Depart. of nursing &
health sciences The Hong Kong Polytechnic
University
To resolve differences in published data on
tear antioxidant levels by comparing the
concentration of water-soluble antioxidants in
human reflex tears collected by capillary tube
and by the Schirmer strip collection method and
in basal and reflex tears collected using the
Schirmer strip method. Yawn-induced reflex
tears (collected simultaneously by capillary
tubes and by Schirmer strips) and basal tears
(by Schirmer strips and using local anesthetic)
were collected from 12 healthy subjects.
Tear cysteine, ascorbate, glutathione,
urate, and tyrosine were measured by
high-performance liquid chromatography within a
few minutes of collection. Cysteine, ascorbate,
glutathione, and tyrosine were 5 to 10 times
higher (P < 0.01) in both reflex and basal
tears collected by Schirmer strip compared with
reflex tears collected by capillary tube from
the same subject. Urate levels were slightly but
nonsignificantly higher in Schirmer strip
samples (P > 0.05).
The conflict in published data on tear
antioxidants is caused by differences in
collection methods. With the exception of urate,
antioxidants accumulate to very high levels in
corneal cells. Spuriously high antioxidant
levels in tears collected using Schirmer strips,
therefore, are most probably caused by
contamination with intracellular constituents.
The capillary tube collection method is proposed
as the method of choice for reflex tear
collection for biochemical studies. This
less-invasive method facilitates the evaluation
of tear antioxidant levels as a biomonitoring
tool for corneal health. Although moderately
increased antioxidant levels may be beneficial,
the authors hypothesize that marked increases
may indicate damage to the ocular surface.
Tear fluid protects the external surface of
the eye. Tears can be described as of two types:
reflex tears, which are
induced by a stimulus such as yawning,
irritation, or bright light, and basal tears,
which are the nonstimulated secretion of the
tear glands. The major function of tears is to
maintain corneal health by diluting, flushing
out, or neutrafizing foreign bodies and
chemicals and reactive oxygen species (ROS).
Owing to its exposed nature, the corneal surface
is at particular risk of oxidative damage by
photo-induced and environmental ROS.
Antioxidants in tears act to oppose such
damage.
Several low-molecular-weight antioxidants
have been found in human tears. These include
endogenous compounds, such as cysteine,
glutathione, urate, and tyrosine and dietary
ascorbate.
There is considerable interest in the
possible role of tears incorneal health, but to
date there are few publications on tear
antioxidants. Most previously published data
were obtained on tears collected using Schinner
strips. This method of tear collection. is well
established for measuring tear volume; however,
the volume of tears collected by Schirmer strip
is very small, and obtaining an accurate
composition analysis is difficult. Evaporation
of water from the smail tear sample captured on
the strip may significantly increase the
apparent concentration of solutes, including
antioxidants.
Schirmer strips are also invasive, and
damage to ocular surface cells by these strips
could occur. It has been reported that the use
of Schirmer strips is associated with elevated
plasmin concentrations in the tear samples,
indicating that cells on the conjunctival
surface are damaged. Vascular fragility caused
by strip-induced irritation in the lower
cul-de-sac of the eye
(where Schirmer strips are usually placed
during tear conection), and injuries to the
conjunctival surface may change the
composition of the tears collecte. Because
many antioxidants are found in blood plasma and
are highly concentrated
within cells, transudation of vascular fluid
and/or leakage from damaged cells at the site of
collection onto the Schirmer strip could lead to
a significant increase in antioxidant
concentrations of the tear fluid collected. This
could help account for the lower levels of
ascorbate and urate recently reported, in which
capillary tubes, rather than Schirmer strips,
were used
for tear collection. The capillary tube
collection method is much less invasive than the
Schirmer strip technique. A
small, disposable glass capillary tube is
placed just above the lower tear meniscus and
with care, minimal contact between the tip of
the capillary tube and the globe can be
achieved. Antioxidant concentrations in tears
collected by capillary tubes, therefore, may
give a more accurate indication of tear
composition.
The main purpose of this study was to
compare the concentration of water-soluble
antioxidants in human reflex tears collected by
Schirmer strips and by callary tubes. A
secondary purpose was to, investigate the
difference between basal and reflex tears
collected using the Schirmer strip method. The
tear components of interest were cysteine,
ascorbate, glutathione, urate, and
tyrosine.
[...]
Results presented here show also that tears
coffected using Schirmer strips contained
significant amounts of intracellular
constituents. We suggest, therefore, that this
method of tear collection be limited to studies
of tear volume. Furthermore, previously
published biochemical data on tears collected
using Schirmer strips should perhaps be
re-examined in fight of the data presented here.
In this present study, we measured the
antioxidants cysteine, ascorbate, glutathione,
urate, and tyrosine in tears. We have reported
previously that the total antioxidant capacity
of reflex tears is approximately 400 µM and
that ascorbate and urate account for
approximately half of this. The small amounts of
cysteine, glutathione, and tyrosine detected in
tears in this present study, however, cannot
account for the remaining 50%, of the total
antioxidant capacity. There are clearly as yet
unidentified antioxidant(s) in tears; thus,
further work is needed in this area.
In summary, vascular fragility and cell
injury or transfer caused by the Schirmer strip
affect tear composition and result in spuriously
high antioxidant levels in tears. The use of a
capillary tube for tear collection is much less
invasive, and is suggested as the method of
choice for reflex tear collection for
biochemical studies. There is currently no
suitable method available for collection of
basal tears for biochemical analysis. Indeed, it
bas been suggested that even when basal tears
are collected using the standard protocol of
Schirmer strips and an anesthetized cornea, some
degree of reflex tearing still occurs. Tears
contains several antioxidants, including
cysteine, ascorbate, glutathione, urate, and
tyrosine, in addition to as yet unidentified
antioxidant(s). The source of tear antioxidants
remains to be established. Depending on the
source (corneal cell leakage or lacrimal gland
secretion), measurement of tear antioxidant
levels may be useful to assess defense status,
or may reflect corneal damage. In the first
scenario, increased levels may be beneficial and
decreased levels would indicate increased
oxidative stress, whereas in the second
scenario, increased levels would act as a
biomarker of damage to the ocular surface.
Further work is needed to clarify this. In
either case, however, measurement of tear
antioxidants may be a useful tool to monitor
corneal health.
Variability of tear
protein levels in normal young adults:
between-day variation
Ng V, Cho P, Mak S, Lee A
Graefes Arch Clin Exp Ophthalmol 2000 Nov
238:892-9
An adequate knowledge of physiological
variation is important for valid comparative
studies of tear proteins. The aim of this study
was to investigate the between-day variation of
the human tear protein levels, including the
total protein concentration (TPC) and the levels
of major protein fractions. Two sampling
methods, the yawn and the eye-flush methods,
were used and compared.
METHODS: TPC was determined by the
Bradford method. The major protein fractions
were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and their levels were determined by
scanning densitometry after SDS-PAGE. The tear
protein levels were monitored for 3 days.
RESULTS: The between-day differences
in the levels of TPC and the individual protein
fractions were not statistically significant in
either sampling method, but the variations of
some proteins were large and would be clinically
significant. Different variations were observed
in different proteins. The variations in serum
albumin were large, up to 61% and 113% in the
yawn and eye-flush methods respectively. The
variations in lactoferrin, tear-specific
prealbumin and lysozyme were relatively small in
the yawn method. The variations in
protein levels obtained by the eye-flushmethod
were generally higher than by the yawn
method.
CONCLUSION: Although the between-day
differences in tear protein levels were not
statistically significant, the variations in
some proteins would be large in magnitude. The
variability of tear protein levels obtained by
the eye-flush method was larger than that by the
yawn method. Therefore, caution should be taken
if the eye-flush method is used for sampling
tears for quantitative analysis of tear
proteins, although it is easier to collect tear
samples using this method. The results will be
useful to exclude normal variation in tear
protein levels when comparing pre- and
post-therapeutic tear protein levels in eyes
treated for tear-related abnormalities.
