Exp. Eye Res.(1990) 50, 251-259

The pH in the

Precorneal Tear Film and Under a Contact Measured with a Fluorescent Probe FRED S. CHEN

Department of Ophthalmology,

AND

DAVID

Lens

M. MAURICE”

Stanford University Medical Center, Stanford, CA 94305, U.S.A.

(Received 6 July 1989 and accepted in revised form 14 September 1989) The reaction of the precornealtear film of the human eyewas determinednon-invasively by instilling pyranine, a pH-sensitivefluorescentdye.The meanvalue was 7.83 (S.D. kO.10) andit takesup this value immediately on opening the eye after the lids had been kept closed.The HCO, systemseemsto be responsible for only a portion of the bufferingpowerof the tear 6lm. When a drop bufferedto pH 6.4 with 0.075 M PO, was instilled,the tearsreturned to their normal value in about 7 min, consistentwith the washout time of solutesin the conjunctival sac. A pH of 73 was establishedin the tear fluid behind contact lenses,either gaspermeableor impermeable,probably asa result of their restricting the lossof CO, from the eye. The rabbit,pre-cornea1tear fihn is more alkaline, at about pH 8.2. Key words : tear film ; pH ; pyranine ; contact lens; human: rabbit.

1. Introduction The pH of the tear fluid is of interest in its own right as well as in connection with the ionization of topically applied drugs and their penetration into the eye, and it has been the object of more than 30 investigations (Murube de1 Castillo, 1981). Only recently (Fischer and Wiederholt, 1982), however, has a study been made in humans of the precorneal tear flm, which is the portion of the fluid most relevant to cornea1 physiology and to drug penetration. These workers brought a glass microelectrode together with a reference electrode into contact with the anesthetized cornea surface and were able to measure the pH within a few seconds. The availability of a slit-lamp fluorometer enabled us to try an alternative, less invasive, method of making these measurements. The tear film is stained with a fluorophore, pyranine, and the comparison of the magnitude of its fluorescence at two different wavelengths of the existing illiiation is sufficient to determine the pH of the fluid. The changes with time of the human and rabbit tear film have been compared both under normal conditions, when buffered eyedrops are instilled, and under a contact lens. A brief account has been given elsewhere (Maurice and Chen, 1986). 2. Materials

and Methods

Dye

Pyranine (8-hydroxypyrene- 1,3,6-trisulfonic acid trisodium salt) has an emission spectrum that peaks at 510 nm and is unaffected by the pH. However, in acidic solution it exists principally in its protonated form, which absorbs most strongly around 405 nm while in alkaline solution it dissociates into its base form, which absorbs most strongly around 470 nm * For correspondence. 00144835/90/030251+09 18

$03.00/O

(Fig. 1). Therefore, when the compound is excited alternately at these two absorption wavelengths, the ratio of the intensities of the emitted light will be characteristic of the pH of the solution (Wolfbeis et al., 1983). Pyranine, with a pK, of 7.3, is most sensitive to pH values ranging from 6.3 to 8.3. For instillation into the eye, pyranine was dissolved in physiological saline or in 01 M phosphate buffer of various pH to give, usually, a concentration of 0.2 5 %. The dye is not particularly toxic on intravenous injection (Lutty, 1978). Instilling nine drops of 1% solution at lo-min intervals into a rabbit’s eye led to no signs of damage either immediately or after 24 hr, and the dye did not stain the cornea. Instilling 1% drops in one subject did not cause discomfort at any tie. This is as expected since the highly charged molecule should not penetrate the epithelial cells of the eye and only their outer membranes would be exposed briefly to a high concentration of the dye. Fluorometer The fluorescent intensity was measured with a slitlamp fluorometer (Maurice, 1987). Broad band interference lllters centered on wavelengths 420 nm (violet) and 450 nm (blue) w&e mounted in front of the light source and every few seconds were switched manually by the observer. The instrument was designed so that similar filters could be incorporated within it, but these were not conveniently located for rapid alternation. The illuminating light was focused as a 2-mm diameter circle on the surface of the cornea. In the photometric microscope, the image of this circle was focused on a mirror and was observed through an eyepiece. A small rectangle of the reflective surface of the mirror, equal in area to about one fifth of the illuminated circle, was missing so that the light passed through it on to a photomultiplier tube. A broad band 0 1990 AcademicPressLimited EER 50

252

F. S. CHEN

Filters

Violet

11

II

Blue

AND

D. M. MAURICE

Green I

I

Absorption

PH9 _-

/

/

/-\J/--

i 450 Wavelength

(nml

FIG. 1. Absorption spectra of the protonated (pH 6) and ionized (pH 9) forms of pyranine, and their emission spectrum. Adapted from Wolfbeis et al. (1983). The band width of the filters used in these experiments is also shown. interference fllter centered on 540 nm (Fig. 1) was placed in this light path. When the instrument was correctly positioned, it measured the fluorescence of the tear illm at the exciting wavelength selected. Once the instrument had been calibrated, the ratio of the fluorescent readings at the two wavelengths was used to determine the tear illm PH. The output of the photomultiplier tube was ampliiled, converted to its logarithm, and plotted against time on a pen and ink recorder. The distance between the recorded tracings when the filters were switched was proportional to the ratio of the corresponding tear film fluorescences and indicated its pH independently of the total concentration of pyranine in the fluid.

Experimenbl

Procedures

The human subjects were students or laboratory workers who were not known to be suffering from any ocular problems other than refractive errors. They were all between 20 and 30 yr old, except for one who was in his early 60’s. The procedure was cleared by the human subject committee of Stanford Medical School, and informed consent was obtained from the volunteers before the experiment. The subject was seated in front of the iluorometer as if it was a normal slit-lamp, and the eye not being observed looked at a fixation light. The instrument was focused on the cornea near its center and background readings were taken. The subject sat back, a warm drop of pyranine solution was instilled into the eye horn a normal dropper, the excess blotted with a tissue and the head returned to the support: at the same time the recorder was started and allowed to mn throughout the experiment. On a few occasions, a

micro drop of O-8 ~1 was wiped onto the bulbar conjunctiva. While the instrument was kept in focus with one hand, the excitation filters could be moved with the other. Normally, they were kept a few seconds in each position to allow the new level to be established on the recorder which was provided with a 06 set time constant; when identification of the peaks was difI%cult, the period of the blue light was made a little shorter than that of the violet. After the Instrument had been focused at the beginning of the measurements little further adjustment was needed. Similar procedures were followed with the experimental animals, New Zealand white rabbits weighing about 2 kg. They were tranquilized with 10 mg kg-l of thorazine and secured on a rack (Maurice and Singh, 1984) which was mounted on a platform which replaced the normal head rest. The upper lid was pulled back and a large drop of the pyranine solution was allowed to fall on the upper bulbar conjunctiva. In humans, the changes in pH in the tear iluid underneath contact lens, both gas impermeable (polymethyl methacrylate) and permeable (Polycon) were followed. The lens diameters were 8-9 mm and their inner curvatures were calculated to give a 4050 pm central clearance. Tight-fitting lenses were also fitted to two subjects. Before a lens was inserted, the inside was blotted dry and a small drop of 025 % unbuffered pyranine solution was placed within it, Examination in the ordinary slit-lamp fitted with a cobalt filter showed that, even immediately after insertion, any dye that escaped onto the surface of the lens was negligible in comparison with that which was trapped beneath it. The dye did not penetrate into the lens material.

THE

pH

IN THE

PRECORNEAL

TEAR

253

FLUID

Calibration Fluorescent intensity ratios were determined for buffered solutions of pyranine placed in cuvettes containing a fluid layer lo-20 pm thick, the order of magnitude of the precorneal tear Urn. They were constructed by glueing together two microscope slides under pressure and a fresh one was made for each solution ; their separation was checked with a micrometer screw gauge. The pH of the bulk solution was measured to an accuracy of 001 units with a glass electrode.

total tear protein concentration is about 6 g 1-l (Murube de1 Castillo, 1981) and the effects of both a 36 g 1-l solution (human plasma diluted with an equal volume of pH 7 buffer) and 3.6 g 1-l solution were tested in the thin cuvettes. The only marked effect was found when the pyranine concentration was dropped to 00125 Y0 with the higher concentration of plasma. This depressed the calibration curve (Fig. 3) and could lead to a serious error in the estimates. With a 0.12 5 Y0solution of pyranine a small depression of the curve could still be detected with the high protein solution, which would not be important over most of the range, but could become serious around pH 8.

3. Results Calibration

Rabbits

Figure 2 shows recordings from cuvettes when filled with pyranine solutions at three different pH values. The ratio of the readings obtained with the two filters as represented by the excursion of the pen is seen to undergo a change of about 10 : 1 between the pH extremes. The calibration curve is shown in Fig. 3 and it was not affected by whether the solutions were buffered with phosphate or Tris. In the most sensitive region, the methods is capable of detecting a difference of about 0.025 pH units, but it becomes insensitive above pH 8 as it would be also in the acid range, which was not of concern in these experiments. The calibration did not change appreciably over the range 2-0*002% pyranine, the lower level being that at which background noise from the tissues became a troublesome factor. Albumin has been reported to cause a shift in the pH fluorescent ratio curve (Thomas et al., 1990) and there is a possibility that the proteins dissolved in the tear illm could lead to an error in the measurements. The effect of human plasma on the calibration curve oj thin films of pyranine was investigated. The tear proteins diier from those in the blood, but they share many components. Several workers agree that the

Observations in animals are simple because they do not move their eye or blii over long periods and the focus of the instrument scarcely needs to be checked. As is well known, spontaneous blii in rabbits are infrequent. The eyes of these animals were artificially bliied at irregular intervals by pulling the upper lid down gently with a finger to cover the cornea and then raising it. After a drop of pyranine had mixed with the tears, in most cases the absolute level of fluorescence in the precorneal tear 6lm did not change until the eye was next blinked, although on some occasions it fell slightly or, more rarely, rose. After a blink, the level could be similar to or several times larger or smaller than what it was before: however, after a series of blinks there was no large shift, but only a gradual decline. An unbuffered drop generally had a pH of 6. The initial reading in the eye was about pH 7 and, if the eye was not blinked, it rose in a minute or two to above pH 8 (Fig. 4). If the observations were carried on for 5 min or more, the pH immediately after a blink was about 75 and then rose over the next 30 set to a value above 8 (Fig. 4). In eight eyes the final level of

100

r

pH 8.9 Blue pH 6.5 Violet

filter Violei

Blue

filter Diluted

I : IO

Blue

Violet

IL

FIQ. 2. Recordings taken on thin films of pyranine in different buffers. Each cycle of the curves corresponds to switching of the excitation filters. The tracing on the extreme right is of the previous solution diluted ten times. Note the logarithmic scale of the ordinate. 18-2

254

F. S. CHEN

AND

D. M. MAURICE

pH lay in the range of 7.9-8.5, mean 8.2, after 5 min. In this range the estimates are not accurate, but in six cases it appeared to be certainly 8.2 or above. When drops buffered to pH 6 were used the tear pH was near this value originally, and its rate of neutralization was irregular, depending probably on how frequently the eye was blinked, but reached pH 7 in about 10 min. A gradual change in pH occurred between blinks that was considerably slower than with unbuffered drops (Fig. 5).

Humans -0.6

I

I 6.5

6

I 7

I 7.5

I 8

The instillation of the dye solution gave rise to no sensations other than those resulting from the saline or buffer in which it was dissolved. Although the procedure is comfortable and the brightness of the light is not disturbing, the constant changing of the color filters was distracting and it was diicult for even an experienced observer to maintain fixation for more than a few minutes at a time. The number of altemations of the filters that can be fitted in between blinks is limited and it is apparent that a most sophisticated signal processing system, such as that used by Bonanno and Poise (198 7) in the cornea, would be an improvement.

I

8.5

PH

3. Calibration curves of ratio of excitation filter readings against PH. The absolute values of the log ratios were adjusted by means of a neutral density filter. Broken line shows effect of presence of protein on a relatively low concentration of pyranine. FIG.

Unbuffered

UnbufSered Drops With either a standard drop of 02 5 % or a micro drop of 2.5 % pyranine solution, the pH of the tear 6Im stabilized after about 30 set and showed no change after blinking (Fig. 6). The absolute amount of dye in the tear film dropped with time as would be expected as a result of basal lacrimation, but as long as the reading was at least ten times the background value from the eye, the recorded pH value remained constant to better than one tenth of a unit during a single experiment. The values were estimated in eight subjects using 8-30 successive filter reversals on each occasion [their mean was pH 7.83 (kO.10 SOD.)].

PH 9

. .*

.

.

:

C.

.

.a

.

I I

71 0

I

*

2 Time

I

I

3

4

(mln)

FIG. 4. Experimental tracing and derived tear f%m pH values after a drop of UnbufFerecIpyranine was instiIIed into the eye of a rabbit. RobbIt

(pH 6-l buffer)

8 PH

7

.

.

.

.

.

l

.

.

.

.*

l

.

.

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.

l *



.**.*

l

0’ l

l

l

.

6 0

3

6

9

12 Time

FIG.

15

18

21

(mm)

5. Changes in pH a!& instilling drop of pyranine, buffered to 61 with 0075

M

phosphate, into rabbit eye.

THE

pH

IN THE

PRECORNEAL

TEAR

255

FLUID Huron

+

0

Eye

closed

2

I

t

3

4 Time

FIG.

(unbuffered)

5

7

6

(mm)

6. Changes of pH in tear f&n after instilling drop of unbuffered pyranine into human eye. Arrows

show occurrence of

blinks. Human

(pli

6.4

buffer)

w \1b

il 4

Eye

closed

t

4

Eye

closed

f

8

l* PH

l *

l .e’ ..

.*

7 .

.

.

.

.*

.*’ 6 0

I

2

3

4

5 Time

6

7

8

9

(mln)

FIG. 7. Changes ofpH in tear film after instilling a drop of pyranine, buffered to pH 6.4 with O-075 M phosphate, into human eye.

Buffered Drop

Open Eye

Immediately after the instillation of a single drop of pyranine in a 0075 M phosphate buffer at pH 64, the value in the tear film was found to be little different from this value. The tear film slowly became more alkaline, reaching pH 7 in about 2 min, and approximately its normal value in a further 5 mm (Fig. 7). The neutralization of the tears could be hindered by closing the eye. In two subjects an attempt was made to change the pH of the conjunctival sac by repeatedly instillmg drops of unstained O-1 M phosphate buffer at pH 64 before the buffered pyranine drop was administered. No marked increase in neutralization time was noted and it did not seem valuable to pursue this approach.

One eye of a subject was lightly anesthetized with two drops of dilute (005%) proparacaine. When the eye was comfortable, a drop of unbuffered pyranine was instilled, and normal blinking was consciously maintained for a few minutes. The eye was then held open for up to 1 min while readings were taken of the tearfilm.ThepHstabiliidatavalueof783 (10.33) in these cases, which is identical with the normal value. In one subject, a stream of warm air was blown over the unanesthetized eye from a hair dryer placed a few feet away at the fastest rate that could be tolerated without lacrimation. No shit in the pH of the tear film could be detected whether the dryer was on or off.

256

F. S. CHEN

Closed Eye

Attempts were made to measure the tear film pH as soon as possible after the eye was opened following a

period of closure. A drop of unbuffered pyranine was instilled into an eye and a minute or two later readings were taken with the eye blinking normally. The subject then closed the eye for one minute and opened it again on signal, when readings were recommenced. Although the instrument remained focused on the cornea, small readjustments and reflex blinking caused a slight delay, and nearly 30 set passed before sufficient readings could be accumulated. In three subjects there was only a 001 pH average difference before and after the period of closure. In an attempt to obtain values more quickly after the opening of the eye, recordings were repeatedly taken using only one excitation filter and at the paper speed of 1 cm set-’ (Fig. 8) with the response time increased to that of the recorder (full scale in 0.2 5 set). The eye was closed only for brief periods of a few seconds in order that the instrument would remain in focus upon the cornea and the filters were alternated between each recording. The tracings showed a rise over about 0.5 set to a plateau that was maintained occasionally up to 5 set, although the measurement was usually terminated before then. A small proportion showed a marked irregularity of the trace probably as a result of blinking or eye movements, but these could be distinguished without ambiguity and rejected. In 16 recordings, taken from five subjects, the change in the value between 1 and 3 set was noted for each of the two filters. This gives a value A log B or A log V, where B

Blue

5

4

Filter

3

Violet

2

I Eye open

Set

4

D. M. MAURICE

or V is the fluorescence reading in blue or violet light, and A log B = [log BIBsec- [log Bll,,,. Then A log B-A log V can be expressed as [log B/VjSsec - [log BIYLC which allows the pH change between 1 and 3 set to be derived from the calibration curve. This amounted to +0.015 units, on the average, which was not significant. Contact Lenses

When the subject inserted steeply fitting PMMA lenses and blinked normally, the concentration of pyranine trapped underneath diminished with time, but allowed satisfactory readings to be taken for lo-20 min. Six subjects showed a pH of 7.0-7.95 under the lens within the ilrst 2 min of insertion, but this declined and became virtually steady and reached an average value of pH 7.3 (range 6.9-7.6) when the recordings were discontinued after 12-18 min. Three of the subjects were normally contact lens wearers, but they did not give rise to readings that grouped separately from the new wearers. Four of the subjects were also measured with gas permeable lenses, but in no case did the pH recording differ by more than 0.1 units from that with the impermeable lenses. Measurements under a tight-fitting lens or toward the edge of a steep-fitting lens where the tear film was thin, though few, did not seem to be appreciably different. Two subjects were instructed to avoid blinking, as far as possible, and in these cases the concentration of pyranine beneath the lens did not drop, so that presumably there was no tear circulation behind them. After 10 min, the pH was at a level of 6.9 and 7.3 in the two cases. In the former, a period of rapid blinking reduced the pyranine concentration about tenfold, whereupon the pH rose to 7.2 and again fell to 7-O after 10 min with slow blinking (Fig. 9). 4. Discussion

Filter

3

AND

2 Set

I

Eye open

FIG. 8. Tracings of tear 6hn fluorescence on opening the eyelids using a single filter in each case.

The use of pyranine has the advantage over other methods that have been described in that it measures the pH of the tears in the precorneal i&n itself, in a non-invasive way. Unfortunately, the alkalinity of the tears results in the measurements being taken near the end of the pyranine titration curve and a dye with similar properties, but a pK of around 8 would result in more accurate estimates of tear film PH. A cause of concern is the interference of macromolecules in the tear film with the absolute value of the estimate. There are two considerations that would suggest that this is not very serious. First, when pyranine was mixed with relatively high concentrations of plasma the pH of the solution was correctly estimated until the dye concentratioh became quite low in comparison to what was used in the eye. Correspondingly, the estimates of tear iilm pH remained constant as the pyranine in it became

THE

pH

IN THE

PRECORNEAL

TEAR

D.M. PMMA

.

.

257

FLUID

.

lens (steep

. .

.

fit)

.

.

.

.

. . .

Time

.

.

.

l

.

l

.

.

.

(min)

FIG. 9. Changes in total fluorescence and pH under a PMMA contact lens in one subject. Note fall in concentration of

pyranine on rapid blinking but no change in PH. diluted, at least over a concentration range of 10 : 1. Second, when a butrered drop was used, the initial pH of the tear illm did not differ from that in the bulk phase by more than 0.1 unit. It might be argued that the instillation of the drop would wash the interfering macromolecules out of the precorneal illm, but experience with iluorescein shows that the precomeal concentration after a single drop is only about one quarter of that in the bulk phase (Maurice and Mishima, 1984) so that it can be supposed that the tear illm will maintain at least three quarters of its original macromolecular concentration. Certainly, the concentration of any ilxed material on the cornea1 surface would not be reduced by the buffer and thus cannot be affecting the pyranine fluorescence. Unfortunately, any protein error would be most serious at the higher pH, where the steady-state levels are found in the human eye, and a question must remain as to how accurate are these absolute values that we report. It may be noted, however, that the pyranine concentrations we used are considerably higher than those studied by Thomas et al. (in press) so that our error would not be as serious as their llnding would suggest. The use of a pH sensitive fluorophore with a higher pK,, when one becomes available, would be valuable in settling the question. Buffering System of Tears The CO, buffering system in the tear 6hn has not been well deilned. The HCO, level in the conjunctival tear fluid is about 25 mu (Yoshimura and Hosokawa, 1963; Milder, 1975). In the lacrimal gland secretion of the rat it was determined to be 20 mu by Alexander, van Lennep, and Young (1972), who, noting a discrepancy between Cl and combined Na and K of 40 mM, concluded that negative charges on proteins in the secretion would account for the 20 mM discrepancy. Charged groups on the cell surfaces, the

mucus, etc., could also exert a buffering power on the tear him. Furthermore, in mildly alkaline solutions CO, can interact directly with proteins to form carbamates (Edsall and Wyman, 1958). The buffering capacity of the isolated tear fluid has been investigated directly by Hill and Camey (19 80) by adding NaOH in a closed system. They found the addition of 2 mEq of alkali shifted the pH irorn 7.4 to 8.6. This is close to pH 9.0 which would be expected if it were a simple HCO, buffered system. On the other hand, Hind and Coyan (1949) bubbled a sample of human tears with pure nitrogen which resulted in a rise of pH to 8.4. The pH should have risen above 9 if this was a true equilibrium at zero CO, tension in a simple HCO, buffered system, which suggests that other systems are also operating in the tear fluid. In any case, the instilled 0.25 % pyranine drop is 5 mi3q and it will be diluted to about 1 mEq when it first mixes with the tears and then will be further diluted by drainage, It is unlikely to have a marked effect on the tear buffering system. Normal Values The value of human tear film pH found by the pyranine method, 7.83, is in accordance with many other estimates (Murube de1 Castillo, 1981) including that of Fischer and Wiederholt (1982) who took measurements with a glass microelectrode directly from the i&n. The S.D. of our values, 0.1 units, indicates a lower variability than suggested by the range of other methods, when this is revealed. Holding the eye open or blowing air over it did not change the tear pH appreciably, and it seems that the tear lllm in humans is more strongly buffered than the isolated tear fluid. The diffusion of CO, from the anterior chamber, pH 74 (Becker, 1959), or comeal stroma, pH 7.5 (Bonanno and Polse. 1987) might have some effect in holding down the tear PH. If we accept that there is a

258

linear gradient of CO, across the cornea and tear illm (Fatt, 1968), the average CO, tension in the film should be raised by 4/500 x 55 = 0.44 mmHg above that in the atmosphere, 0.25 mmHg. The buffering system of the tears is not well enough understood to calculate if a O-69 mmHg CO, level could reduce the pH to 7.8 from the value 8.4 in the absence of CO,, found by Hind and Goyan (1949). Changes in pH Although the precorneal tear 6lm is alkaline, one could expect the freshly secreted lacrimal fluid and that under the lids to be in hydrogen ion equilibrium with the blood and interstitial fluid. In fact, several workers including Hind and Goyan (1949), Yoshimura and Hosokawa (1963), and Hill and Carney (1980) have found collected human tears to be pH 74. Immediately upon opening the eye, after the lids have been closed for a time, the tear him should have a neutral pH but then should rapidly lose CO, to the air and become alkaline (Fischer and Wiederholt, 1982). If the air in front of the cornea were stagnant it might be thought that the CO, concentration in front of it could build up and decrease the tear PH. It is not clear how much air flow occurs across the cornea1 surface. Lewis et al. (1969) showed that there was a 05 m see-’ air stream up across the face which clears the cornea by a few mm, which suggests that the tear7 could be in contact with a thick stagnant boundary layer. However, calculations make it evident (Cussler, 1984) that this would introduce a negligible build up of CO, at the cornea1 surface because of the rapidity with which it diiuses in air. Even if the layer were 1 cm thick its diffusional resistance should be about one-fiftieth that of the cornea. The rate at which the tear film equilibrates with the air can be estimated. When the eye opens and the tear surface is exposed to a low pC0, level the gas can be expected to leave without hindrance so that the surface layer of the tear film is at the same level. A rapid change in the ionization of HCO, and of pH follows in which the rate controlling step is the dehydration of H&O, to CO, (Edsall and Wyman, 1958). This has a rate constant of about 80 set-’ in the absence of carbonic anhydrase, and will be even faster if any is present, so that the pH change will be virtually instantaneous. The build-up of HCO, will be held back by the diiusion of CO, into the tear flhn from the cornea. Diffusional movements of the HCO, as well as H, Na and other ions from the tear film will be restricted by the barrier presented by the surface of the epithelium. If the average CO, concentration in the tear 6lm is 95 % of its way to equilibrium in time t, theory requires (Crank, 1975) that erf (x/2 z/IX) = 005, where x is the half flhn thickness, 4 x 10e4 cm, and D is the diffusion constant of CO, which can be assumed to have a uniform value of O-5 x 10m5 cm2

F. S. CHEN

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D. M. MAURICE

set-l across the tears and cornea (Fatt, Hill and Takahashi, 1964). Then t will be about 3 sec. No sign of pH change was detectable between 1 and 3 set which, perhaps, is not surprising in view of this calculation. Moreover, the eyelids were shut for only a few seconds at a time so that the tissue under them might not have had enough time to equilibrate with the blood. A puzzling phenomenon is the slow rise in pH, lasting about 1 min, that occurs when a slightly acidic unbuffered drop is instilled in the rabbit eye (Fig. 4). Observations with fluorescent dyes show that the precorneal film remains in place and isolated from the marginal tear strips under these conditions ; exchanges can only take place across the surfaces of the tear film, therefore. Although the pH shift is in a direction compatible with a loss of CO, it is difficult to identify a rate limiting process that is so slow. Perhaps a mechanism involving ion transfer across the epithelial cell membranes is involved. The time constant of transfer of small ion such as Na across the entire epithelium, permeability 6 x lOA cm hr-’ (Maurice, 1955), is of the order of 1 hr ; a faster exchange with the cytoplasm across the outer cell membranes could be envisaged. Buffered Solutions After the instillation into the human eye of a drop of 75 mM phosphate buffer the tears returned to their normal pH in a few minutes. For the pH to regain its normal value the buffer molecule must be removed from the tear film. This will be affected by the normal turnover of the tears, which in humans drops the concentration of a solution in the lacrimal fluid ten times in about 15 min, on the average, although there are considerable differences between individuals. Loss across the conjunctival and cornea1 epithelium is probably small compared to that by tear flow for the phosphate ion. The 75 mM phosphate in the drop that was instilled should be rapidly diluted to about 2 5 mrvr by the fluid previously present in the conjunctival sac, and in the following 5 min it will be further reduced to around 8 mM, which is low compared to the bicarbonate and other buffering systems that are present at 40 mM. Thus, wash-out by freshly secreted tears probably controls the persistence of the influence of an instilled buffer. Contact Lens

Physiological pHs were measured in the tear film under contact lenses, both PMMA and gas-permeable. Thus, the disturbances to the epithelial and endothelial cells of the cornea that are sometimes found in longterm wearers cannot be ascribed to an acid environment on the tissue surface. The lenses were generally fitted steep, so that a thickness of about 40 pm of fluid was enclosed above

THE

pH

IN THE

PRECORNEAL

TEAR

the cornea at the lens center. The apparent pH here did not differ meaningfully from that at the edge of the lens where it was nearly in contact with the eye nor from that under a close fitting lens. It is not probable, then, that the measured value is affected by fixed components of the tear film. The 0, level under a lens will be depressed but this does not affect pyranine fluorescence (Wolfbeis et al., 1983). Fatt (1968) showed that because no gas could escape from the cornea1 surface, the CO, tension under a hard contact lens should build up, but only to 58 mmHg as compared to 55 in the aqueous humor. Holden, Ross and Jenkins ( 19 8 7) found that a hydrogel lens simiiarly forms a barrier to CO, loss. Such a small change in CO, tension is in accordance with the physiological pH we determined under both impermeable and permeable lenses.

Fischer,F. H. and Wiederholt,M. (1982). Humanprecorneal tear i%n pH measuredby microelectrodes.AZbrecht von Gruefe’s Arch. Klin. Exp. Ophthalmol. 218, 168-70. Hill, R. M. and Camey,L. G. (1980). Human tear responses to alkali. Invest Ophthalmol. Vis. Sci. 19, 207-10. Hind, H. W. and Goyan, F. M. (1949). The hydrogen ion concentration and osmoticpropertiesof lacrimal fluid. 1. Am. Pharm. Assoc. 38, 477-9. Holden. B. A., Ross,R. and Jenkins, J. (1987). Hydrogel contact lensesimpedecarbon dioxide efflux from the human cornea. Cur-r. Eye Res. 6, 1283-90. Lewis,H. E.,Foster,A. R., Mullan, B. J., Cox,R. N. and Clark, R. P. (1969). Aerodynamics of the human microenvironment.Luncet 1, 1273-7. Lutty, G. A. (1978). The acute intravenous toxicity of biologicalstains,dyes,andother fluorescentsubstances. Tax. Appl. Pharm. 44, 225-49.

Maurice, D. M. (19 55). Influence on cornealpermeabilityof bathing with solutionsof differingreaction andtonicity. Br. J. Ophthalmol.

This research was supported by NIH grant EY04863. We would like to thank MS Chantal Malkin for her assistance at one stage of the experiments. References Alexander, J. H., van Lennep, E. W. and Young, J. A. (1972). Water and electrolyte secretion by the exorbital lacrimal gland of the rat studied by micropuncture and catheterization techniques. Pjhiger’s Arch. 337, 299-309.

Becker,B. (1959). Carbonicanhydraseand the formation of aqueous humor. Am. J. Ophthalmol.47, 342-61. Bonanno,J. A. and Polse.K. A. (1987). Measurementof in vivo human cornea1stromapH: openand closedeyes. Vis. Sci. 28, 522-30.

Crank, J. (1975). The Mathematics of @@usion (2nd ed.). ClarendonPress: Oxford. Cussler, E.L. (1984). Diflusion; Mass Transfer in Fluid Systems.CambridgeUniversity Press:Cambridge,U.K. F&all, J. T. and Wyman, J. (1958). Carbon dioxide and carbonic acid. In Biophysicul Chemistry. Vol. 1. Pp. 550-89. AcademicPress:New York. Fatt, I. (1968). Steady-state distribution of oxygen and carbon dioxidein the in vivo cornea.II. The openeyein nitrogen and the coveredeye.Exp. Eye Res. 7,413-30. Fatt. I., Hill, R. M. andTakahashi,G.(1964). CarbonDioxide efflux from the human corneain vivo. Nature203, 738.

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Brancato, R. and Coscas,G.). Pp. l-3. Kugler and Ghedini: Amsterdam. Maurice, D. M. and Chen,F. (1986). The pH of the tear film. Proc.lnt. Sot. Eye Res. 4, 122. Maurice, D. M. and Mishima, S. (1984). Ocular pharmacokinetics.In Handbook of Experimental Pharmacology.Vol. 69. (Ed. Sears, M. L.). Pp. 19-116. Springer-Verlag,Berlin. Maurice, D. M. and Singh, T. (1984). An improvedmethod for restrainingrabbitsfor examinationof the eye.Invest. Ophthalmol. Vis. Sci. 25, 1220-l. Milder, B. (1975). The lacrimal apparatus. In Adler's Physiology of the Eye (Ed. Moses,R. A.). Pp. 18-37. Mosby: St Louis,MO. Murube de1 Castillo, J. (1981). DucriologiaBasicu. Sot. Espaiiolade Oftalmologia:LasPalmas. Thomas,J. V., Brimijoin. M. R., Neault, T. R. and Brubaker, R. F. (1989). The fluorescent indicator pyranine is suitablefor measuringstromaland camera1pH in vivo. Exp. Eye Res. 50. 241-9. Wolfbeis,0. S.. Fiirlinger, E., Kroneis,H. and Marzoner, H. ( 1983). Fluorometricanalysis.1. A study on fluorescent indicators for measuringnear neutral ’ Physiological’ pH-values.Fresenius Z. Anat. Chem. 314, 119-24. Yoshimura, H. and Hosokawa,K. (1963). Studieson the mechanismof salt andwater secretionfrom the lacrimal gland. Jpn. 1. Physiol. 13, 303-18.

The pH in the precorneal tear film and under a contact lens measured with a fluorescent probe.

The reaction of the precorneal tear film of the human eye was determined non-invasively by instilling pyranine, a pH-sensitive fluorescent dye. The me...
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