EPA-650/1-75-005
March 1974
Environmental Health Effects Research
AIR POLLUTION EXPOSURE
AND
IMMUNOGLOBULIN LEVELS
U.S. Environmental Protection Agency
Office of Research and Development
Washington, D.C. 20460
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EPA-650/1-75-005
AIR POLLUTION EXPOSURE
AND
IMMUNOGLOBULIN LEVELS
by
Meloy Laboratories, Inc.
Biological Products Division
6715 Electronic Drive
Springfield, Va. 22151
Contract No. 68-02-0586
Program Element No. 1AA005
EPA Project Officer: Dr . Carl Hayes
Human Studies Laboratory
National Environmental Research Center
Research Triangle Park, N. C. 27711
Prepared for
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
WASHINGTON, D. C. 20460
March 1974
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EPA REVIEW NOTICE
This report has been reviewed by the National Environmental Research
Center - Research Triangle Park, Office of Research and Development,
EPA, and approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or commercial
products constitute endorsement or recommendation for use.
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environ-
mental Protection Agency, have been grouped into series. These broad
categories were established to facilitate further development and applica-
tion of environmental technology. Elimination of traditional grouping was
consciously planned to foster technology transfer and maximum interface
in related fields. These series are:
1. ENVIRONMENTAL HEALTH EFFECTS RESEARCH
2. ENVIRONMENTAL PROTECTION TECHNOLOGY
3. ECOLOGICAL RESEARCH
4. ENVIRONMENTAL MONITORING
5. SOCIOECONOMIC ENVIRONMENTAL STUDIES
6. SCIENTIFIC AND TECHNICAL ASSESSMENT REPORTS
9. MISCELLANEOUS
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating
to the tolerances of man for unhealthful substances or conditions. This
work is generally assessed from a medical viewpoint, including physio-
logical or psychological studies. In addition to toxicology and other
medical specialities, study areas include biomedical instrumentation
and health research techniques utilizing animals - but always with in-
tended application to human health measures.
This document is available to the public for sale through the National
Technical Information Service, Springfield, Virginia 22161.
Publication No. EPA-650/1-75-005
11
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TABLE OF CONTENTS
Page
Objective 1
Scope 1
Experimental Design 2
Methods and Materials 3
Results 8
Conclusions 15
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1. Obj ective
The objective of this effort was to determine whether or
not air pollution affects the serum levels of IgA, IgG, IgM
and C3 to a different degree in smokers than in nons.mQk.e.r sv.
The null hypothesis that there is no significant difference
in the levels of these proteins in the serum was used as the
basis of the -study.
To test the null hypothesis, it was necessary to determine
the serum labels of the four proteins in question by the method
of single radial immunodiffusion described by Fahey and McKelvey
and to analyze the resulting data statistically. The serum tested
were obtained from 147 nonsmokers and 517 smokers. These two
categories were further broken into geographic area and the
presence of cough and phlegm.
2. Work Scope
The task consisted of two parts: (1) the quantitative
determination of the levels of IgA, IgG, IgM and C3 in 800
samples of sera (517 smokers, 147 nonsmokers and 136 unkeyed) ;
and (2) analysis of data. The quantitative determinations were
performed in a manner that most assured the technical correctness
and unbiasedness of the results.
The analysis of the data was performed using standard
statistical techniques utilizing a digital computer for the
computations because of the large number of samples involved.
1. Fahey, J.L., and McKelvey, E.M., J. Immunol., ££:84-90 (1965).
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3. Experimental Design
Originally, the Environmental Protection Agency was to
supply the 800 serum samples necessary for the task. The initial
experimental design and analysis were based on the fact that
there would be 400 smokers and 400 nonsmokers. The actual samples
received were not as evenly distributed as originally intended.
In fact, Meloy Laboratories was supplied with 855 samples. These
855 samples had thirteen character code numbers. The key to this
code was supplied by the EPA, but was incomplete. In addition,
of the 855 samples, only 664 appeared on the key list. The 664
were divided into groups by a key category number ranging from
1 through 9 and distributed as follows:
Code
2
3
4
5
6
7
8
9
Area
Chicago
Suburbs
Rural
Chicago
Suburbs
Rural
Chicago
Suburbs
Rural
Characteristics Number Supplied
Smokers; cough and phlegm
Smokers; cough and phlegm
Smokers; cough and phlegm
Smokers; no cough or phlegm
Smokers; no cough or phlegm
Smokers; no cough or phlegm
Nonsmokers
Nonsmokers
Nonsmokers
23
131
126
66
92
79
38
88
21
Of these 664 samples, only 22.13% were nonsmokers Because
of this deviation from the original distirbution, the analysis
of data had to be reorganized (Section 4). The quantitative analysis
of the 664 samples and 136 unkeyed samples was performed using the
method originally proposed.
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4. Methods and Materials
The study utilized quantitative immunodiffusion plates and
the techniques described by Fahey and McKelvey1. To insure the
consistency from plate to plate, one hundred 24-determination
plates of each protein type were acquired and reserved for the
>•>
study .
The samples were randomly divided into 15 groups of 51 samples
and one group of 35 samples. One group of samples was run per day.
A daily analysis consisted of setting up five plates of each protein
type yielding a total of twenty plates per day.
On one plate of each protein type each day, four reference
standards, a control serum, and seven samples were run in duplicate.
The configuration of the other four plates consisted of the control
serum and 11 samples in duplicate per plate. The sample locations
were randomized using a random number generator to eliminate biased
readings.
The plates were incubated at room temperature for 18 hours
and resulting diameters determined using a micro-comparator. The
diameters were read to the nearest O.lmm.
The standards for IgG, IgA and IgM used for this study
were calibrated against purified proteins and against the World
Health Organization International Reference Preparation 67/95. The
results of these determinations are expressed in milligrams per
milliliter and International Units, (I.U) .
1. Fahey, J.L., and McKelvey, E.M., J. Immunol., 9_4_:84-90 (.1965).
2. Meloy Laboratories, Biological Products Division, 6715 Electronic
Drive, Springfield, Virginia 22151.
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In the case of C3, there is no W.H.O. standard available, and
the C3 level of the standard was established by calibration
against purified proteins only.
The control serum that was utilized in the study was a
well characterized pool of normal human serum obtained from
normal donors and calibrated against the secondary standard in
more than 40 determinations.
Daily results were analyzed by regression techniques. The
standard curve of each protein type was subjected to linear
regression analysis using a digital computer. The resulting
equation was used to compute the concentrations of the control
points and the samples.
The control points were compared for each of the five plates
and more than a 10% difference in any plate resulted in retesting
all samples on that plate. Duplicate values computed for the
samples were compared also. Deviations of more than 10% between
duplicate values were marked for retesting.
Results from the calculations, including the control points
and the standard curves for protein type were stored on magnetic
tape for reference during the final analysis at the completion of
the study.
At the completion of the 16 days of quantitative analysis,
the resulting 16 standard curves for each of the four proteins
were used to compute the mean regression equation for each protein.
The concentrations of the sera from each group and for each
protein were subjected to standard statistical computations for
the mean, variance, standard deviation, standard error of the
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mean, coefficient of variation, skewness, and kurtosis. In
addition, the range of values was determined for each protein
in each group.
The regression equation for each daily group and each
protein was used to compute the mean standard curve for each
protein. The 95% confidence for each mean curve was used to
determine if entire daily groups were to be repeated. All curves
fell within the confidence limits and no repeats were necessary.
The concentrations in mg/ml and International units (I.U.)
for IgG, IgA and IgM and the concentration in mg/ml for C3
resulting from the daily analysis were used to compute the mean
concentrations for the groups. These values were utilized
in the t-test to establish the significance of the differences
between smokers and nonsmokers. The t-test was set up between
groups as:
tn-2 = *i - *2
"1 n2
Where: n and n are the numbers in the groups being compared;
X and X~
. , .
are the mean concentrations being compared;
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TABLE 1
Group Standard Curve Coefficients
(Section A.I)
The general equation is: log Y = A +b (diameter)
IgG
A b
IgA IgM
A b A b
C3
A b
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
Mean
Confidence Limits
Intercept (A)
Slope (b)
-.433 +.286
-.406 +.265
-.493 +.286
-.392 +.267
-.367 +.253
-.372 +.249
-.342 +.250
-.394 +.264
-.418 +.277
-.392 +.266
-.321 to -.493
.237 to .295
-.423
-.466
-.551
-.496
-.349
-.383
-.516
-.469
-.374
-.448
-.308
.219
+ .316
+ .273
+ .362
+ .329
+ .275
+.262
+ .335
+ .253
+ .268
+ .297
to -.585
to .375
-1.43 +.633
-1.51 +.619
-1.18 +.546
-1.21 +.579
-.996 +.531
-1.19 +.544
-1.00 +.467
-.805 +.277
-.827 +.288
-1.12 +.498
-.64 to -1.609
..234 to .762
-1.08
-1.14
-1.00
-.977
-.939
-1.05
-.989
-.744
-.868
-.976
-1.21
.558
+ .511
+ .543
+ .484
+ .484
+ .485
+ .502
+ .488
+ .410
+ .465
+ .486
to -.742
to .414
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TABLE 2
Compilation of Daily Standard Curve Coefficients,
Mean Standard Curve and Confidence Limits
IgG
Day A b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
-1.455
-1.362
-1.241
-1.165
-1.136
-1.345
-1.127
-1.178
-1.236
-1.296
-1.058
-1.026
-1.183
-1.086
-1.172
-1.138
+ .263
+ .257
+ .252
+.234
+.232
+ .254
+.225
+.238
+.246
+.246
+.224
+.222
+.243
+.227
+.244
+.233
IgA
A b
-1.955
-1.569
-1.703
-1.911
-1.865
-1.877
-1.812
-1.794
-1.704
-1.700
-2.043
-2.004
-2.113
-.546
-2.052
-1.825
+ .256
+.218
+.228
+.248
+.235
+.247
+ .237
+.246
+.230
+ .233
+ .270
+.267
+ .277
+.226
+.274
+ .240
IgM
A b
-1.949
-2.052
-1.891
-1.990
-1.944
-2.059
-1.997
-2.057
-2.100
-2.006
-1.792
-2.018
-2.270
-1.891
-1.804
-1.814
-.354
+ .364
+ .347
+ .377
+ .359
+.375
+ .369
+ .380
+ .382
+ .374
+ .296
+ .370
+.428
+ .357
+.340
+ .332
C3
A b
-2.419
-2.522
-2.602
-2.156
-2.258
-2.218
-2.113
-2.398
-2.293
-2.395
-1.960
-2.286
-2.366
-2.204
-2.115
-2.215
f.407
f.406
f.406
f.353
f.367
f.374
+.332
+ .395
+.369
+.379
+.280
+ .368
+ .394
+ .359
+.350
+ .361
Mean Regression
Coefficients
Confidence Limits
Intercept (A)
Slope (b)
-1.209 + .241
-.935 to 1.482
.208 to .274
IgA
-1.842 + .245
-1.540 to -2.18
.202 to .286
IgM
-1.985 + .356
C3
-2.276 + .37
-1.621 to -2.3 -1.919 to -2.63
.269 to .437 .28 to .426
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5. Results
5.1 Immunochemical Evaluation of Samples
In order to establish the validity of the quantitative deter-
mination and the comparisons to be made from these determinations,
it was necessary to establish that the proteins being measured in
each of the sample groups were the same antigenically and that
enzymatic breakdown of the proteins to be measured had not taken
place during the four to five year storage period.
The degree to which the proteins were antigenically related
between groups was established by the examination of the slopes,
intercepts and comparison of the standard errors of the slopes in
a parallel line assay. From each group, one serum was taken at
random. Six two-fold dilutions in physiological saline were made
of each of the nine sera selected, and the concentrations of each
dilution for each protein were determined in duplicate using radial
immunodiffus ion. For each protein, the diameter was plotted against
the logarithm (base 10) of the concentration for each dilution.
The resulting 9 lines were compared by computing the 95% confidence
limits and the mean regression equation. The results of the parallel
line assays show that the antigens were the same in each of the
groups.
The integrity of the proteins that were measured were eval-
uated on the undiluted nine samples used in the parallel line test.
These samples were assessed by immunoelectrophoresis against
appropriate specific antisera. Breakdown by any of the four
proteins would have been readily determined by this method. There
was no evidence that the integrity of any of the four proteins in
any of the nine samples had been disturbed as shown in Figures 1-3.
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FIGURE 1
Immunoelectrophoresis of 'Groups 1-3
Control
Sample
Control
Sample
Control
Control
Sample
Control
•Sample
-Control
Control
Sample
Control
Sample
Control
Anti-IgG
Anti-IgA
Anti-IgM
Anti-C3
Anti-IgG
Anti-IgA
Anti-IgM
Anti-C3
Anti-IgG
Anti-IgA
Anti-IgM
Anti-C3
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FIGURE 2
Immunoelectrophoresis of Groups 4-6
Control
Sample
Control
Sample
Control
Control
Samole
Control
-Sample
Control
Control
Sample
Control
Sample
Control
Anti-IgG
Anti-IgA
Anti-IgM
Anti-C3
Anti-IgG
Anti-IgA
Anti-IgM
Anti-C3
Anti-IgG
Anti-IgA
Anti-IgM
Anti-C3
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FIGURE 3
Immunoelectrophoresis of Groups 7-9
Sample
Control
Sample
Control
Control
Sample
Control
Sample
-Control
Control
Sample
Control
Sample
Control
Anti-IgG
Anti-IgA
Anti-IgM
Anti-C3
Anti-IgG
Anti-IgA
Anti-IgM
Anti-C3
Anti-IgG
Anti-IgA
Anti-IgM
Anti-C3
11
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5.2 Statistical Analysis of Groups
The data stored as daily results on magnetic tape were
sorted into groups 1 through 9 using a computer. These groups
were subjected to standard statistical computations which resulted
in the range of values, mean concentration, standard error of the
mean, variance, standard deviation, coefficient of variation,
skewness and kurtosis for each group and each protein. These
calculations were carried out for concentration both in
milligram/milliliter and International Units/milliliter (Table 3).
In order to test the significance of the resulting differences
in the mean concentration of each protein and each group, the t-test
was formulated and applied to the groups, as discussed in section
4. The results of these inter-group comparisons are shown in
Table 4. A t-value of less than 2.0 indicates that the differences
in means are not significant. No group was shown to deviate
significantly from any other group when this test was applied.
12
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TABLE 3
Mean Concentrations of Proteins by Group
Group
1
2
3
4
5
6
7
8
9
n
22
121
123
64
91
80
34
86
19
IgG
mg/ml lU/ml
10.48 128
10.42 127
11.05 134
11.35 138
11.13 136
11.15 136
11.99 146
11.09 135
11.09 135
IgA
me/ml lU/ml
1.69 112
1.67 111
1.78 118
1.99 132
1.78 118
1.69 112
1.79 119
1.74 115
1.68 111
IgM
mg/ml lU/ml
1.28 210
1.45 238
1.35 220
1.51 247
1.46 239
1.49 244
1.48 242
1.44 236
1.53 250
C3
mg/ml
1.66
1.44
1.44
1.54
1.42
1.43
1.43
1.43
1.58
13
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TABLE 4
Results of T-Test for Groups of
Smokers vs. Non-Smokers
Group
1 vs 7
2 vs 8
3 vs 9
4 vs 7
5 vs 8
6 vs 9
lEG
.184
.036
.005
.038
.002
.004
leA
.041
.014
.022
.052
.008
.003
IeM
.106
.002
.072
.010
.004
.016
C3
.148
.003
.089
.046
.004
.092
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6. Conclusions
Normal levels of the three immunoglobulins, IgG, IgA and
IgM, have been reported by Allansmith as 7.1-15.4 mg/ml ,
0.6-4.9 mg/ml, and .37-2.04 mg/ml respectively, and by Stiehm,
et al4 as 5.69-19.2 mg/ml, 0.61-3.3 mg/ml, and 0.47-1.47 mg/ml
respectively. A comparison of the results from each of the 9
groups to these ranges certainly places them in the normal
population.
In addition, the t-test establishes that no significant
differences exist between the mean concentrations when smokers
were compared to nonsmokers by geographic location. Additionally,
inspection indicates no significant differences exist between
smokers with cough and phlegm and those without.
Raw data required under this project was submitted in a
Preliminary report.
^Allansmith, M. J. Pediat. 7_2_:276 (1968).
Stiehm, E.R., and Fudenberg, H.H. Pediat. 37:715 (1966)
15
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TECHNICAL REPORT DATA
(Please read instructions on the reverse before completing)
1. REPORT NO.
EPA-650/1-75-005
4. TITLE AND SUBTITLE
Air Pollution Exposure and Iramunoglobulin Levels
3. RECIPIENT'S ACCESSION"NO.
5. REPORT DATE
March 19714
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Meloy Laboratories , Inc.
Biological Products Division
6715 Electronic Drive
Springfield, VA 22151
10. PROGRAM ELEMENT NO.
1AA005
11. CONTRACTK3HBJ6DCTJOC
68-02-0586
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final - 8/23/72 to 6/30/73
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The objective of the study was to determine whether air pollution affects
serum levels of immunoglobulins to a different degree in smokers than in
nonsmokers. The sera tested were obtained from army recruits in the Chicago
area, 517 smokers and 1^7 nonsmokers. The smokers and nonsmokers were further
categorized by geographic location and by the presence of cough and phlegm.
Quantitative determination of levels of IgA, IgG, IgM, and C3 in the samples
of sera were by the method of single radial irnmunodiffusion described by Fahey
and McKelvey. The results indicated that no significant differences existed
between the mean concentrations of immunoglobulins when smokers were compared to
nonsmokers by geographic location, nor were there significant differences between
smokers with cough and phlegm and those without.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Air Pollution
Blood serum
Smoking
Immunoglobulin
Cough
Phlegm
06F
18. DISTRIBUTION STATEMENT
Unlimited
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
17
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
I
EPA Form 2220-1 (9-73)
16
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