Ascorbic acid concentration and total antioxidant activity of human tear fluid measured using the FRASC assay

PURPOSE: To evaluate a novel method (FRASC) for total ferric reducing (antioxidant) activity and ascorbic acid concentration applied to human tears, to investigate the stability of ascorbic acid, and to determine the antioxidant status of human reflex tears. METHODS: Linearity, sensitivity, and precision of FRASC and ascorbic acid loss during 7 days' storage were assessed; total antioxidant activity and ascorbic acid and uric acid concentrations of reflex tears from 47 healthy subjects were measured. RESULTS: FRASC has good precision, linearity, and sensitivity. Ascorbic acid is stable for at least 7 days at moderately acidic pH (pH 3.6) and low temperature. Total antioxidant activity and ascorbic acid and uric acid concentrations (mean +/- SD) in reflex tears were 409 +/- 162, 23 +/- 9.6, and 68 +/- 46 microM, respectively. Ascorbic acid and uric acid constituted around half the total antioxidant activity measured. There was a significant correlation between uric acid and total antioxidant activity (r = 0.754; P: < 0.0001). Men had significantly (P: = 0.0045) higher tear ascorbic acid concentrations than women. CONCLUSIONS: FRASC is suitable for measuring total antioxidant activity and ascorbic acid in human tears. Further clinical study is needed to investigate the male-female difference seen, to characterize the remaining 50% antioxidant activity, and to investigate the effects of environmental conditions, antioxidant supplementation, age, and ocular disease on tear antioxidant status.     

Water-soluble antioxidants in human tears: effect of the collection method.

PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.     

Clinical evaluation of a measurement method for secretory IgA in tears

PURPOSE: To evaluate the efficacy of measurement of secretory IgA (sIgA) in tears with tear sampling methods using filter paper, and to review sIgA measurement method for clinical application. SUBJECTS AND METHODS: Subjects were divided into the following 4 groups: a healthy control group, 29 eyes of 29 subjects; a contact lens group, 15 eyes of 15 subjects; a dry eye group, 13 eyes of 13 subjects; and a herpes group, 6 eyes of 6 subjects. In all subjects the sIgA value in tears was measured. In addition, in eighteen eyes of 18 healthy control individuals, the tear sIgA value was measured three times, morning, noon, and night in one day, and the variation of tear sIgA value was checked. The tears were sampled by the Schirmer 1 method. Schirmer papers were eluted in 200 microl of 0.5 M NaCl and 0.5% Tween 20 in 0.05 M phosphate-buffered solution (pH 7.2). Tear sIgA value was measured by ELISA (enzyme-linked immunosorbent assay). RESULTS: Tear sIgA value was 1249.0+/-1025.0 microg/ ml in the healthy control group; 1057.4+/-1583.3 microg /ml in the contact len groups; 197.8+/-91.3 microg/ml in the dry eye group; and 759.7+/-467.8 microg/ml in the herpes group. There was no significant difference between the healthy control group and the contact lens group, or the control group and the herpes group. There was a significantly (p<0.0001) low value in the dry eye group in comparison with the healthy control group. In the 18 healthy control individuals, there was a tendency for the tear sIgA value collected at noon to be higher. CONCLUSION: Measurment of the changes in tear sIgA values caused by inflammation of the lacrimal gland is useful as a clinical test of lacrimal gland function.     

Antioxidants in tears and plasma: Inter-relationships and effect of vitamin C supplementation

PURPOSE: To investigate inter-relationships between total antioxidant capacity and ascorbate concentration in plasma and tears, and the effect of antioxidant supplementation with reference to these variables. METHODS: Twenty-one subjects were studied in this placebo-controlled, cross-over intervention trial. Fasting plasma and tear ascorbate concentrations and total antioxidant capacity (as Ferric Reducing/Antioxidant Power (FRAP)) were measured pre- and post-supplementation with vitamin C (1 g/day). RESULTS: Mean +/- SD ascorbate in tears and plasma at entry were 17 +/- 6 and 52 +/- 13 micro M, respectively; FRAP values were, respectively, 273 +/- 94 and 1101 +/- 168 micro M. There was no significant correlation between tear and plasma levels (r = -0.068; P = 0.771 for ascorbate; r = 0.418; P = 0.059 for FRAP). Neither was significant correlation seen between the two variables in plasma (r = 0.162; P = 0.483) or tears (r = 0.353; P = 0.117). Acute responses (up to 3 hours) showed a similar pattern of increase in both fluids, however, peak response in tears (33 +/- 4 micro M) was much smaller and slightly later than in plasma (125 +/- 13 micro M). After 4 weeks' supplementation, ascorbate increased (P < 0.001) in both fluids, however, the increase in tear ascorbate was small (5 micro M), compared to plasma (38 micro M). The increase in tear ascorbate appeared to plateau after 2 days' supplementation; plasma levels were still increasing. Higher tear ascorbate at entry was associated (P < 0.05) with smaller supplementation-related response. No significant changes in FRAP were seen in either fluid (P > 0.05). CONCLUSIONS: Ascorbate concentration in both plasma and tears increased with vitamin C supplementation, but the total antioxidant capacity of these fluids did not. Furthermore, the increase in tear ascorbate was modest in comparison to that in plasma, and is suggestive of a "ceiling" for tear ascorbate of under 40 micro M. Results support the concept of a control mechanism for an integrated antioxidant defense system, and suggest that the amount of ascorbate in tears is both actively controlled and purposefully limited.     

Lipid,lipase and lipocalin differences between tolerant and intolerant contact lens wearers

PURPOSE: Tear volume is reduced in symptomatic contact lens wearers, evaporation of the ocular tear film may be a cause. In this study we have focussed on symptomatic or intolerant subjects and compared their tear film lipid-related features to those tolerant to soft contact lens wear. METHOD: Fourteen tolerant and 10 intolerant to lens wear subjects were recruited for this study. Intolerance to lens wear was defined as experiencing dryness symptoms in the first 6 hours of lens wear and consequently not being regular lens wearers. Lipid layer appearance was graded on a 0-5 scale, meibomian gland obstruction was observed, and the McMonnies questionnaire completed. Tears were collected without reflex stimulation. Degraded lipid (tear aldehyde content), secretory phospholipase A2 enzyme (sPLA2) concentration and activity and lipocalin concentration were analysed using spectrophotometry to quantify colour reactions and enzyme linked immunosorbent assays. Statistical results were calculated using non-parametric tests (median +/- interquartile range) or chi-squared test. RESULTS: Degradation of polyunsaturated fatty acids and related esters leads to the by-products, malondialdehyde and 4-hydroxy-2(E)-nonenal. Intolerant subjects were found to have significantly (p = 0.004) higher concentrations of these by-products in their tears (0.85 +/- 1.0 microM; n = 9) compared to tolerant subjects (0.15 +/- 0.15 microM; n = 10). Intolerant subjects (1.86 +/- 0.05 ng/microl; n = 9) had significantly more (p = 0.047) sPLA2 enzyme in their tears compared with tolerant subjects (1.80 +/- 0.08 ng/microl; n = 12) and significantly more enzyme activity (p = 0.012). Intolerant subjects had significantly higher amounts of lipocalin in their tears (2.40 +/- 1.5 microg/microl; n = 10, p < 0.001) compared to tolerant subjects (0.45 +/- 0.85 microg/microl; n = 13). CONCLUSION: Changes to the components of the tear film, however small, can disturb the nature and dynamics of the tear film. Increased lipases, degraded lipids and lipocalins in the aqueous tear film potentiates intolerance to contact lens wear and was associated with increased McMonnies dry eye history scores and symptoms scores.     

Substance P and its metabolites in normal human tears

PURPOSE: To determine amounts and biochemical characteristics of substance P-like immunoreactivity (SPLI) in tears of normal human subjects. METHODS: Forty-three healthy subjects (16 males and 27 females; age range, 17-80 years) participated. Ten microliters of unstimulated tears were collected with a micropipette from one eye of all subjects. Tear samples were partially purified by C-18 cartridges. SPLI concentrations in purified samples were measured by enzyme immunoassay (EIA). For biochemical characterization of SPLI, tear extracts were fractionated by high-performance liquid chromatography (HPLC). Each fraction then was subjected to EIA. To determine the metabolism of substance P in tears, synthetic substance P was incubated in medium containing pooled tears and then analyzed by HPLC with the detector set at a 210-nm wavelength. RESULTS: The SPLI concentration in normal human tears was 306.0 +/- 96.5 pg/mL (mean +/- SD; range, 148-555 pg/mL). SPLI did not significantly vary by age or gender. HPLC analysis indicated that SPLI in tears consisted of five different substances and that substance P was converted to several fragments, including SP(8-11) by enzymes present in tears. CONCLUSIONS: Substance P, a normal component of human tears, presumably is released from the nerve endings in the ocular surface and converted to fragments by degradative enzymes in tears.     

Is ascorbate in human tears from corneal leakage or from lacrimal secretion?

Background: This study investigated whether fresh main lacrimal gland secretion contains ascorbate, with a view to providing indirect evidence of an immediate source of this antioxidant in tears. Our hypothesis was that, if the source is corneal leakage, continuous tearing or rinsing of the eye will result in a marked decrease, by dilution, in ascorbate concentration in the reflex tears collected. Alternatively, the ascorbate concentration will be relatively constant if the main lacrimal gland secretion is the main immediate source. Methods: Five successive samples of yawn‐induced reflex tears were collected from the same eye of each of 42 subjects. In 36 of these volunteers, the testing eye was then flushed with 10 ml of saline and a sixth tear sample (‘post‐flush’) was collected immediately. Tear ascorbate concentrations were measured using a validated high performance liquid chromatography method. Results: The ascorbate concentration of the first sample (baseline) was slightly but significantly (P < 0.0001) lower (17.3 ± 8.9 μM) than the four subsequent samples in all subjects (average 21.4 μM). Ascorbate concentrations of post‐flush samples were very similar to pre‐flush values. Mean ± SD ascorbate concentrations of pre‐ and post‐flush samples were, respectively, 22.5 ± 10.9 and 17.3 ± 5.8 μM. Discussion: Results show that ascorbate is present in fresh reflex tears. Data do not support the view that the cornea is the source of tear ascorbate in healthy eyes. Rather, results indicate that ascorbate is present in main lacrimal gland secretion and that this antioxidant is depleted in basal tears. Measurement of tear ascorbate may offer useful information regarding antioxidant supply to and protection of the cornea.     

HPLC analysis of closed, open, and reflex eye tear proteins

Changes in the closed, open and reflex eye tear proteins of normal subjects were compared and analysed. Tear proteins were resolved by high-performance liquid chromatography (HPLC) utilising both gel filtration (P-300 SW) and reverse-phase (C-18) columns and the HPLC fractions were further analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. The protein composition of the closed-eye tear was significantly different from that of the open and reflex-eye tear. Secretory IgA (sIgA) was the predominant protein in closed eye tears constituting 49% of the total protein compared to 11% in reflex tears, whereas lysozyme was the predominant protein (53%) in reflex tears. Levels of lactoferrin, lipocalin and lysozyme were relatively constant in both open and reflex tears. HPLC profiles of the closed-eye tears, upon continuous stimulation of lacrimal glands indicated that sIgA was significantly reduced whereas lactoferrin, lipocalin, and lysozyme were significantly increased. These results indicate that the tear composition upon waking attains that of the open eye within 4 to 5 minutes, and upon continuous stimulation this reflects the reflex-eye tear composition. It also indicates that mechanisms responsible for changes in concentration of constitutive and regulated tear protein with stimulus can be studied successfully using non-invasive methods to collect human tears.     

Cystatins in human tear fluid

The activities of cysteine proteinases which include several lysosomal cathepsins are controlled by naturally occurring inhibitory proteins termed cystatins. Cystatins occur both intracellularly and extracellularly in various tissue fluids including tears. Tears were collected by the Schirmer paper strip method from healthy volunteers who had no history or signs of external ocular disease. The tear components were extracted from the filter papers, and used to determine the apparent free cystatin activity and cystatin levels of tears, and for immunoblots. Tears were also collected using capillary tubes for the measurements of cystatins. By titrating papain, a cysteine proteinase, of known specific activity with tear fluid, relatively high levels of apparent free cystatin activity were demonstrated in tears: 28.8 +/- 3.47 (S.E.M.) pmols papain inhibited per mg tear protein (n = 9). The concentrations of cystatins in tear samples were measured by an indirect enzyme-linked immunosorbent assay (ELISA) using antibodies against human salivary cystatin S and purified cystatin S as standard. The ELISAS revealed that tears contain high levels of cystatin-like immunoreactive material, amounting to about 10% of tear proteins. In microgram cystatin S/mg protein the values were: right eye: 94.7 +/- 9.9; left eye: 115.5 +/- 14.8; n = 12. Cystatin levels of tears collected using capillary tubes were comparable: 120.7 +/- 19 micrograms/mg protein (n = 10). Immunoblots of tear fluids revealed a protein of about 14,000 molecular weight which reacted with antihuman cystatin SN monoclonal antibodies. Protein(s) of similar molecular weight were visualized using antibodies against human cystatins S and C. Less abundant additional cystatin-like immuno-reactive proteins were detected by using the two latter antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of connective tissue growth factor (CTGF) in human tear fluid: preliminary results

PURPOSE: Connective tissue growth factor (CTGF) is one of the main regulators of fibrosis. We aimed to evaluate its presence in the human tear fluid of healthy individuals. METHODS: A total of 70 tear fluid samples were collected from eight volunteers prior to and after stimulation of reflex tears with onion vapour. Specific ELISA analysis was performed with goat IgG against human CTGF. RESULTS: Connective tissue growth factor was detected in seven samples (10%), with maximum levels of 17 ng/mL in basal tears. Induction of reflex tearing resulted in a fast and significant decrease of CTGF concentrations (r = - 0.95). No CTGF was detected in 90% of the samples. CONCLUSION: Connective tissue growth factor may occur in tear fluid in healthy human eyes. This indicates a possible role for tear fluid CTGF in ocular surface fibrosis and wound healing.     

Basal tear turnover and topical timolol in glaucoma patients and healthy controls by fluorophotometry.

To assess the effect of glaucoma and timolol on tear secretion, basal tear turnover was measured with fluorophotometry in 13 open-angle glaucoma patients not using any ophthalmic medication, 24 patients using timolol medication daily, and 41 healthy control subjects. Basal tear turnover is defined as the tear turnover at the lowest level of reflex lacrimation possible under physiologic conditions. Tear turnover was calculated from the decay of the tear fluorescence after instillation of fluorescein. Minimal influence of reflex lacrimation was obtained by instilling 1 microliter of 2% fluorescein without touching the eye and by discarding measurements performed in the first 5 min. Minimization was confirmed by a monophasic decay of tear fluorescence. The values of patients who used timolol and those who did not use timolol were significantly lower than those of healthy control subjects (mean values in percent/minute +/- standard deviation: 10.1 +/- 3.2, 12.3 +/- 4.1, and 15.6 +/- 5.4, respectively; Student's t-test: P < 0.02). The values of patients who used timolol were significantly lower compared to those of patients who did not use timolol (P = 0.03). The tear film break up time values of patients who used timolol were significantly shorter than those of patients who did not use timolol and healthy control subjects (Fisher exact test: P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)     

Group IIA phospholipase A(2) content in tears of patients having photorefractive keratectomy

PURPOSE: To study the effect of photorefractive keratectomy (PRK) on the concentration of group IIA phospholipase A(2) (GIIAPLA(2)) in tears. SETTING: Departments of Ophthalmology and Pathology, University of Turku, Turku, and Helsinki University Eye Hospital, Helsinki, Finland. METHODS: Tear samples were collected from 25 eyes of 23 patients (mean age 32.3 years +/- 8.6 [SD]) preoperatively and 2 and 7 days after PRK. The GIIAPLA(2) concentration in the tears was measured by time-resolved fluoroimmunoassay. RESULTS: The GIIAPLA(2) concentration was significantly lower and the tear fluid flow rate significantly higher 2 days after PRK than preoperatively. At 7 days, the GIIAPLA(2) concentration and the tear fluid flow-corrected excretion of GIIAPLA(2) were significantly higher than preoperatively and at 2 days. The tear flow rate was also significantly higher than preoperatively. CONCLUSIONS: The GIIAPLA(2) content in tears decreased 2 days after PRK due to dilution of the GIIAPLA(2) content during hypersecretion of reflex tears. Photorefractive keratectomy caused an increase in the tear flow rate, GIIAPLA(2) concentration, and tear fluid flow-corrected excretion of GIIAPLA(2) in tears 7 days after surgery, enhancing the protection of tears against bacterial infections.     

Relationships between central tear film thickness and tear menisci of the upper and lower eyelids

PURPOSE: To investigate the relationship between central tear film thickness (TFT) and tear menisci of the upper and lower eyelids using real-time optical coherence tomography (OCT). METHODS: Both eyes of healthy subjects were imaged with a real-time OCT to obtain height, curvature, and area of upper and lower tear menisci simultaneously. Central TFT was indirectly measured by calculating the difference between baseline measurements of the central corneal thickness plus tear film and the true corneal thickness obtained after instillation of artificial tears. Results from two normal blinks were obtained from one eye at each visit and repeated the next day. RESULTS: The average central TFT was 3.4 +/- 2.6 microm. The upper tear meniscus curvature, height, and area were 239 +/- 112 microm, 268 +/- 68 microm, and 22,732 +/- 11,974 microm2 respectively. There were no significant differences in curvatures, heights, or areas between upper and lower tear menisci, nor were there any differences in measured variables between the two blinks at each visit or between the two repeated visits in the right and left eye groups (P > 0.05). The upper and lower tear menisci in each eye group on each day correlated strongly with curvature, height, and area (all P < or = 0.03). However, no tear meniscus variable was a significant predictor of TFT (all P > 0.44). CONCLUSIONS: OCT is a promising tool in the measurements of TFT and dimensional variables of tear menisci. Upper and lower tear menisci have nearly identical dimensions.     

Group IIA phospholipase A2 content of basal,nonstimulated and reflex tears

PURPOSE: To determine the concentration of group IIA phospholipase A2 (GIIAPLA(2)) in basal, nonstimulated and reflex tears of young normal subjects. METHODS: The GIIAPLA(2) content of tears was measured in left eye of 16 healthy subjects (mean age 24.5 +/- 2.2 years). Five samples were taken from each subject: nonstimulated sample; basal sample in a dark room after topical anesthesia; and reflex tear samples immediately on stimulation by bright light and breathing of the vapor of onions, and 1 and 3 minutes after the onset of tear flow. RESULTS: The GIIAPLA(2) content of reflex tears was statistically significantly lower than the GIIAPLA(2) content of basal tears, whereas there was no statistically significant difference in the GIIAPLA(2) content between nonstimulated and basal tears. CONCLUSIONS: The current results indicate that the basal and nonstimulated tears are similar in their GIIAPLA(2) content, and that the GIIAPLA( 2) content of tears decreases with stimulation, possibly due to dilution of GIIAPLA(2) content during reflectory hypersecretion of tears.     

The Fall and Rise of Tear Albumin Levels: A Multifactorial Phenomenon

Albumin in tears is used as a diagnostic marker of ocular insult and inflammation, but whether its presence in tears is responsive or part of an adaptive reaction remains unresolved. A review of the literature on tear albumin concentration emphasizes that variables such as collection method, stimulus, assay technique, and disease state influence the quoted values to different extents. Influence of assay technique is negligible in comparison to variation in sampling conditions. Ocular disease increases albumin concentrations but not in a specific manner. The literature review also highlighted that little systematic research has been carried out on the daily cycle of tear albumin levels. In order to remedy this shortcoming, we investigated variations in tear albumin concentration during the waking day. The concentration of albumin in 400 tear samples collected from 13 subjects was assessed at 2-hourly intervals throughout the waking day. Highest daytime albumin concentrations were obtained within 10 minutes of waking, with a mean concentration of >50 ± 22 μg/ml. Albumin levels were at their lowest, but most consistent, 2-6 hours post-waking. This pattern was followed by a progressive increase in albumin concentration during the latter part of the day. Although individual subject-to-subject concentration differences were observed, this distinctive pattern of diurnal variation was found in all subjects. The results presented suggest a regulated, not random, pattern of variation within the period of study.     

Factors influencing the quantitative determination of tear proteins by high performance liquid chromatography

HPLC analysis of human tears allows tear protein profiles to be obtained within ten minutes. A tear protein profile normally consists of 4 major peaks: IgA, lactoferrin, protein G and lysozyme. Although it is a rapid method, the use of High Performance Liquid Chromatography in the (quantitative) determination of proteins in tears is influenced by various factors. The day to day variability of the quantitative use, ranges between 7 and 9% for the various tear proteins. Combining the HPLC method with a convenient collection method such as sponges or Schirmer strips, showed that the sponges and some of the Schirmer strips used in this study eluted significant material absorbing light at 280 nm. No statistical difference was observed in the HPLC protein profiles of tears collected with Schirmer strips or with sponges. Using sponges has the advantage that they can absorb almost twice as much tears in a same period of time as Schirmer strips. HPLC analysis of human tear proteins is not accurate when there is albumin leakage as in traumatic sampling with Schirmer strips or in inflammatory states. The I-125 column which was used in our study is not able to separate lactoferrin and albumin, which may cause an overestimation of lactoferrin in inflammatory conditions. The study presented here indicates that for quantitative use of HPLC in epidemiological tear protein research better separating protein gel filtration columns are needed.     

Reflex and steady state tears in patients with latent stromal herpetic keratitis.

PURPOSE: To compare tear production in patients with stromal herpetic keratitis with that in healthy control subjects. METHODS: After instillation of 2 microL fluorescein into both eyes, the tear-fluorescein concentration was measured by fluorophotometry. During the first 10 minutes, steady state tear turnover (TTO-1) was determined. After a nasal alcohol stimulus to induce reflex tears, a second steady state tear turnover (TTO-2) was obtained during 15 minutes. The index of reflex lacrimation (IRL) was calculated as the percentage decrease in tear fluorescein concentration directly after the stimulus. TTO-1, TTO-2, and IRL were determined in the patients' affected eyes (n = 12), in the patients' healthy contralateral eyes, if possible (n = 9), and in one eye of healthy control subjects (n = 24). RESULTS: The TTO-1 in the affected and healthy eyes of patients was approximately two times lower than the TTO-1 in eyes of healthy control subjects (P = 0.012 and P = 0.024, respectively) and almost equal to the TTO-2 in eyes of healthy control subjects (P = 0.32 and P = 0.40). There were no significant differences in the values of TTO-1, IRL, and TTO-2 between affected and healthy eyes of patients (P > 0.5). IRL and TTO-2 did not differ significantly among the three groups (P > 0.5). CONCLUSIONS: Both eyes of the patients were dry. The dryness could be due to a defective reflex lacrimation under physiological conditions that can still be induced by nonphysiological nasal excitation. The cause of this may be demyelination of both trigeminal root entry zones as a result of a unilateral eye infection by the herpes virus. Another possibility is that dryness predisposes to herpetic infection or recurrent inflammation.     

What does the phenol red thread test actually measure?

PURPOSE: This study attempts to resolve whether the phenol red thread test (PRT) is a test of tear volume or tear production through comparisons with other techniques. METHODS: Twenty asymptomatic subjects (10 men and 10 women; average age 30.6 +/- 10.8 years) had PRT (Zone Quick, Menicon) results compared with tear turnover rate (by fluorophotometry; Fluorotron Master, OcuMetrics) and tear volumes (from tear meniscus height and back extrapolation from fluorometric data). RESULTS: PRT wetting was not correlated with either tear turnover or volume (by fluorophotometry or tear meniscus height) on a Pearson product moment correlation test (p > 0.05). CONCLUSIONS: No clear experimental evidence in favor of the PRT being a measure of tear production or volume was found. It is probable that the PRT measures uptake of a (small) amount of fluid residing in the eye, stimulates a low degree of reflex tearing, and reflects the absorption characteristics of the thread dependent on the biophysics or composition of tears.     

Protein levels in nonstimulated and stimulated tears of normal human subjects

Atraumatically collected nonstimulated (less than 1 microliter/min) and stimulated (greater than 50 microliters/min) tears from 30 clinically normal subjects were fractionated by size exclusion high-performance liquid chromatography (SE-HPLC). Enzyme-linked immunosorbent assay (ELISA) and kinetic assays were applied to relevant HPLC fractions to quantitatively identify 12 tear proteins. Secretory IgA levels were much higher in nonstimulated than in stimulated tears, and a similar disparity was seen also with IgA1 and IgA2 in the HPLC fraction containing secretory IgA. IgM levels were also higher in nonstimulated tears. Levels of the primary lacrimal gland proteins, lactoferrin, tear specific prealbumin, and lysozyme were similar in both types of tears. Significantly higher concentrations of the major serum proteins, IgG, transferrin, and serum albumin were measured in nonstimulated tears. Overall, 8 of the 12 proteins assayed were present at significantly higher concentrations in nonstimulated tears. These results show that tear flow rate strongly influences the protein profile obtained. Therefore, to allow valid comparisons of tear protein profiles within and between studies that use atraumatic collection procedures, an indication of flow rate during collection should be reported.