only 0.25:0.75 mixture with respect to that of meibum ( fig .
4b ) . the average molecular area of 0.70:0.30 mixture did not change much and that of 0.97:0.03 mixture was reduced substantially in comparison with meibum ( fig .
the excess area of mixing calculated for mixed films of sebum and meibum at different surface pressures ( fig .
5 ) showed that in 0.25:0.75 mole fraction mixture sebum caused an expansion effect ( as indicated by the positive values of aex ) .
the 0.70:0.30 mole fraction mixture also showed slight expansion but the highest mole fraction of sebum ( 0.97:0.03 ) caused a condensation effect on the lipid mixture at the pressure of 15mn / m ( as indicated by the negative values of aex ) .
the baseline pressure for 0.97:0.03 mixture was 15 mn / m , so aex values below this pressure could not be calculated for this mixture .
similarly , the maximum surface pressure for meibum was 15 mn / m , so aex values above this pressure could not be calculated for mixtures . in spite of these technical difficulties , the data indicated that sebum can cause expansion or condensation of meibum depending on its concentration in the mixture .
excess area of mixing of the mixed films of sebum and meibum at different surface pressures .
the data indicate that sebum exhibits an expansion effect on meibum in 0.25:0.75 mole fraction ratio , a slight expansion effect in 0.70:0.30 mole fraction ratio , but a condensation effect in 0.97:0.03 mole fraction ratio .
ftir was used to measure the biophysical properties of meibum , sebum , sq and meibum mixtures .
the changes in hydrocarbon chain conformation with temperature ( phase transition ) of sebum or sq have never been measured .
6a ) centered at 27c ( table 2 ) . as expected from the melting temperature of sq ( 75c ) , sq was 87% disordered and did not undergo a phase transition between 20 and 80c ( fig .
the slight increase ( 0.4 cm ) in the ch2 symmetric stretching frequency of sq between 20 and 80c may be due to increased bond rotational motion upon heating .
the infrared ch2 symmetric stretching band center of mass ( frequency ) was measured to calculate trans / gauche rotamer ratios as a lipid order parameter .
( a ) ( ) lipid phase transition of sebum from a 61-year - old caucasian male .
( ) lipid phase transition of meibum from children mixed with sebum ( 20% by weight ) .
( b ) no order - disorder transition was detected for squalene through the temperature range measured .
( d ) phase transitions for a pool of human meibum pooled from adults and squalene ( 3:1 , weight : weight ) . (  ) curve fit of data from figure 3c for comparison .
lipid phase transition parameters the lipid phase transition parameters of meibum from children ( table 2 ) were characteristic of those measured previously for children .
meibum from children was much more ordered than meibum from adults ( table 3 ) in agreement with a previous study . at physiological temperature , 33.4c ,
sebum was less ordered than meibum from children ( table 2 ) and more ordered than meibum from adults ( table 3 ) .
meibum from children was mixed with sebum to test if sebum had an effect on the phase transition parameters of meibum .
when meibum from children was mixed with sebum ( 20% sebum by weight ) , the mixture became much less ordered compared with meibum alone and all of the phase transition parameters were significantly different ( fig .
sebum lowered the enthalpy of the phase transition and lowered the phase transition temperature of meibum ( fig .
confirmation of h and c nmr resonance assignments for squalene and human sebum sebum contains 28% sq ( table 4 ) so we tested if sq could contribute to the disordering of meibum by sebum .
squalene has a much lower melting point ( 75c ) than the transition temperature of both sebum ( table 2 ) and meibum ( 2835c ) .
a ratio of sq well above expected physiological levels was used to clearly delineate an effect should one occur .
squalene caused significant ( p < 0.05 ) changes in the phase transition parameters for a pool of human meibum ( figs .
although sq caused a small 5c change in the phase transition temperature of meibum , because the phase transition temperature of adult meibum is close to physiological temperature , sq caused a large ( 21% ) decrease in meibum lipid order from 30% to 23.8% .
the phase transition temperature , enthalpy , entropy and order at 34c all decreased with sq ( table 3 ) .
the = ch resonance region ( figs . 7a , 7ab , 7ba , 7bb ) , and ch2 and ch3 regions ( figs .
7ac , 7ad , 7bc , 7bd ) of the nmr spectra of human sebum extracted from tape ( cuderm corp . ; figs .
7aa , 7ac ) were almost identical to those observed in the spectrum of human sebum collected directly using an extractor tool ( fig .
however , there were slight differences in the resonances at 2.38 and 0.847 ppm that were more intense in the nmr spectrum of human sebum extracted from sebutape compared to sebum extracted directly .
( a ) h nmr spectra of sebum from a 59-year - old caucasian donor .
the = ch resonance regions of ( a ) sebum extracted from tape ( cuderm corp . ) ; ( b ) sebum collected directly .
the ch2 and ch3 resonance regions of ( c ) sebum extracted from tape ( cuderm corp . ) ; ( d ) sebum collected directly .
( b ) h nmr spectra of : ( a , c ) sebum collected directly from a 59-year - old caucasian donor ; ( b , d ) squalene . the = ch resonance region accounts for 6 of the 50 protons of sq ( fig .
7bb ) that are characteristic of terpenoids ( fig . 8) . the close correspondence between the nmr resonance intensities ( table 2 ) and chemical shifts ( table 3 ) of human sebum ( figs .
7bb , 7bd ) strongly suggests that the resonances in this region for human sebum are due to terpenoids such as sq .
vicinal coupling constants ( jhh ) , the frequency difference between the split peaks , are larger for anticlinal than for synclinal isomers .
the vicinal coupling constants for the peaks near 5.15 are 7.6  0.7 hz , thus confirming the proposed anticlinal isomer assignment .
the largest resonance in this region was observed at 5.32 ppm with a shoulder at 5.35 ppm ( figs .
these partially overlapped resonances are assigned to protons of the = ch moieties from hydrocarbon chains and to the proton attached to carbon # 6 of cholesterol esters , respectively .
the spectra hsqc confirm the resonance assignments for this region of the h and c spectra of human sebum ( fig .
h nmr spectra of human sebum collected directly from a 59-year - old caucasian donor atop the hsqc spectra .
7bc ) and hsqc spectra confirm the resonance assignments for the h and c spectra of human sebum ( fig .
9b ; table 3 ) for this region . from the intensities of the nmr ester resonances in figure 7ab
, we calculated the mole fractions of sq , cholesteryl , and wax esters and triglycerides in a sample of human sebum ( table 4 ) .
as stated previously , sebum could mix with meibum on the eyelid margin ( fig .
the major aim of our study was to determine if sebum affected the surface properties of meibum .
using langmuir trough technology , we found that the surfactant properties of a mixture of meibum and sebum were enhanced compared with meibum alone .
meibum itself forms a highly compressible liquid film that is suitable for repeated compression and expansion during blinking .
the baseline pressure and surface activity of sebum films were much higher than that of meibum films .
the estimated molecular weight of sebum is 490 g / mole and it is rich in triglycerides , which contribute to the high spreading pressure of its film , while the estimated molecular weight of meibum is 720 g / mole with a relatively lower level of triglycerides .
so the presence of triglycerides in sebum might explain the high baseline spreading pressure observed in our sebum - meibum mixtures .
the molecular areas of mixtures at highest compression , calculated by estimated molecular weights of two components and assuming all molecules were on the surface , were too small for a lipid molecule .
these unusually small molecular areas were indicative of multilayer formation in which molecules continued to stack vertically under increasing compression but did not collapse even at high pressures .
this would explain high compressibility and high surface pressures in the sebum - meibum mixtures .
our infrared spectroscopic study showed that at physiological temperature , sebum was 30% less ordered ( more fluid ) than meibum with weaker lipid  lipid interactions .
lipid interactions would be expected to aggregate into  islands  and not to exhibit strong spreading pressures .
conversely , sebum that is less ordered would be expected to exhibit a high spreading pressure .
this hypothesis was confirmed in our langmuir trough study . in another langmuir trough / infrared spectroscopy study we showed that the major factor affecting the conformation ( fluidity ) of many lipid systems including meibum was hydrocarbon chain saturation . as confirmed in this study , meibum from adults were less ordered than meibum from children largely due to hydrocarbon chain saturation .
although hydrocarbon chain saturation is likely to contribute to the difference in lipid order between sebum and meibum from children , it has yet to be determined .
sebum decreased the phase transition temperature and hydrocarbon chain order when mixed with meibum resulting in a more fluid film .
this is in agreement with the langmuir trough study which showed that sebum expanded meibum films at lower concentrations , fluidizing the meibum .
furthermore , sebum caused meibum to be more compressible and more surface active at higher pressures .
a variety of techniques have been used to confirm squalene is present in sebum . in this study , using hsqc nmr spectroscopy , we confirmed that the resonances near 5.2 ppm tentatively assigned to sq in human sebum are correct .
heteronuclear single quantum correlation , an inverse heteronuclear two - dimensional technique , constitutes one the most powerful methods available for tracing out the carbon skeleton of organic compounds . in our sebum sample ,
the molar percentage of sq was 28% identical with the average values reported ( table 1 ) . based on the observed nmr resonance intensities and shifts observed , we can confidently confirm that squalene is present on the eyelid and in this study , sebum .
however , it is possible that other terpenoids and derivatives of squalene such as epoxides and mono to tetramethylsqualene could be present as wide variety of terpenoids exist in nature . as our nmr spectra are complicated by a wide variety of lipids , nmr alone can not be used to confidently identify the many types of terpenoids that exist , especially if they are not abundant .
we propose to determine if other terpenoids are present in sebum , tears , and meibum using gas chromatography / mass spectrometry along with nmr analysis as was done for other systems .
using infrared spectroscopy , we found that 20 weight percent sq had a disordering effect on meibum .
this is opposite the effect of sq in other phospholipid systems where sq may have a condensation or area - expansion effect in a lipid mixture depending on its content and film pressure .
it exerts area condensing effects on polar lipids above 10 mole percent and in dense liquid - expanded or liquid - condensed phase .
although sq causes the hydrocarbon chains of meibum lipid to be more fluid , sq in sebum is unlikely to influence the rheology of meibum ; in another study , sq alone did not possess surfactant properties and when mixed with human meibum , it was shown that at a higher degree of film area compression sq did not affect the surface pressure of the films . at low surface pressures , sq localized over thinner regions of meibum films .
we found that sebum expanded meibum films at lower concentrations and condensed meibum films at higher concentrations .
it is likely that only a small quantity of sebum , less than 28% , is expected to mix with the tear film , so physiological levels of sebum would be expected to expand or fluidize meibum , making it spread better and be more surface active , the qualities beneficial for the tear film lipid layer .
sebum caused meibum to be more stable at higher pressures ( greater maximum surface pressure ) .
so sebum could stabilize meibum , allowing it to withstand the high shear pressure of a blink . from this study
, we see sebum - meibum interaction appear to be relevant . in future studies , we hope to measure meibum - sebum interactions related to age , sex , race , and disease .
 purposesebum may contribute to the composition of the tear film lipid layer naturally or as a contaminant artifact from collection . 
 the aims of this study were to determine : if sebum changes the rheology of meibum surface films ; if the resonance near 5.2 ppm in the 1h - nmr spectra of sebum is due to squalene ( sq ) ; and if sebum or sq , a major component of sebum , interacts with human meibum.methodshuman meibum was collected from the lid margin with a platinum spatula . 
 human sebum was collected using lipid absorbent tape . 
 langmuir trough technology was used to measure the rheology of surface films . 
 infrared spectroscopy was used to measure lipid conformation and phase transitions . 
 we used 1h - nmr to measure composition and confirm the primary structure of sq.resultsthe nmr resonance near 5.2 ppm in the spectra of human sebum was from sq which composed 28 mole percent of sebum . 
 both sebum and sq lowered the lipid order of meibum . 
 sebum expanded meibum films at lower concentrations and condensed meibum films at higher concentrations . 
 sebum caused meibum to be more stable at higher pressures ( greater maximum surface pressure).conclusionsphysiological levels of sebum would be expected to expand or fluidize meibum making it spread better and be more surface active ( qualities beneficial for tear film stability ) . 
 sebum would also be expected to stabilize the tear film lipid layer , which may allow it to withstand the high shear pressure of a blink . 
Materials and Methods
Materials
Collection and Processing of Human Meibum
Clinical Assessment
Collection of Sebum for NMR and Langmuir Studies
NMR Spectral Measurements
Fourier Transform Infrared Spectroscopy (FTIR)
Langmuir Trough Studies
Statistics
Results
Human Material
Langmuir Trough Studies
FTIR Studies
NMR Studies
Discussion