British Journal of Ophthalmology, 1984, 68, 549-552
`
`Dynamics of ocular surface pH
`
`WILLIAM H. COLES AND PATRICIA A. JAROS
`From the Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia, USA
`
`SUMMARY We studied ocular surface pH in 161 subjects. The mean pH for 133 normal volunteers
`was 7*11, SD 1*5. We found that older women had a more alkaline pH than other subjects, that the
`pH shifted from acid to alkaline during the day, that one hour of eyelid closure caused an acid shift
`in pH, and that pH recovered to baseline values within 40 minutes after acid drop instillation. We
`explored the mechanism of pH regulation, and we believe that pH changes could affect contact lens
`toleration, drug effectiveness, and clinical signs in disease processes.
`
`and rinsed with distilled water after use on each
`subject.
`The ocular surface pH was tested in 133 health
`volunteers. One hundred and seventeen were selected
`from Grady Memorial Hospital and 16 were subjects
`not seeking eye care. Patients with inflammation due
`to allergy, conjunctivitis, lid abnormalities, or pro-
`longed eyelid closure were excluded from this group.
`The subjects were divided into eight groups by age
`and sex. Effects on pH of age, sex, order or readings,
`first eye measured, and time of day were studied.
`Six patients without ocular disease, who had been
`admitted to an orthopaedic ward, were tested eight
`times throughout one day. The measurements were
`taken every hour in both eyes from 0900 to 1700.
`Effects on pH of time of day were studied.
`Fourteen healthy
`volunteers were tested
`to
`determine the effects of eyelid closure on ocular
`
`Using the direct contact microelectrode we have
`studied ocular surface pH in the inferior lateral fornix.
`Shifts in pH could have therapeutic and diagnostic
`significance. Both antibiotic effectiveness'' and cell
`viability' are affected by pH.
`pH changes may also help in understanding mech-
`anisms of disease processes, since rosacea,4 allergy,
`and bacterial infection5 alter pH. Even toleration of
`soft contact lenses might be related to pH, because
`lens hydration decreases in acid pH."
`We present pH findings on 322 eyes (161 subjects)
`and explore mechanisms for the maintenance of, as
`well as deviations from, the steady state pH.
`
`Materials and methods
`
`Subjects' age, sex, race, topical and systemic medica-
`tion, and time of day were recorded. Patients taking
`substances known to be excreted in tears (metho-
`trextate, aspirin, ampicillin, bacampicillin7 ") were
`excluded from the study. Informed consent was signed
`by all subjects.
`Both eyes were tested. The eye to be measured first
`was selected at random by coin toss. A digital readout
`(Corning 125) pH meter was standardised with pH 7
`and pH 10 solutions and adjusted to read samples at
`32°C"' before each measurement. A microelectrode
`pH probe (Microelectrodes, Inc. 410) was placed in
`the lateral inferior fornix (Fig. 1), and after a 5-10-
`second stabilisation period for the digital readout the
`pH was measured. Three consecutive readings were
`taken in each eye. The probe was cleaned with alcohol
`
`Correspondence to William H. Coles, MD, Emory Univcrsity Clinic,
`25 Prcscott Strcet, Room 3417, Atlanta, Gcorgia 30308, USA.
`
`Fig. 1
`fornix.
`549
`
`Placement ofpH microelectrode in lateral inferior
`
`APOTEX 1009, pg. 1
`
`

`
`55()
`
`William H. Coles and Patricia A. Jaros
`
`surface pH. The eye to be patched was selected at
`random by coin toss..A cotton ball placed on the lid in
`combination with an eye patch assured lid closure.
`After one hour the patch was removed and the pH
`was immediately measured, followed by measure-
`ments in the unpatched eye.
`Eight subjects were measured before drop instilla-
`tion (phenylephrine 10%, pH 4-80, and Mydriacyl
`(tropicamide) 1%, pH 6.00), and subsequently until
`two measurements were found at preinstillation
`values. Effects on ocular surface pH after instillation
`of dilating drops with regard to pH recovery were
`studied.
`
`Results
`
`For each of the normal subjects three separate pH
`readings were taken. As the three readings were not
`significantly different from one another, the mean of
`the three readings was obtained. Table 1 presents the
`means, standard deviation, and sample sizes for each
`of the eight groups. The mean pH values for the two
`age groups differed for males and females (analysis of
`variance F=5-52, p<0-03). The pH increased
`significantly with age in women.
`The pH shifted from acidic to alkaline values when
`six individuals were tested throughout one day
`(p<0.05) (Fig. 2). There was a significant acidic shift
`in pH following one hour of lid closure. The patched
`eyes had a mean pH 7 20 before patching and pH 7-06
`after one hour of eyelid closure (SE=0-16). The
`control eyes had a mean pH 7-19 before and pH 7-19
`after one hour (SE=0. 16).
`Ocular surface pH recovered to predrop instillation
`levels within 40 minutes in six subjects (Fig. 3).
`
`Discussion
`
`As regards pH on the eye, the ocular surface is more
`than the interface between eye and air or eye and
`conjunctiva. At any point on the conjunctiva or
`cornea the pH, measured by the direct contact micro-
`electrode, is probably affected by all the layers of tear
`film and epithelial cell metabolism. In addition the
`
`Table I
`Ocular surface pH measurements on subjects
`<40 and B40
`
`Rac-e
`
`B
`
`W
`
`B
`
`W
`
`Sex
`
`M
`
`M
`
`F
`
`F
`
`Age
`<40)
`4()
`<40
`¢4()
`<4(0
`4()
`<40
`4()
`
`Mea(i SD
`7-070 11
`7-15 0-16
`7 140 13
`712 0-23
`7-03 0)09
`7-13 0-19
`7 0600(7
`728x0(14
`
`,,
`
`17
`14
`16
`5
`24
`27
`18
`12
`
`7.50
`
`74040
`
`7.30
`
`6
`
`7.00
`
`6.90
`
`9:00
`
`11:00
`
`1:00
`TIME
`Fig. 2
`Changes ofpH to alkaline as measuredfrom 0900 to
`1700 h. Six subjects.
`
`3:00
`
`5:00
`
`opposing surface, air or lid conjunctiva, also varies
`conditions on the ocular surface. Although we might
`define the ocular surface as tear film layers and epi-
`thelium, the many mechanisms that contribute to
`alterations in surface pH may have little correlation
`with exact structural definition.
`Previous studies on pH have been limited by an
`anatomical approach to measurements. Those studies
`looked at variation in tear film pH by collecting
`samples at the meniscus,"I2 a technique known to
`induce an alkaline error. '('
`The only studies which
`have used the direct contact microelectrode have not
`investigated the variability of the readings at a specific
`point on the surface. Our data show that the pH on
`the conjunctival surface varies in ways that probably
`
`7.60-
`
`7.10-
`
`Iar 6.10
`5.60-
`
`5.10-
`
`4.60-
`
`0
`
`30
`
`40
`
`20
`10
`TIME (minutes)
`Fig. 3 pH values beforeand afteradministration ofdrops.
`A pH reading was taken as baseline. One drop of
`tropicamide (pH 6-0) and one drop ofphenylephrine (pH
`4-8) were instilled in the lowerfornix. 0 represents time of
`drop instillation. pH was measured until recovery to baseline
`values. Six subjects.
`
`APOTEX 1009, pg. 2
`
`

`
`Dynamics ofocularsurfacepH
`
`contribute to contact lens intolerance and could
`influence both drug effectiveness and inflammatory
`responses.
`We are not sure that ocular precorneal tear film
`and the pH means at various locations on the surface
`might not vary from the area we studied. Differences
`have been measured between upper and lower
`fornices. I4 We chose to measure ocular surface pH in
`the inferior lateral fomix because it is easily accessible
`to the tip of the probe, and the lateral fornix provides
`consistent,coverage of the probe with lid conjunctiva
`where variation in temperature and atmospheric
`exposure are minimal. Our aim was to decrease
`stimulation and tearing; measurements in the fornix
`avoid the sensitive corneal surface. We found that no
`change occurs among three consecutive pH readings
`and that there is no difference between eyes. This
`indicates that no stimulation occurs that affects the
`readings with the technique as described.
`pH as measured on the ocular surface is not simply
`a reflexive regulation of the tears but an important
`physical measurement influenced by the interaction
`of many factors. To categorise the complexities, our
`data suggest a steady state (with predictable fluctua-
`tions around a mean) which is maintained by (1) the
`influences oftear buffer capacity; (2) cellular products
`and secretions on the ocular surface; and (3) factors
`such as age and sex, which affect the mean pH.
`A major stabiliser to the pH on the surface of the
`eye is the bicarbonate buffer system in the aqueous
`portion of tears.'5 Our data and other data 14 15
`strongly support this concept. The slow rise in pH
`during the day can best be attributed to the diffusion
`of CO2 to the atmosphere. With loss of CO2 the
`concentration of H + decreases and an alkaline shift in
`pH results.
`Equilibrium: C02+H20± H2C03?,
`H++HC0O-
`Eyc open: I C02+H20 *- H2CO3 <- I H++HC03-
`(pH T )
`After an hour of eyelid closure, CO2 is retained in the
`aqueous tears.
`Eyesclosed: T C02+H20-- H2CO3-+ t H++HC03-
`(pH 4.)
`A rapid drop in pH occurs as shown by our results.
`The changes in pH associated with lid closure and
`atmospheric exposure are almost surely related to
`CO2 concentration primarily involving this buffer
`system. '5
`The bicarbonate buffer system is most important in
`the maintenance of a consistent pH. But there are
`protein buffers and cellular secretions that contribute
`to changes and fluctuations in pH.
`There are two tear proteins that exert a buffer
`
`551
`
`capacity in the aqueous portion of tears. Lysozyme is
`a basic protein and specific tear albumin is acidic. A
`decrease in lysozyme, which is known in keratocon-
`junctivitis sicca and smog eye irritation, may decrease
`tear buffering capacity. 6
`Meibomian glands secrete lipids as a stable compo-
`sition of cholesterol and cholesteryl esters on to the
`surface of the tear film. A low-grade infection, how-
`ever, changes these Meibomian lipids.'7 8 Bacteria
`generate enzymes that lyse long-chain components of
`these lipid secretions, forming free fatty acids that are
`irritating to the tear film and corneal surface, creating
`an unstable tear film'7 and probably altering pH on
`the ocular surface.
`Mucin, which is alkaline and secreted by goblet
`cells, is dissolved within the overlying aqueous layer
`and interacts with the outer lipid layer of the tear
`film. '7 Changes in the quality and quantity of mucin,
`as seen with dry eye or acid burns, could influence pH
`on the ocular surface.
`Sex and age affect the mean pH. Our studies show
`that females over 40 have an alkaline shift in the
`mean. We also find a steady increase in the mean
`surface pH with age in all subjects.
`Tear fluid changes that occur with aging include a
`higher incidence of dry eye. This is especially true of
`elderly females. Perhaps mechanisms involved in dry
`eye, such as an increase in mucin or other non-specific
`aging changes, could participate in pH alterations on
`the ocular surface.
`Ocular surface pH means are also affected by time
`of day. We have shown on the ocular surface, as
`others have in tears,'2 that pH becomes more alkaline
`as the day progresses. The bicarbonate buffer system
`probably has the most important influence on this
`alkaline shift in pH. But, since the tear constituents
`of aqueous, mucin, and oils are secreted, other factors
`may also be important. The bodily secretions of
`saliva'9 and urine2"' as well as blood2' 22 show daily
`changes in pH. Known influences include hormonal
`in some cases, metabolic in others. One may speculate
`that the secretions comprising tears could be similarly
`affected.
`If the ocular surface pH is made acid, the return to
`the steady-state levels is probably by reflex tearing.
`Our data showed that lowering the pH with 2 drops
`(Mydriacyl, pH 4-80, and phenylephrine, pH 6-00)
`results in a rapid return within 20-40 minutes to
`initial pH, probably secondary to the tearing induced
`by irritation from the acid substances. The dilution
`effect of the reflex tears is probably most important to
`the return of the pH to its level before the drops were
`given.
`Deviations from the steady state pH can also occur
`from disease processes. pH is acid in rosacea patients,
`as measured by direct contact microelectrode.' An
`
`APOTEX 1009, pg. 3
`
`

`
`552
`
`allergic reaction in the eye is seen to cause an alkaline
`shift in pH' when studied with a pH sensitive dye,
`bromthymol blue. Further confirmation is needed in
`allergy and other conditions which might alter ocular
`surface pH, such as dry eye and bacterial infection,
`by direct-contact microelectrode measurement.
`The hydration of soft contact lenses can be
`decreased by acid changes in pH. Permalens water
`content decreases 8% when the pH goes from 7-4 to
`6.6 This has been thought to be a reason for contact
`lens intolerance and possibly cause of the tight lens
`syndrome.6 Previous reports4 have shown acid
`changes in rosacea on the conjunctival surfaces that
`could be in ranges that would make rosacea patients
`poor contact lens candidates.
`Antibiotic effectiveness in vivo changes with pH.'
`The aminoglycosides cannot penetrate tissues at pH
`above 7-4.' Gentamicin is the most commonly used
`aminoglycoside in ophthalmology, and it has been
`shown that conditions of reduced pH may decrease
`the antibacterial effect of this drug.' The effectiveness
`of gentamicin as well as other ophthalmic amino-
`glycosides, such as neomycin and tobramycin, needs
`to be investigated when the ocular surface is altered,
`since an alkaline pH, as shown by our studies, is
`common in older females.
`Cell viability in vitro is best at pH 7-4-7-8; pH
`lower than 6-8 or higher than 7-6 slows growth.3 Cells
`involved in ocular healing are also affected in vitro by
`pH changes. For example, the optimum growth of
`fibroblasts occurs when the pH is in the range of 7-4 to
`
`7.823
`
`We conclude that ocular surface pH varies signifi-
`cantly in both healthy and disease states. Understand-
`ing of the surface pH could have therapeutic and
`diagnostic significance and might help in the under-
`standing of complexities of disease processes and
`intolerance of eyes to contact lenses.
`
`We acknowledge the help of the Dcpartment of Biomctry, Emory
`University, in the statistical analysis.
`
`This study was supported in part by National Institutes of Health;
`National Eye Institute Grant No. EY03923 (Dr Coles); and in part
`by a Departmcntal Grant from Rescarch to Prevent Blindness.
`
`William H. Coles and Patricia A. Jaros
`
`References
`
`I Vandaux P. Peripheral inactivation of gcntamicin. J Antimicrob
`Chemother 1981; 8 (suppl A): 17-25.
`2 Barza M. Principles of tissue penetration of antibiotics. J Anti-
`microb Chemother 1981; 8 (suppl C): 7-28.
`3 Fischer A. Growth of fibroblasts and hydrogen ion concentration
`of the medium. J Exp Med 1921; 34: 447-54.
`4 Jaros PA, Coles WH. Ocular surface pH in rosacea. Contact
`Lens Assoc J 1983; 9: 333-6.
`5 Norn MS. Hydrogen ion concentration of tear fluid. Acta Oph-
`thalmol (Kbh) 1968; 46: 189-200.
`6 McCarey BE, Wilson LA. pH, osmolarity and temperature
`effects on the water content of hydrogel contact lenses. Contact
`Intraocul Lens Med J 1982; 8: 158-67.
`7 Doroshow JH, Gershon YL, Gaasterland DE, Hubbard SP,
`Young RC, Myers EE. Ocular irritation from high-dose metho-
`trexate therapy: Pharmokinetics of drug in tear film. Cancer
`1981; 48:2158-62.
`8 Valentic JP, Lcopold IH, Dea FJ. Excretion of salicylic acid into
`tears following oral administration of aspirin. Ophthalmology
`1980; 87: 815-20.
`9 Simon C, Malercyzyk V, Klaus M. Absorption of bacampicillin
`and ampicillin and penetration into body fluids (skin blister fluid,
`saliva, tears) in healthy volunteers. ScandJ Infect Dis 1978; suppl
`14: 228-32.
`10 Abelson MB, Udell IJ, Weston JH. Normal human tear pH by
`direct measurement. Arch Ophthalmol 1981; 99: 301.
`11 Hill RM. Laboratory studies. Int Ophthalmol Clin 1981; 21:
`223-36.
`12 Carney LG, Hill RM. Human tear pH. Arch Ophthalmol 1976;
`94:821-4.
`13 Norn MS. Human tear pH. Arch Ophthalmol 1977; 95: 170.
`14 Fischer FH, Wiederholt M. Human precorneal tear film pH
`measured by microelectrodes. Albrecht von Graefes Arch Klin
`Ophthalmol 1982; 218: 168-7t).
`15 Hill RM, Carney LG. Human tear responses to alkali. Invest
`Ophthalmol Visual Sci 1980; 19: 207-10.
`16 Pretsch RC, Pearlman ME. Human tear lysozyme variables.
`Arch Ophthalmol 1973; 90: 94-6.
`17 Lemp MA. Precorneal fluid and blinking. mnt Ophthalmol Clin
`1981;21:55-64.
`18 Brown SI, Dervichian OG. The oils of the Meibomian glands.
`Arch Ophthalmol 1969; 82: 537-40.
`19 Dawes C. Circadian rhythms in the flow rate and composition of
`unstimulated and stimulated human submandibular saliva. J
`Physiol (Lond) 1975; 244: 535-48.
`20 Elliot JS, Sharp RF, Lewis L. Urinary pH. J Urol 1959; 81:
`339-43.
`21 Shock NW, Hastings AB. Studies of the acid-base balance of the
`blood. J Biol Chem 1934; 104: 585-601.
`22 Hastings AB, Eisele CW. Diurnal variations in the acid-base
`balance of the blood. Proc Soc Exp Biol Med 1940; 43: 308-12.
`23 Hosford GN, Hicks AM. Hydrogen ion concentration of tears.
`Arch Ophthalmol 1935; 13: 14-25.
`
`APOTEX 1009, pg. 4