mise à jour du 20 février 2003
Invest Ophthalmol Vis Sci 2001; 42; 13; 3130-4
Water-soluble antioxidants in human tears: effect of the collection method
Choy CK, Cho P, Chung WY, Benzie IF
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.