c
Validation of Human Eye
Irritation by Tear Lysozyme
Levels
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FINAL CONTRACT REPORT
Title of Study:
Contract Number:
Time Period:
Date Submitted:
Primary Contractor:
Validation of Human Eye Irritation by Tear Lysozyme
Levels
68-02-0096
June 1, 1971 - June 30, 1972
August 29, 1972
Duke University
Durham, North Carolina 27706
Project Director:
Richard L. Pietsch, M.D.
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TABLE OF CONTENTS
,
Final Report Summary Page
Purpose of Contract 2
Methods 2
Results 4
Discussion 7
Summary 9
References 10
Attachment I 12 t
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Tables I, II, III, IV, & V 13-17 i
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Purpose of Contract
I
Eye irritation is" a common non-specific complaint 6f our country's
urban population.
Previous subjective studies suggested that acrolein,
formaldehyde, peroxyacetyl nitrate (PAN), and perhaps the oxides of
. nitrogen are the components of photochemical smog responsible for eye
, 't t' 1,2,3,4,5,6,7
~rr1 a 10n.
Although good correlations between repeated
subjective observer responses and oxidant concentrations have been
found, these studies are open to criticism as the. subjects were well
aware that their eye irritation was being assessed by those desiring
to control air pollution.S
Human tear lysozyme has been said to
decrease in subjects having acute eye irritation and in various ocular
d' de 11 no.' .
- :L5.0l" ;rs. .~n:n'g -the pr.ecedingyear, -w.e set .up .a .method0f-measuring
human tear lysozyme concentration and analyzed variables such as age.,
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sex, 'race~ temporal factors and ~he presence or absence of eye irritation.
Only age was found to have a significant ef:f;ect on normal persons.
Eye
irritation such as from corneal ulcers has' been 'known for many years to
. 9
cause a decrease in lysozyme concentrat~on.
During the current year we set up a field study in Los Angeles and
sampled volunteers in low, medium, and high air pollutant locations to
determine the feasibility of using tear lysozyme levels as an objective
indicator of eye irritation.
Nethods
The field study was conducted in Los ~ngeles and Riverside Counties
during the Week August 31, 1971 through September 3, 1971.
Through the
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assistance of Dr. Louis ~bhoney of the Los Angeles Health Department,
tear sampling was done at specific health department clinics which were
near air pollution monitoring stations where ambient pollutant samplings
have revealed consistent levels.
Dr. Pietsch, Dr. Pearlman, and ~~s.
Reed arrived in Los Angeles the day prior to sampling with all necessary
eq~pment including 1500 plates for lysozyme analyses.
A total of 358 volunteers were easily found at the health clinics,
local police and fire stations and at the County Health Department in
Riverside (with Dr. Erickson's assistance).
Permission slips were signed
prior to sampling by each volunteer and questionnaires were completed
(see attachment #1).
Volunteers were specifically asked their age, length
and place of residence, number of hours out of doors, use of air
conditioning, presence of subjective eye irritation and use of eye
medications.
All eyes were examined for evidence of objective irritation
prior to sampli.ng.
The method of Bonavida and Sapse was used ior all lysozyme
determinations.10
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Topical anesthesia was not used.
Tear strips were
placed beneath the medial 1/3 of the lower lid of each eye and allowed
to remain for five minutes.
The moistened 5 rom strips were immediately
incubated at 37°C for 24 hours, following which the zone of lysis was
measured,
Ambient air pollutant levels were monitored in each area
during the time of our sampling.
Statistical analyses were completed
on the data obtained during the remainder of the year.
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Results
Overall Effects
The overall average lysis in each area was taken as an index of the
chronic effect of oxidants on lysozyme concentration (Table 1).
In the
areas of high pollutant exposure. Azusa and Riverside. mean oxidant
levels during an eleven hour period during the day were only .13 ppm
while hourly maxima reached only 0.24 ppm.
Al though there was a tendency
for lower mean lysis in the high oxidant exposed groups. this was not
statistically significant.
Mean age was similar in all populations sampled
ranging from 27.2 years in the control to 29.2 years in Azusa to 36.2 years
in Riverside.
Mean length of residence was also comparable ranging from
10.8 years in the control to 15.6 years in Azusa to 12,9 years in Riverside.
A cor:relllti-on. o.f. .ave:rage l¥sis. ,wi.th..ag.e . or .w..t.h hourly .oxidants, carbon
monoxide. and oxides of nitrogen and sulfur revealed no relationship.
Overall mean lysis was 29.0 mm for those with residence less than two
years and 29.1 mm for those with residence greater than two years.
This
difference was not statistically significant.
When individual groups were
compared. no significant difference existed between them.
In a correlation
matrix (Table II). no relationships appeared between age and lysis and
pollutant concentrations for those with residence of two or. more years.
Acute Effects
A regression analysis was performed on all groups using mean lysis
as the dependent variable.
Time of measure~ent. age. length of residence.
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length of time spent outdoors, and hourly oxidant were the independent
, .
variables.
Time of measurement was significant for the high exposure group
in Riverside and the low exposure group in Los Angeles.
In a correlation
matrix, mean lysis could not be correlated with oxidant concentration one
or two hours previous to sample collection.
In addition, mean lysis did
not correlate with carbon monoxide, oxides of nitrogen, or sulfur oxide
measurements for the day of collection.
Because of the correlation with time, a comparison of subjects
measured in the morning with subjects measured in the afternoon was made.
Average mean lysis was 28.8 rom in the morning versus 29.2 mm in the
afternoon.
In the morning group any oxidant reading--maximum oxidant,
oxidant two hours prior, oxidant one hour prior, or oxidant at the time
. of,tl-le.te-s.t--were "corre:lat-e'd 'with lysis.
The posi'ti ve correlation was
significant at the 0.05 level.
Overall mean lysis and oxidant concentrations
at the time of one or two hours previous to the sample collection do not
correlate in the correlation matrix.
Average age was 33.3 years for the
morning subjects and 27.5 for the afternoon subjects.
Average residence
was 14.5 years for the morning subjects and 11.8 years for the afternoon
subjects.
The effects of being indoors and having air conditioning'were
evaluated.
People working in non airconditioned buildings were considered
to be ''outside'' for the purposes of this .study.
However, since an enclosed
area might provide some protection from pollutants, several groups working
outside were analyzed separately (Table III).. One group of firemen in ,the
high exposure area with the highest oxidant concentration exposure of .19ppm
had a mean lysis of 27 mm.
TI~is group was also subject to high oxidant
exposure for the two hours preceeding testing:
.16 and .12 ppm.
Another'
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. ..outside group measured the preceeding hour exposed to .16 ppm at the time
of testing and .12 and .08 ppm in the two preceeding hours demonstrated
29 mm of lysis.
.,
Two groups who had been mostly in buildings rather than working
-outside, exhibited 29.4 and 29.8 mm of lysis despite exposure to .21 and
.20 ppm at the time of testing and the aforementioned high levels in the
preceeding hours.
Effects of Air Conditioning
The chronic effect of air conditioning on mean lysis was investigated
by selecting all volunteers sampled with air conditioning in both home
and office.
Forty-six eyes with a mean lysis of 29.0 mm were found in Azusa
(high exposure) while 103 eyes with a mean lysis of 28.9 mm were found in
.. "Ri'VeTsi'de (hi-gh "exp'osure). . "Pour 'eye's 111 the cbl'l'trol group "had a mean lysis
of 29.1 mm.
Conversely, those with no air conditioning in their home or
office had 28.8 mm of lysis in. Azusa, 29.0 mm in Riverside, and 29.2 mm
in the control area.
No effect of air conditioning in lysis could be
demonstrated.
The effect of subjective and objective evidence of eye irritation
on lysis levels was evaluated (Table IV).
The subjective presence of eye
irritation had no effect on lysis levels.
In fact, mean lysis was slightly
higher in this group (29.1 mm).
No subjects complained of severe eye
i rri ta tion .
1nose with objective evid~nce of eye irritation (infection
or mild conjunctivitis) had only slightly lower lysis as one would anticipate
(28.69 mm).
Mean lysis was higher (28.89 mm) among those with neither
subjective nor objective irritation.
Table V reveals that the prevalence
of volunteers who appeared irritated or appeare~ and felt irritated was
lower in the control area.
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Mean lysis was not lower among those working outside complaining
of subjective and objective eye irritation (Table III).
Only in the
previously mentioned group of 18 firemen in Azusa was lower mean lysis
found among the four subjects complaining of irritation, 26.3 mm, and
the two subjects \dth subjective eye irritation, 26.5 nun.
Discussion
Bonavida and Sapse reported lower tear lysozyme levels in patients
11
complaining of eye irritation at oxidant concentrations of .15 to .25ppm
In attempting to confirm such findings, our results are disappointing.
OVerall lysis was not significantly lower in our high exposed group.
However, mean daily pollutant concentrations in Azusa 'and Riverside did
not .:reach .the l-evels '"S,aps-e .aad 13&navid'a'efl'eount-e-red in'1:'neir report of
susceptible persons.
A further study done during a smog alert might be
more helpful in elucidating the value of the lysozyme technique.
No chronic differences in lysis could be demonstrated between those
living in air conditioned areas most of the time and those without this
convenience.
In addition, subjects measured in air conditioned buildings
in Riverside did not show different lysozyme levels than those outside.
Lysis levels were some\vhat lower in subj ects having both subj ecti ve
and objective evidence of eye irritation.
Any cause of prolonged eye
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irritation lowers tear lysozyme levels.
Perhaps Bonavida and Sapse
selected a population of susceptible individuals with measurable
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differences not present on a day to day basis in the population at l~rge.
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It should be emphasized that there was no correlation between only
subjective complaints and decreased lysis.
A threshold effect must be considered.
Firemen exposed to .19 ppm
of oxidant did show lower mean lysis.
However. other firemen exposed
to .16 ppm exhibited mean lysis similar to that among controls.
This
may represent a threshold effect.
Ei ther the preceding three hours of high
oxidant levels or the hourly oxidant level reached could have triggered
lower lysozyme levels.
For this relationship to be valid. one must-
assume that the higher mean lysis among the group exposed to .20 ppm
was due to a protective effect from being indoo~s even without air
conditioning. A subjective study by Richardson and Middleton 12 suggested
a threshold of oxidant concentration of .10 ppm for "barely noticeable"
'i:rrit'a't.i:on .
irritation".
.18"1'pm-fo'r -'-'moderate - i-rrt't'a tion'" .
13
However, Hammer ~ ~ noted no
-and.25 1>pm for-"-'-severe
increase in subjective
complaints on a day when an oxidant leve~ of .23 ppm was reached.
Further information might be obtained by training several field
technicians in the Los Angeles area to collect tear samples.
A short
study of subjects out of doors during a smog alert could verify or
disprove the value of lysozyme assays as an objective index of eye
irritation.
Higher levels of oxidants might be necessary to get a lysis
effect.
Summary
Tear samples were collected from 358 volunteers in Southern
California in smog polluted areas.
No consistent variation of lysozyme
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concentration with oxidant concentration was demonstrated.
Subjects
exposed to .19 ppm of oxidant in the open air did show decreased lysis.
A threshold effect is possible.
No correlation was established between
lysis and mild subjective complaints of irritation.
Objective indica-
tions of eye irritation showed a mild decrease
in lysis levels.
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References
1.
Hamming, W., ~ncPhee, R.:
Relationship of nitrogen oxides in auto
exhaust to eye irritation - further results of chamber studies.
In Atmospheric Environment Vol. I, 577-586, Pergamon Press,
Great Britain, 1967.
2.
Romanovsky, J., Ingels, R., Gordon, R.:
Estimation of smog effects
in the hydrocarbon-nitric oxide system.
J. Air Pollut. Control
Ass. .!2.: 454-459, 1967.
3.
Schuck, E., Renzetti, N.:
Eye irritants during photooxidation of
hydrocarbons in the presence of oxides of nitrogen.
J. Air
Pol1ut. Control Ass. ~: 389~392, 1960.
4.
Schuck, E., Stephens, E., Middleton, J.:
Eye irritation respo~se at
low concentrations of irritants.
Arch. Envi ran. HealtJl 13:
570",,575, 1966.
s.
Merrifield, P., Graham, T.:
The statistical evaluation of eye
irritation.
APCA Proceedings of the semi-annual technical
conference, San Francisco, Cal., November 18-19, 1957.
6.
Hamming, W., Dickinson, Jr.:
Control of photochemical smog by
alternation of initial reactant ratios.
J. Air Pollut. Control
Ass. ~: 317-323, 1966.
7.
Altshuller, A., Kpoczynski, 5., Lonneman, W., Wilson, D.:
Photochemical
reactivities of e~lausts from 1966 model automobiles equipped to
reduce hydrocarbon emissions.
J. Air Pollut. Control Ass. 17:
734-.737, 1967.
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8.
Renzetti, N., Schuck, E.:
Preliminary observations of the relationship
between eye irritation in synthetlc systems and in the atmosphere.
J. Air Pollut. Control Ass. ll: 121-124, 1961.
9.
Lysozyme: An antibacterial body present in great
Ridley, F.:
concentration in tears, and its relation to infection of the
human eye.
Proc. R. Soc. Med. ~: 1495-1506, 1928.
10.
Bonavida, B., Sapse, A.:
Human Tear Lysozyme: II. Quantitative
determination with standard Schirmer strips.
Am. J. Ophthal.
66: 70-80, 1968.
11.
Sapse, A., Bonavida, B., Stone, W., et a1.:
Human Tear Lysozyme: III.
Preliminary study on lysozyme levels in subjects with smog eye
irri tation.
Am. J. Ophthalmol. 66: 76-80, 1968.
Richardson" N. A.., Niddleton. l'{' C.:
EV.aluati.on of blters for
removing irritants froffl poll~ted air.
Uniyersity of California,
Department of Engineering, Lo~ Angeles, Report Number 57-43,
June, 1957.
13.
Hammer, D., Portnoy, B. ~ Mass.ey, F. ~ et a1.;
--.-. -
Los Angeles air pollution
and respiratory symptons - relationship during a selected 28-day
period.
Arch. Environ. Health 10: 475-480, 1965.
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ATTACHMENT I
Name
Address
Location: Max
Med
Min
/
/
Do te :
Time:
8 - 9 - 10 - 11 - 12 - 1 - 2 - 3 - 4
Ase:
How long have you been outdoors or in an open building today?
hours
Air conditioned:
Home:
Office:
Yes
Yes-
No
No-
How long have you lived in this area? - years
mon ths
Do you often take vitam ins?
Yes
No
Do you use eye drops ? Yes-
No
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If yes, type and time of last dro?:
Eyes irritated? Yes-
No
length of time:
Less thon 1 hour
1 hour - 1 day
-
1 -5 days
= Chronically
Eyes appear irritated:
Yes
1 + red
2+ teor
3+ red and tear
No
Comments regarding eye disease (other than 9Iass~s)
L Y5 15:
O.D.
0.5.
mm.
mm.
WETTiNG:
O.D.
0.5.
Mm.
Mm.
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Table I
Mean Lysis vs Maximum Pollutant Levels
;
Sample Daily M~imum Pollutants
Station Mean L sis Ozone ( m) CO .Nitric Oxide Sulfur Oxides Oxidant (11 hrs.)
Azusa, 8/30 28.71 0.16 6 .22 .04 .08
Riyerside J 8/31 28.98 0.24 .13
Azusa, 9/1 28.76 0.23 7 .24 .05 .13
Inglel'lood Imperial
Heigh ts, 9/2 .29.17 0.02 9 .30 .04 .01
Inglewood, 9/3 29.23 0.04 15 .47 .06 .01
.....
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Table II
Correlation ~oefficients of Mean Lysis with Other Variables
(Correlation of
.42 is Significant)
Factor
All
Participants
Azusa & Riverside
(High Exposure)
Time
.10
.23
- Age
-.13
-.19
Oxidant 2
hours before
-.01
.24
Oxidant 1
hour before
-.03
.14
Oxidant at time
of sample
collection
-.03
.16
14
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Table III
Summary df Outdoor Group
Group Sampled Numb er Oxidant Mean Number with Mean Number with Mean
.Eyes Lys is Subj ecti ve Lysis Objective Lysis
Sampled I~ri tation Irritation
Azusa 8/30 22 .10 28.8 1 28.0 1 30.0
11 a.m.
Azusa 8/30 18 .14 29.4 4 29.3 5 29.6
3 p.m.
{
Azusa 8/30 8 .11 28.5 0 0 '1
4 p.m.
'
Riverside 8/31 23 .13 28.96 6 28.8 5 29.2
12 p.m.
Azus a 9/1 20 .16 29.0 1 29.0 1 28.5
11 a.m.
Azusa 9/1 i8 .19 27.0 4 26.3 2 26.5
12 p.m.
....
VI
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Table IV
Effect of Subjective and Objective Eye Irritation on Lysis
Lysis Levels Neither
Date Exposure Level Mean S 0 $+0 $ nor 0
8/30 High 28.71 28.92 28.71 29.11 28.64
8/31 High 28.98 28.97 28.17 29.17 28.93
9/1 High 28.76 . 28.25 . 28.20 26.50 29.11
2 -Low -29...17 ....29.22 .2.8...45 29.21
9/3 Low 29.23 29.31 29.08 30.0 29.24
Overall 28.95 29.13 28.72 28.69 28.89
1 = Subjective eye irritation
2 = Objective eye irritation
3 = Both subjective and objective evidence of eye irritation
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Table V
Prevalence of Eye Irritation Among Subjects Sampled (per cent)
Appeared Felt Looked & felt
Sample Station Irritated Irri tated Irritated
Riverside 16.1 28.4 10.9
Azusa 29.3 21.0 9.1
Imperial Heights (control) 6.9 18.1 0.9
Overall 29.6 45.3 13.7
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