Retinol Secretion by the Lacrimal Gland
John L. Ubels,*t Kevin M. Foley,* and Vivian Rismondo*
In order to determine the source of the retinol which has been identified in the tear fluid, the lacrimal
gland ducts of rabbits and rats were cannulated and the collected lacrimal gland fluid was analyzed by
high performance liquid chromatography. Retinol was identified in the lacrimal gland fluid of rabbits
and rats, and it is concluded that the lacrimal gland is the source of retinol in the tears. Dose-response
studies show that intravenously administered pilocarpine and intra-arterial acetylcholine stimulate secretion of retinol by the lacrimal gland. Intravenous administration of vasoactive intestinal peptide (VIP)
also stimulates retinol secretion in a dose-response manner. These observations are similar to the effects
of cholinergic drugs and VIP on protein secretion by the lacrimal gland. Invest Ophthalmol Vis Sci 27:
1261-1268, 1986
rimal glands of rats and rabbits. 910 Therefore, the effects
of several cholinergic agonists and VIP on retinol secretion by the lacrimal gland were also investigated.
Analysis of tear fluid of rabbits, cynomologous
monkeys, and humans has shown that vitamin A is
present in the tears as all-trans retinol. 12 This finding
suggests that the tear fluid may serve as a nutrient
source supplying retinol to the avascular cornea which
has an absolute requirement for this vitamin. In testing
this hypothesis, it is essential that the source of retinol
in the tear fluid be identified since the tears consist of
a mixture of mucin from goblet cells, lipids from meibomian and Harderian glands (rabbit), and the aqueous
secretion containing electrolytes and proteins from the
lacrimal glands. It has also been reported that some
tear components, such as transferin and IgG, may be
present as the result of extravasation of material from
conjunctival capillaries or breakdown of exfoliated epithelial cells.3 Since lacrimal gland fluid (LGF) uncontaminated by other secretions can be collected from
the lacrimal gland ducts of rabbits4"6 and rats7 and
analyzed by high pressure liquid chromatography
(HPLC),1 these methods were chosen as a first step in
identifying the source of retinol in tears. It will be shown
in this paper that the lacrimal gland is a source of retinol
in tear fluid.
The lacrimal gland is known to be under parasympathetic control with cholinergic stimulation causing
secretion of fluid, electrolytes, and protein.5'6'8 It has
also been shown that vasoactive intestinal peptide (VIP)
stimulates secretion of enzymes and fluid by the lac-
Materials and Methods
Experimental Animals
The animals used in these experiments were New
Zealand White rabbits of either sex weighing 2.5-3.2
kg and either Sprague-Dawley or WKY rats weighing
200-400 g. Rabbits were anesthetized with an initial
dose of ketamine HC1 (30 mg/kg) and xylazine (5 mg/
kg) given intramuscularly, unless otherwise noted, and
anesthesia was maintained by additional doses every
30 min throughout the experiment. Rats were anesthetized with sodium pentobarbital (50 mg/kg) given
intraperitoneally. At the end of each experiment, the
animals were euthanized with a sodium pentobarbital
overdose.
Vitamin A-deficient rabbits were prepared as previously described. Briefly, weanling rabbits weighing
0.5 kg were placed on a casein base diet deficient in
vitamin A (Teklad Test Diets, Madison, WI) fed ad
libitum for 14-20 weeks.1'1' The animals were used for
experiments when their weight had plateaued (1.5-1.8
kg) and the corneas had reached stage 3 to 4 of keratinization. Animal use procedures conformed to the
ARVO Resolution on the Use of Animals in Research.
Experimental Protocols
From the Departments of *Physiology and fOphthalmology,
Medical College of Wisconsin, Milwaukee, Wisconsin.
Supported by grants R23 EY04069, R01 EY05640, and P30
EY01931 from the National Institutes of Health and by a grant from
Alcon Laboratories, Fort Worth, Texas.
Submitted for publication: October 21, 1985.
Reprint requests: John L. Ubels, PhD, Department of Physiology,
Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226.
The opening of the excretory duct of the lacrimal
gland of the rabbit is located in the palpebral conjunctiva near the lateral canthus. The duct opening was
cannulated with a tapered glass cannula as described
by Botelho and co-workers.4"6 The cannula measured
0.3-0.5 mm in outside diameter at the tapered end,
with an inner diameter of 1 mm at the open end, and
1261
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1262
INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / August 1986
was 6-10 mm long. As LGF filled the cannula, it was
collected using a tapered, L-shaped glass pipette. 1213
Initial experiments were designed to allow collection
of large amounts of LGF for HPLC analysis to determine the presence of retinol. LGF flow was therefore
maximally stimulated by injection of 5 /ig of carbamylcholine chloride (carbachol) into the marginal ear
vein of normal and vitamin A-deficient rabbits. The
drug was injected every 15 min for up to 3 hr, and
LGF samples were pooled to allow analysis of as much
as 100>lofLGFbyHPLC.
In a second series of experiments, tears were sampled
from three vitamin A-deficient rabbits as previously
described1 and analyzed by HPLC to confirm the absence of retinol. The animals were then given a 100,000
IU dose of retinyl palmitate orally and placed on a
normal diet (Purina Rabbit Chow). Additional 25,000
IU doses of retinyl palmitate were given every other
day for 4-6 days. When the coraeal pathology had
cleared, tears were again sampled and analyzed to confirm the presence of retinol. The animals were then
anesthetized, the lacrimal gland duct was cannulated,
and LGF was collected following carbachol stimulation.
Because of the long-acting, widespread systemic effects of carbachol, further experiments on the effect of
cholinergic stimulation on retinol secretion by the rabbit lacrimal gland were conducted using pilocarpine
and acetylcholine. Pilocarpine was administered intravenously via the marginal ear vein or the femoral vein.
The dose-response characteristics of the effect of pilocarpine on retinol secretion were studied in five animals using the following protocol. The flow rate of
LGF was minimized by application of two drops of
proparacaine HC1 to the cornea. After 5 min, a 100400 Mg/kg dose of pilocarpine14 was administered and
10-25 ix\ of LGF was collected in a pre-weighed pipette
for a precisely timed period of no more than 5 min.
The volume of LGF collected was immediately determined gravimetrically and the flow rate was calculated.
The sample was transferred to a 25 /xl Hamilton syringe
and injected onto the HPLC column. When the LGF
flow rate had returned to basal levels (usually after
about 10-15 min), proparacaine was again applied to
the cornea and the procedure was repeated until the
animal had received two injections at each dosage of
100, 200, 300, and 400 /xg/kg of pilocarpine. Doses
were administered in randomized order.
The effect of acetylcholine (Ach) on retinol secretion
by the lacrimal gland was studied on five animals by
injecting Ach directly into the arterial supply of the
gland, The animals were anesthetized with an initial
dose of 35 mg/kg sodium pentobarbital. Catheters were
placed in the femoral artery and vein for monitoring
Vol. 27
arterial pressure and administration of drugs. The animal was ventilated through a tracheostomy tube using
a Harvard 607 respirator. The internal maxillary artery
which supplies the lacrimal gland was isolated by ligation of the posterior occipital, superficial temporal,
and external maxillary arteries. The lingual artery was
catheterized in the retrograde direction5 and infused
with lactated Ringer's solution at 0.6 ml/min. Acetylcholine introduced into the infusate was carried to the
lacrimal gland by blood flowing from the common carotid artery into the internal maxillary artery. The effect
of Ach on retinol secretion by the lacrimal gland was
studied by infusion of bolus doses of 20-60 fig Ach6
following the same protocol used with pilocarpine.
Prior to each infusion of Ach, the rabbit received a
0.35 mg dose of d-tubocurarine chloride intravenously
to prevent movement due to the effect of Ach on skeletal muscle.
LGF secretion was also stimulated by intravenous
infusion of vasoactive intestinal peptide (VIP) at doses
ranging from 2-8 jig/kg.10 Dose-response effects were
studied in six animals using the same protocol used in
the pilocarpine study except that LGF was collected
for 10 min following each VIP injection. Because of
the cardiovascular effect of VIP, which causes vasodilation, after each dose the arterial pressure was allowed to return to control levels and remain there for
15 min before a subsequent dose was administered.
The duct of the exorbital lacrimal gland of the rat
was cannulated in similar fashion to the rabbit, as described by Thorig et al.7 A tracheostomy tube was
placed in the trachea so that salivary secretions in response to cholinergic stimulation would not interfere
with respiration. Pilocarpine (50 Mg/kg) was infused
into the femoral vein and the LGF secreted was collected. The LGF secreted in response to repeated doses
of pilocarpine was pooled until a 20 /x\ volume sufficient
for HPLC analysis was obtained.
High Pressure Liquid Chromatography (HPLC)
Samples of LGF were analyzed for retinol on a
Beckman (Fullerton, CA) Model 334 Gradient Liquid
Chromatograph equipped with a Beckman Model 165
Variable Wavelength UV Detector, a Dupont (Wilmington, DE) Zorbax ODS reversed-phase column,
and a Rheodyne 7302 column inlet filter (Rainen Instrument Co., Woburn, MA). The mobile phase for
most experiments was 90% methanol:10% water containing 10 mM ammonium acetate (NH4Ac) at a flow
rate of 1 ml/min. This is a standard method for separation of retinoids and has previously been used in our
laboratory for separation of retinoid metabolites extracted from cornea and identification of retinol in
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No. 8
RETINOL SECRETION DY THE LACPJMAL GLAND / Ubels er ol.
tears. 151 This system is capable of resolving isomers of
retinol and separating retinol from retinaldehyde16 as
was confirmed during the course of the present study
using a retinaldehyde standard and a retinol isomer
produced by UV irradiation of all-trans retinol. A second solvent system consisting of 90% acetonitrile:10%
water containing 10 mM ammonium acetate17 was also
used to confirm the results obtained using methanol:
water. The detector was set at a wavelength of 330 nm
and a sensitivity of 0.005 absorbance units full scale.
The scanning channel of the 165 detector was set at a
sensitivity of 0.005 absorbance units full scale, and absorbance spectra were obtained by scanning peaks from
300-400 nm as they passed through the flow cell of
the detector.
All-trans retinol and retinaldehyde (Sigma Chemical,
St. Louis, MO) standards were dissolved in HPLC grade
methanol and stored under nitrogen at -80°C. Standard curves were generated and retinol in LGF was
quantified based on peak height as previously described. ' The solvents used were HPLC grade and were
filtered through a 0.2 ^m filter and degassed before use.
All experiments were conducted in a room equipped
with amber lights to protect the retinoids from exposure
to light with a wavelength of less than 380 nm.
Results
1263
Retinol Standard
20 ill 1 0 * M
20 ul licrlmil Gland Fluid
14
ie 1 ;
Fig. 1. Chromatograms of a retinol standard and the retinol peak
in two samples of rabbit lacrimal gland fluid collected during stimulation with carbamylcholine. Mobile phase, methanol:water, 10 mM
NH4Ac.
analyzed for retinol. The retinol peak detected using
the methanokwater system was also detected by this
method (Fig. 5).
Retinol was also detected in the LGF of three rats
(Fig. 6); however, since it took 3-4 hr to collect the 20
jul of LGF needed for analysis, this model has not been
pursued.
Dose-Response Experiments
Administration of increasing doses of pilocarpine
had no effect on retinol concentration in LGF. However, the LGF flow rate increased from 1.8 ± 0.27 ix\/
Retinol Standard
A.
100 n\ LGF
Identification of Retinol in Lacrimal Gland Fluid
Chromatographic analysis of samples of LGF collected during cholinergic stimulation resulted in elution
of a peak with the same retention time as a retinol
standard (Fig. 1). Scanning of this peak from 300-400
nm revealed an absorption spectrum corresponding to
that for retinol with an absorbance maximum of 330
nm, as shown in Figure 2 for a 100 /A LGF sample.
The retinol peak was absent from the LGF of vitamin
A-deficient rabbits (Fig. 3). In agreement with our previous study, retinol was absent from the tear fluid of
vitamin A-deficient rabbits but returned after repletion
of the animals with vitamin A. Subsequent collection
and analysis of LGF from the same animals showed
that this retinol was secreted by the lacrimal gland (Fig.
4). Mean retinol concentrations in LGF collected during maximal cholinergic stimulation from normal rabbits and three vitamin A-repleted rabbits were 62.3
± 5.9 ng/ml (n = 10) and 130 ± 17.5 ng/ml (n = 7),
respectively.
These results were confirmed using the acetonitrile:
water mobile phase. In order to detect retinol in LGF
using this system, it was found to be necessary to first
extract the LGF with methanol in a 1:1 ratio. This
resulted in precipitation of protein which was sedimented by centrifugation. The supernatant was then
10
ml
B.
Scan of Retinol
D.
Scan of Retinol Peak
in LGF
460
350
Fig. 2. A, Chromatogram of retinol standard. B, Absorbance spectrum from 300-400 nm of the retinol standard peak. C, Chromatogram of 100 fil of rabbit lacrimal gland fluid with retinol peak at 14
ml. D, Absorbance spectrum from 300-400 nm of the retinol peak
in the lacrimal gland fluid sample. The peak absorbance of both
spectra is 330 nm. Mobile phase, methanol:water, 10 mM NHy Ac.
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1264
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1986
LGF (16 ul)
Vitamin A Deficient Rabbit
.001 AU
B. Scan of 7 ml Peptide Peak
Vol. 27
ple. A range from 67.1 ± 11 pg/min at 200 fig/kg to
458.8 ± 72.8 pg/min at 400 ixg/kg (Fig. 7B) was measured.
A similar response was observed with stimulation of
LGF secretion by injection of bolus doses of Ach into
the arterial supply of the lacrimal gland. Flow increased
from 3.5 ± 0.4 /il/min in response to a 20 ng dose to
8.4 ± 0.9 /ul/min in response to a 60 ng dose. Retinol
concentration remained constant at 45.7 ± 3.5 ng/ml
(n = 35) over this range of flow rates (Fig. 8A). Calculation of the retinol secretion rate from these data
resulted in a rate of 156.9 pg/min at 20 ng Ach to 437.0
pg/min at 60 ng Ach (Fig. 8B).
Intravenous administration of VIP also results in
stimulation of LGF secretion. Flow rates ranged from
1.22 ± 0.11 /il/min for a 2 jtg/kg dose to 2.06 ± 0.28
/xl/min for an 8 jug/kg dose. Again, the retinol concentration remained constant over this range offlowrates
although at a higher level than that observed with cholinergic stimulation, 85.9 ± 5.5 ng/ml (n = 33) (Fig.
9 A). Calculation of the retinol secretion rate over this
range of VIP doses resulted in values ranging from
109.8 ± 28.0 pg/min at 2 ^g/kg VIP to 181.8 ± 41.7
pg/min at 8 jtg/kg (Fig. 9B).
A.
Vitamin A Delicient Rabbit
10 Ml Tears
200
Vitamin A Repleted
6 Days — 13 M' Tears
Fig. 3. A, Chromatogram of lacrimal gland fluid from a vitamin
A-deficient rabbit. Note lack of retinol peak at 14-15 ml. B, Absorbance spectrum from 200-400 nm of the peak which elutes from
lacrimal gland fluid at 7 ml. The absorbance maxima at 210 nm and
280 nm suggest that the peak which elutes at 7 ml in this and other
rabbit lacrimal gland fluid samples is a peptide. Mobile phase, methanol:water, 10 mM NH 4 Ac.
2
6
10
14
18
ml
Vitamin A Repleted — 6 Days
D.
min in response to a 100 ng/kg dose of pilocarpine to
11.2 ± 1.3 Atl/min in response to a 400 ng/kg dose of
pilocarpine. Retinol concentration remained constant
at 40.3 ± 2.1 ng/ml (n = 30) over this range of flow
rates (Fig. 7A). In order for retinol concentration to
remain constant with increasing LGF flow rate, the
rate of retinol secretion by the gland must also increase
with increasing levels of stimulation. Therefore, the
retinol secretion rate (pg/min) for each dose of pilocarpine (tig/kg) was calculated from the LGFflowrate
and corresponding retinol concentration for each sam-
17 Ml LGF
Fig. 4. Effect of repletion of a vitamin A-deficient rabbit on retinol
levels in tears and lacrimal gland fluid. Mobile phase, methanol:water,
10 mM NH4Ac.
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1265
RETINOL SECRETION DY THE LACRIMAL GLAND / Ubels er ol.
No. 8
50/JI Retinol Standard
0.5x10"^
2O;JI
5 0 / J I Methanol Extract
of LGF
20/il Rat LGF
Retinol Standard
0.25xl0" 6 M
6
i
10
S
ml
A—N>
6
10
i
IO
ml
Fig. 6. Chromatograms of a retinol standard and the retinol peak
in a sample of rat lacrimal gland fluid. Mobile phase, methanol:water,
10 mM NH 4 Ac.
14
ml
Fig. 5. Chromatograms, obtained using a mobile phase of acetonitrile:water 10 mM NH4Ac, of a retinol standard and the retinol
peak in rabbit lacrimal gland fluid. The lacrimal gland fluid was extracted with methanol (1:1) before analysis.
Discussion
Analysis of rabbit lacrimal gland fluid by HPLC
shows that vitamin A is present in this secretion as alltrans retinol. This conclusion is based on the following
evidence: (1) the presence of a chromatographic peak
with the same retention time as a retinol standard, (2)
an absorbance spectrum and absorbance maximum
(330 nm) which corresponds to that for retinol, (3) the
absence of this peak from the LGF of vitamin A-deficient rabbits, and (4) the reappearance of the peak in
the LGF of vitamin A-repleted rabbits.
The alternative explanation of the data would be
that the chromatographic peak is a retinol metabolite
such as retinoic acid, retinyl phosphate, a retinyl ester,
retinaldehyde, 3,4 didehydroretinol (vitamin A2), or a
retinol isomer. Retinoic acid and retinyl phosphate are
more polar than retinol and elute from the column at
or near the solvent front in our HLPC system.151718
Using a mobile phase of 80% methanol:20% water, we
have been unable to detect retinoic acid in LGF or
tears. A retinyl ester such as retinyl palmitate would
have a retention volume of at least 100 ml in our system. Metabolism of retinol to retinaldehyde is known
to occur in liver, retina, and intestine;19 however, retinaldehyde has not been identified in other tissues. Our
HPLC system gives baseline separation of retinol (elution volume 14 ml) and retinaldehyde (elution volume
16 ml). Vitamin A2 and retinol isomers have been
identified in fish; however, they do not appear to be
important in mammals.19 Our HPLC system is able to
separate a retinol isomer produced by UV irradiation
which elutes at 13 ml from all-trans retinol (14 ml). In
chromatograms of LGF, there is always a single peak
corresponding to all-trans retinol. It is, therefore, concluded that the lacrimal gland is the source of the retinol
B
500
Fig. 7. A, Effect of pilocarpine on rabbit lacrimal
gland fluid secretion rate and
retinol concentration. B, Effect of pilocarpine on rate of
retinol secretion by the rabbit
lacrimal gland.
— 400
*E
?300
100
X+SE(n)
100
200
300
400
Pilocarpine (pg/Kg)
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100
200
300
400
Pilocarpine (pg/Kg)
1266
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1986
Vol. 27
B
10
9
8
7
6
5
4
3
2
I
500
400
O
+60 g
0^
Or
T30.I
—I
(14)
(12)
Fig. 8. A, Effect of acetylcholine on rabbit lacrimal
gland fluid secretion rate and
retinol concentration. B, Effect of acetylcholine on rate
of retinol secretion by the
rabbit lacrimal gland.
O
5 200
*
100
X + SE(n)
X+SE(n)
20
40
60
20
Ach (pg)
40
60
Ach (pg)
which has previously been reported to be present in
tears.' In preliminary studies, we have also cannulated
the duct of the Harderian gland of rabbits and extracted
the collected Harderian gland fluid with chloroform/
methanol. Analysis of these extracts gave no evidence
of the presence of retinol, indicating that this lipid secreting gland is not a source of retinol in tears.
The retinol concentration in LGF of normal rabbits
(Figs. 7-9) is within the same range previously reported
for rabbit tears.' The significantly higher levels of retinol
in the LGF of vitamin A-repleted rabbits probably reflects the relatively high doses of vitamin A which these
animals received. The rapid response of the animals
to vitamin A repletion shows that the retinol secretion
mechanism of the lacrimal gland is not affected by the
deficient state and that the gland will begin to secrete
retinol soon after it is again made available. This finding
is in agreement with previous observations of the rapid
recovery from the effects of vitamin A deficiency upon
administration of large doses of retinyl palmitate.20"22
With the detection of retinol in the fluid from the
exorbital lacrimal gland of the rat, it has now been
shown that retinol is present in the tears or LGF of
four species: rat, rabbit, cynomologous monkey, and
human. 1 ' 2 The secretion of retinol into the tear fluid
appears to be a general phenomenon in mammals and
may serve a nutritive role in delivery of vitamin A to
the ocular surface epithelia.
Cholinergic and peptidergic (VIP) stimulation
caused an increased rate of LGF secretion in the present
study, as has also been previously reported by other
investigators.4'510 Increasing levels of cholinergic and
B
2.5
do)
I
2.0
250
1.5
Fig. 9. A, Effect of vasoactive intestinal peptide on
rabbit lacrimal gland fluid
secretion rate and retinol
concentration. B, Effect of
vasoactive intestinal peptide
on rate of retinol secretion by
the rabbit lacrimal gland.
200
o
1.0
—
o 150
0.5
90 —
100
X±SE(n)
2
4
6
VIP (pg/Kg)
X±SE(n)
8
8
VIP (pg/Kg)
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No. 8
RETINOL SECRETION BY THE LACRIMAL GLAND / Ubels er ol.
peptidergic stimulation also result in an increasing rate
of retinol secretion by the lacrimal gland. The increase
in retinol secretion rate parallels the increase in LGF
flow rate, resulting in a constant retinol concentration
over the range of flow rates reported in this study.
Cholinergic drugs and VIP also stimulate protein
secretion by the lacrimal gland. Dartt and Botelho6
have shown that, with injection of Ach, protein concentration remains nearly constant over the same range
of flow rates observed in the present study. Protein
concentration, however, is lower at basal flow rates than
at stimulated flow rates in rabbits and guinea pigs. 623
Retinol concentration at basal LGF flow has not been
measured since the minimum volume required for
HPLC analysis is approximately 15 /xl. Using rat lacrimal gland slices and isolated acini, it has been shown
that carbachol stimulates protein and enzyme secretion
in a dose-dependent manner and that this protein secretion is an exocytotic process which is calcium dependent. 824
Protein secretion by the lacrimal gland is also stimulated in a dose-dependent manner by VIP, and peptidergic neurons containing VIP appear to innervate
the exorbital lacrimal gland of the rat.9 VIP is thought
to stimulate protein secretion via a cAMP-dependent pathway which converges with the cholinergic
pathway.925-26
The similarities between the effects of cholinergic
and peptidergic stimulation on protein and retinol secretion by the lacrimal gland suggest that retinol and
protein may be secreted by the lacrimal gland via
similar mechanisms. Since retinol is a hydrophobic
molecule which is unstable in an aqueous medium, it
is unlikely that it would be present in free form in
the LGF.
Retinol is transported in the blood by serum retinol
binding protein,27 and it would not be expected to reach
the LGF via an extracellular route as has been proposed
for water and sodium.28 Retinol binding protein binds
to specific target cell surface receptors but does not
enter cells, and it is reported to be absent from the tear
fluid of monkeys.29 Our finding that it is necessary to
extract LGF with methanol before HPLC analysis using
acetonitrile indicates that the retinol in LGF is associated with protein. Acetonitrile is not an extraction
medium for protein-bound retinoids17 and, therefore,
apparently will not separate retinol when LGF is applied directly to the column. The methanol mobile
phase, on the other hand, effectively separates retinol
when LGF is applied to the column. If retinol is secreted
with the protein component of the LGF, it may be
bound to a unique retinoid binding protein synthesized
by the acinar cells. Alternatively, it may be secreted
and transported in LGF in non-specific association with
1267
LGF protein as is the case for the relationship between
retinoic acid and serum albumin. 27 The relationship
between retinol and protein in LGF is currently being
investigated.
Key words: retinol, vitamin A, lacrimal gland, tears, cholinergic agonists, vasoactive intestinal peptide
Acknowledgments
The authors thank Dr. Darlene A. Dartt of the Eye Research Institute, Boston, MA, for teaching us the lacrimal
gland fluid collection method, Dr. Henry F. Edelhauser for
his critical review of the manuscript, and Mrs. Barbara Olson
for secretarial assistance.
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15. Ubels JL and Edelhauser HF: In vivo metabolism of topically
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