Experiments were performed to determine the water-soluble
antioxidant components in tear fluid from healthy young adults and to
compare the antioxidant levels in reflex tears collected by capillary
tubes and by Schirmer strips (without use of local anesthetic) and in
basal tears collected by Schirmer strips (with use of local
anesthetic). Twelve Chinese subjects (seven men, five women), aged from
22 to 29 years, were recruited with their informed consent. All
subjects had apparently normal general and ocular health. None were
smokers or users of vitamin supplements, and none wore contact lenses.
Subjects in this self-controlled study attended our clinic on one
occasion only between the hours of 9 AM and 3 PM. Reflex tears were
collected simultaneously from both eyes of each subject (one by
capillary tube and the other by Schirmer strip). Basal tears were
collected, from the eye previously used for reflex tear collection by
capillary tube, 30 minutes after reflex tear collection. This study was
approved by the ethics subcommittee of the university, and all
procedures involving human subjects complied with the Declaration of
Helsinki, as revised in 2000.
A preweighed Schirmer strip (weighed in a clean, sealed Eppendorf tube;
Eppendorf, Fremont, CA) was placed onto the outer canthus of one eye,
randomly selected, of each subject, and reflex tears were collected for
5 minutes. Simultaneously, a disposable capillary tube (Drummond
Scientific Co., Broomall, PA) was used to collect tears from the other
eye, according to the procedure of Callender and
Morrison.
23 Within 30 minutes of reflex tear collection,
basal tears were collected as follows: One drop of local anesthetic
(0.5% benoxinate HCl, Alcon Ltd., London, UK) was instilled into the
eye from which tears had been collected by capillary tube. After 10
seconds, a preweighed Schirmer strip was placed on the outer canthus,
and basal tears collected for 5 minutes. After tear collection, the wet
Schirmer strip was immediately put into the same tube as before, the
tube was sealed to avoid evaporation, and the strip was reweighed to
determine the weight of tears collected. This was translated into
volume by using the tear density, which was calculated by weighing a
known volume of the reflex tears collected by capillary tube from the
same subject. Tears were eluted from strips using 50 μl phosphate
buffer (HPLC mobile phase), and approximately 30 μl eluate was
transferred into the sample cup for measurement. Approximately 30 μl
of reflex tears collected by capillary tube were transferred directly
into a sample cup. The sample cups were immediately placed in the
autosampler compartment of an HPLC system (Millennium; Waters Alliance,
Milford, MA), and the antioxidants of interest (cysteine, ascorbate,
glutathione, urate, and tyrosine) were measured concurrently, according
to the HPLC method of Gogia et al.
3
To check whether there was any antioxidant or contamination of the tear
sample due to contact with the capillary tubes, Schirmer strips, or
local anesthetic, three blank samples were prepared by collecting
mobile phase into a capillary tube, absorbing mobile phase onto a
Schirmer strip, eluting as described earlier, and adding 10 μl of
local anesthetic to 55 μl mobile phase. In addition, recovery of
added antioxidants was determined by measuring freshly prepared
combined standard (containing all five antioxidants of interest), with
the same procedures used to prepare the three blank samples. Combined
standard (100 μM) was prepared freshly on each testing occasion by
mixing equal parts of 500 μM standards of each antioxidant of
interest (cysteine, ascorbate, glutathione, urate, and tyrosine). The
500 μM standards were prepared fresh from stock solutions as follows:
cysteine (20 mM) was prepared from solid (BDH Chemicals Ltd., Poole,
UK) in extra pure water, and dissolving was facilitated by the addition
of a few drops of 0.2 M HCl; glutathione (10 mM) was prepared from
solids (Sigma Chemical Co., St. Louis, MO) in extra pure water; urate
(2 mM) and tyrosine (20 mM) were prepared from solids (BDH Chemicals
Ltd.) in extra pure water to which a few drops of 1 M NaOH had been
added to facilitate dissolving. The stock solutions of cysteine,
glutathione, urate, and tyrosine were aliquoted (separately), stored at−
70°C, and thawed just before use. Ascorbate (10 mM, from D-L extra
pure crystals; Merck, Darmstadt, Germany) was prepared in extra pure
water just before use.
The HPLC system consisted of a solvent pump and injector with sample
cooling function (Waters Alliance), a C18 precolumn (5 μm, 3.9 × 20 mm; Guard-Pak Waters Sentry), and a reversed-phase C18 analytical
column (5 μm, 5 × 250 mm; Resolve, Isco Inc.). The detector
(996 PDA; Waters), UV detector, and electrochemical detector were
connected in series. The output was calculated on computer (Millennium
software, version 3.05.07; Waters Alliance) and the peak areas were
recorded. The mobile phase was 0.2 M
KH2PO4-H3PO4 (Merck, Darmstadt, Germany) at pH 2.7, delivered at 1 ml/min. The
injection volume was 20 μl, and the retention times for cysteine,
ascorbate, glutathione, urate, and tyrosine were, respectively, 3.0,
4.2, 5.0, 9.4, and 10.7 minutes, indicating good separation and clear
identification of these antioxidants. The injection interval used was
12 minutes. Calibration and precision were performed on each occasion
using 2.5, 5.0, 7.5, and 10.0 μl injection volumes of the freshly
prepared combined standard.
The distributions of the data were not significantly different from
normal (one-sample Kolmogorov-Smirnov D tests, P >
0.05), and parametric tests were therefore used. Results were
statistically analyzed using the paired t-test to detect
differences between tears collected by the two methods and differences
between reflex and basal tears collected using Schirmer strips. P < 0.05 was regarded as statistically significant.