United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600 1-79-032
August 1979
Research and Development
Biochemical
Changes in
Humans Upon
Exposure to Sulfuric
Acid Aerosol and
Exercise
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine 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 (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-79-032
August 1979
BIOCHEMICAL CHANGES IN HUMANS UPON EXPOSURE
TO SULFURIC ACID AEROSOL AND EXERCISE
Suzanne Chaney
Wendy Blomquist
Keith Muller
George Goldstein
Clinical Pathology Branch
Clinical Studies Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Chapel Hill, North Carolina 27514
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
u.s. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects.
These studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory participates in the development
and revision of air quality criteria documents on pollutants for which
national ambient air quality standards exist or are proposed, provides
the data for registration of new pesticides or proposed suspension of
those already in use, conducts research on hazardous and toxic materials,
and is primarily responsible for providing the health basis for non-
ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation
of affidavits as well as expert advice to the Administrator to assure
the adequacy of health care and surveillance of persons having suffered
imminent and substantial endangerment of their health.
Since sulfuric acid is a part of our modern environment, it becomes
necessary to establish if any responses are observed in humans under
controlled exposure conditions. This study was designed to further
define the possible, irritant effects of sulfuric acid mist on human
health.
F. G. Hueter, Ph.D.
Di rector
Health Effects Research Laboratory
m
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ABSTRACT
A total of 18 human subjects were exposed to ambient air for four hours
on the first day of exposure and to four hours of 100 ug/m (0.033 uM) sul-
furic acid aerosol (0.5 urn mean mass diameter) on the second day. A total of
17 human subjects were exposed to four hours of ambient air on both exposure
days.
Six biochemical blood parameters were measured pre and post exposure:
glutathione, lysozyme, glutathione reductase, serum gultamic oxaloacetic acid
transaminase, serum vitamin E and 2,3-diophosphoglyceric acid. The results
indicate no significant effect of one four hour exposure of humans to sulfuric
acid aerosol (100 ug/m3).
One significant effect did occur indicating an increase in glutathione
reductase post exposure for both the control group and acid group.
This report covers a period from October 23, 1978, to December 15, 1978,
and work was completed as of May 1, 1979.
IV
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INTRODUCTION
Studies have shown that sulfuric acid and particulate sulfates are
formed by the oxidation of a portion of the sulfur dioxide emitted into
the atmosphere. Experimental toxicology studies have suggested that
these oxidation products have a greater irritant potency than sulfur
2
dioxide gas per se. Human sulfuric acid exposure studies have con-
centrated mainly on the retention of inhaled acid mist as a function of
particle size and concentration and the magnitude of the response as
measured by pulmonary function effects. ' ' ' ' Few studies have been
reported that evaluate effects of sulfuric acid mist on biochemical
Q
blood parameters. Alarie et al. exposed cynomolgus monkeys and guinea
pigs to sulfuric acid mist for 78 weeks and 52 weeks, respectively. No
deleterious effects due to sulfuric acid mist on the measured biochemical
blood parameters could be detected. Since sulfuric acid is a part of
our modern environment, it becomes necessary to establish if any responses
are observed in humans under controlled exposure conditions. This study
was designed to further define the possible irritant effects of sulfuric
acid mist on human health. Six biochemical blood parameters were evaluated
in humans exposed to sulfuric acid mist.
METHOD
Subjects
A total of 35 healthy (as determined by a physical examination and
completion of the Duke University Computer History form and the Minnesota
Multiphasic Personality Inventory), non-smoking, Caucasian, male, university
students characterized by an average height of 179.8 cm, a standard
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deviation of 7.2 cm, a range of 160.0-195.0 cm, an average weight of
72.6 kg, a standard deviation of 8.6 kg, a range of 56.6-95.5 kg, and an
average age of 28.0 years, a standard deviation of 3.5 years, a range of
21.7-34.4 years, were used in this study. A total of 17 subjects were
exposed to air only (group 1, controls) and a total of 18 subjects were
exposed to sulfuric acid aerosol (group 2, experimental).
Procedure
Each subject served for 2 days. On each day blood was drawn immediately
proceeding a 4 hour exposure and immediately following the exposure.
During the exposure, a pulmonary function battery was administered at 0,
2, and 4 hours. During the first 15 minutes, minute ventilation was
recorded. At 30 minutes and 90 minutes, the subjects engaged in a 15
minute exercise period. This consisted of walking 4 mph on a treadmill
inclined at 10°. Subjects were tested in groups of three.
Experimental Design
All subjects were exposed to ambient air on the first day of exposure
o
and 18 of the subjects were exposed to 100 ug/m (0.033 uM) H^SO. aerosol
(0.5 urn mean mass diameter, HMD) on the second day. The remaining 17
subjects received ambient air on the second exposure day. Data were
collected in a counterbalanced fashion. During week one, a group of 3
acid exposure subjects was tested on Monday and Tuesday and a group of 3
air exposure subjects was tested on Wednesday and Thursday. The following
week the order was reversed. Exposures began at approximately 8:30 a.m.
each morning. This alternation scheme was continued throughout the
experiment. This scheme helped control any day of week effects and time
of year effects.
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The chamber atmosphere was maintained at a temperature of 22°C, a
relative humidity of 40% and an air flow of 227 m /min.
A total of six serum and red blood cell biochemical measures were
chosen as dependent variables: serum glutathione (GSH), lysozyme,
2,3-diphosphoglycerate (2,3-DPG), serum glutamic oxaloacetic acid trans-
aminase (SGOT), serum vitamin E, and red blood cell glutathione reductase.
Measurements were taken on all six variables preceeding and following
both exposure days. Each measurement was treated as a dependent variable
in the analysis. Use of multivariate analysis of variance (MANOVA)
allowed evaluating differences between (1) the air and acid groups, (2)
day 1 and day 2 measurements, (3) pre and post measurements, and (4) all
interactions of these effects.
Blood Analysis
SGOT was analyzed on the Centrifichem 400 autoanalyzer by a modified
9 10
Karmen technique. ' Red blood cell glutathione reductase was assayed
by the method of Nichoalds modified such that the enzyme was pre-
incubated at 0°C for 30 minutes in the presence of 60 uM FAD and the
reaction was initiated by the simultaneous addition of 2 mM oxidized
glutathione (GSSG) and 0.24 mM NADPH. 2,3-DPG in erythrocytes was
12
measured by the Nygaard and Rorth method. Reduced glutathione in
13 14
serum was measured by the method of Patterson and Lazarow. ' Serum
vitamin E was measured by the method of Chaney et al. Lysozyme was
measured by the method of Shugar.
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Materials
A Centrifichem Model 400 centrifugal analyzer and Centrifichem
autopipetter (Union Carbide Corporation, Clinical Diagnostics, Rye, New
York 10580) were used for assaying SCOT. Manual assays for glutathione
reductase, GSH, and 2,3-DPG were run on a Gary 118 spectrophotometer.
The lysozyme assay was run on a Varian 635 spectrophotometer. SGOT
reagent kits were purchased from Union Carbide Corporation, Clinical
Diagnostics, Rye, New York 10580. 2,3-DPG reagent kits were purchased
from Calbiochem, La Jolla, California 92037. Lysozyme reagent kits were
purchased from Worthington Diagnostics, Freehold, New Jersey 07728.
D,L-ortocopherol, GSSG (free acid, grade III), FAD (disodium salt,
grade III), NADPH (tetrasodium salt, Type 1), alloxan monohydrate, and
GSH (reduced form, 98-100% purity) were purchased from Sigma Chemical
Company, St. Louis, Missouri 63178.
RESULTS
Table 1 provides summary statistics for all six dependent variables.
Data from both the air-air and air-acid treatment groups are included.
None of the variables appear to pose any problems in terms of violating
assumptions necessary for the analysis of variance.
Table 2 summarizes the MANOVA computed for these data. Each line
in this table tests for an effect on any of the six dependent variables,
or any combination of the six simultaneously. The important hypotheses
tested involve the air-acid difference. None of those effects were
significant. The overall pre-post effect, however, was significant
(p < 0.05). Of the six univariate tests only glutathione reductase had
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a significant effect as indicated by a p value of 0.001. No other
variable had a pre-post p value less than 0.14. The mean for glutathione
reductase pre-exposure was 7.27 umoles/gm Hgb/min and the post-exposure
mean was 7.76 umoles/gm Hgb/min. This pre to post increase produced the
significant p value.
Table 3 provides mean response for all six dependent variables at
each point in time for each exposure group. These means were estimated
as part of the MANOVA. The same data are presented graphically in
Figures 1 through 6. All variables except RBC glutathione indicate
nonsignificant changes. These figures support this conclusion, indicating
that the exposed group and control group started close together and
remained close together during the experiment. The significant pre-post
effect for glutathione reductase is easily seen in Figure 3. When
viewing the graph it should be remembered that day 1 was an air exposure
for both groups.
DISCUSSION
The results indicate no effect on the blood parameters measured of
3
one 4 hour exposure of humans to 100 ug/m (0.033 uM) H^SO^ aerosol (0.5
MMD). This study considered biochemical blood parameters that are
involved in maintaining cellular reductive detoxification ability which
in turn protects cellular components from oxidation and the blood lyso-
zyme level which is an indicator of lung tissue damage.
One significant effect did occur indicating an increase in the GSH
reductase post-exposure. This effect was seen in both the air and acid
exposure groups for each day of the exposure. Consequently acid exposure
-------
is eliminated as a possible cause of the increase. A plausible hypothesis,
not testable with this data, is that subjects were responding to the
moderate level of exercise required during the exposure periods.
These results are consistent with the available animal studies.
What few effects that have been seen have been at much higher levels of
o
sulfuric acid (0.38 to 4.79 mg/m ). Furthermore these effects have been
pulmonary function effects. In fact, the effects may be confined to the
respiratory system. Petering and Shih have suggested that the conversion
of sulfites to sulfates probably is a protective mechanism which occurs
in the respiratory tract.
The collection schedule for this study was determined by the associated
pulmonary function testing schedule. The scheme used would not allow
detection of an effect unless it were detectable immediately post-exposure.
With this one disadvantage, this research does not support the existence
of any effect of exposure to sulfuric acid aerosol on any of the biochemical
blood parameters measured.
Theoretically, sulfuric acid might be expected to initiate a response
in the respiratory system due to its low pH (less than pH 1). However,
the ammonia released by the respiratory system may well eliminate any
effect of inhaled sulfuric acid aerosol through partial or complete
18 3
neutralization. Ammonia concentrations ranging from 29 ug/m to
3
approximately 2,200 ug/m have been measured in exhaled air of healthy
3
human adults. Stoichiometrically, ammonia at 1 ug/m can convert sulfuric
o
acid at a concentration of 5.8 ug/m to ammonium bisulfate and at a con-
3 iq 3
centration of 2.9 ug/m to ammonium sulfate. Thus 35 ug ammonia/m
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o
would convert 100 |jg/m sulfuric acid to ammonium sulfate and only 17 pg
o
ammonia/m would convert this concentration of sulfuric acid to ammonium
bisulfate. Consequently the amount of acid aerosol presented was probably
neutralized in the respiratory tract. In recent studies on several
animal species, on healthy humans and on human asthmatics, there has
been no convincing evidence of functional changes upon exposure to
ammonium bisulfate and ammonium sulfate. No effects were observed even
20
at concentrations up to several milligrams per cubic meter. Thus it
appears that conversion of sulfuric acid to ammonium bisulfate or to
ammonium sulfate does constitute an effective defense mechanism.
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REFERENCES
1. Air Quality Criteria for Sulfur Oxides. Public Health Service
(1969).
2. Amdur, M. 0. Arch. Environ. Health, 23, 459-468 (1971).
3. Amdur, M. 0., L. Silverman and P. Drinker. Arch. Industrial Hyg.
and Occ. Med., 6, 305 (1952).
4. Wilson, I. B. and V. K. LaMer. J. Indust. Hyjf. and Tox., 30,
265-280 (1948).
5. Amdur, M. 0. J. Air PoVL Cont. Assoc., 19, 638 (1969).
6. Amdur, M. 0. Arch. Envir. Health, 23, 459 (1971).
7. Williams, M. K. Br. J. Ind. Med.. 27, 61 (1970).
8. Alarie, Y., W. M. Busey, A. A. Krumm and C. E. Ulrich. Arch.
Environ. Health, 27, 16 (1973).
9. Karmen, A. J. Clin. Invest., 34, 131 (1955).
10. Henry, R. J., N. Chiamori, 0. J. Golub and S. Berkman. Am. J.
Clin. Path., 34, 381 (1960).
11. Nichoalds, G. E. Clin. Chem.. 20, 624 (1974).
12. Nygaard, S. F. and M. Rorth. Scand. J. Clin. Lab. Invest., 24, 399
(1969).
13. Patterson, J. W. and A. Lazarow. Methods of Biochem. Anal. D. Geich,
ed., N.Y. Interscience, 2, 259 (1955).
14. Patterson, J. W., A. Lazarow and S. Levey. J. Blol. Chem., 177,
197 (1949).
15. Chaney, S. Q., P. J. DeWitt, W. Blomquist, K. Muller, R. M. Bruce
and G. M. Goldstein. Manuscript in preparation.
8
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16. Shugar, D. Biochem. Biophys. Acta, 8, 302 (1952).
17. Petering, D. H. and N. T. Shin. Environ. Res., 9, 55 (1975).
18. Larson, T. V., D. S. Covert, R. Frank and R. J. Charlson. Science,
197, 161 (1977).
19. Report of Committee on Sulfur Oxides, Board on Toxicology and
Environmental Health Hazards, Assembly of Life Sciences, The National
Research Council, p. 7-27 (1978).
20. Ibid, p. 7-61.
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Table 1
Summary Statistics for All Six Dependent Variables
Time Mean Std. Dev. Min. Max. Skewness
Glutathione mg/100 ml
Pre Day 1
Post Day 1
41.53
40.83
4.91
5.27
33.60
30.00
54.80
52.30
0.74
-0.03
Lysozyme |jg/ml
Pre Day 1
Post Day 1
10.97
10.84
2.89
2.71
5.49
6.01
17.94
17.22
0.34
0.33
Glutathione Reductase umole/gm Hgb/min
Pre Day 1
Post Day 1
Pre Day 1
Post Day 1
Pre Day 1
Post Day 1
Pre Day 1
Post Day 1
7.08
7.70
13.2
12.9
7.40
7.43
4.60
4.79
1.19
1.20
SCOT IU/1
3.44
2.97
Serum Vitamin E
2.23
2.71
2,3 DPG umole/ml
0.63
0.79
4.95
5.98
7.0
9.0
ug/ml
3.28
3.05
RBC
3.23
3.86
10.21
11.52
21.0
21.0
15.90
18.14
6.16
7.72
0.51
0.98
0.70
1.05
0.20
1.76
0.04
2.07
10
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Table 2
MANOVA Summary for All Six Dependent Variables
Source
Air/ Acid (A)
Day (D)
Pre-post (P)
A x D
A x P
D x P
A x D x P
Likelihood
Ratio
.935
.766
.652
.770
.926
.886
.737
F
.32
1.43
2.49
1.39
.37
.60
1.67
Num
df
6
6
6
6
6
6
6
Den
df
28
28
28
28
28
28
28
P
.919
.239
.046*
.252
.890
.727
.166
*Signifleant at the .05 level.
11
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Table 3
Mean Response on All Six Dependent Variables
Estimated from MANOVA
Variable
Treatment
Pre
Day 1
Post
Pre
Day 2
Post
Glutathione
mg/100 ml
Lysozyme
Glutathione Reductase
[jmole/gm Hgb/min
SCOT
IU/1
Serum Vitamin E
2,3 DPG
jjmole/ml RBC
Air-Air
Air-Acid
Air-Air
Air-Acid
Air-Air
Air-Acid
Air-Air
Air-Acid
Air-Air
Air-Acid
Air-Air
Air-Acid
40.93
42.10
11.33
10.63
6.86
7.29
13.4
13.1
7.36
7.43
4.53
4.66
41.85
39.87
11.21
10.48
7.63
7.77
13.0
12.7
7.39
7.47
4.95
4.63
42.08
41.36
11.44
11.02
7.38
7.56
13.3
12.9
7.28
6.86
4.93
4.66
38.99
42.54
11.42
11.12
7.84
7.81
13.5
12.7
7.74
6.68
5.08
4.89
12
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44
43
AIR EXPOSURE
H2SO4 EXPOSURE
12 16
TIME, hr.
28
Figure 1. Acid by day by pre-post interaction means for GSH.
13
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11.6
AIR EXPOSURE
EXPOSURE
10.4
8 12 16
TIME.hr.
20 24 28
Figure 2. Acid by day by pre-post interaction means for Lysozyme.
14
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8.0
1 1
1 1 1 1
AIR EXPOSURE
H2SO4 EXPOSURE
12 16
TIME, hr.
20
24
28
Figure 3. Acid by day by pre-post interaction means for RBC GSH
Reductase.
15
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AIR EXPOSURE
H2SO4 EXPOSURE
0
8
12 16
TIME, hr.
24 28
Figure 4. Acid by day by pre-post interaction means for SOOT.
16
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AIR EXPOSURE
H2SO4 EXPOSURE
4
8
12 16
TIME, hr.
20
24
28
Figure 5. Acid by day by pre-post interaction means for Serum
Vitamin E.
17
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8
AIR EXPOSURE
H2SO4 EXPOSURE
I I
12 16
TIME.hr.
20
24
28
Figure 6. Acid by day by pre-post interaction means for 2,3-DPG.
18
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORT NO.
EPA-600/1-79-032
2.
3. RECIPIENT'S ACCESSION NO.
-I-
4. TITLE AND SUBTITLE
Biochemical Changes in Humans Upon Exposure to Sulfuric
Acid Aerosol and Exercise
5. REPORT DATE
August 1979
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Suzanne Chaney, Wendy Blomquist,
George Goldstein
Keith Muller, and
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
Clinical Studies Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
10. PROGRAM ELEMENT NO.
1AA816
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
RTP, NC
13. TYPE OF REPORT AND PERIOD COVERED
In house
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A total of 18 human subjects were exposed to ambient air for four hours on the first
day of exposure and to four hours of 100 ug/m3 (0.033 uM) sulfuric acid aerosol exposed
to four hours of ambient air on both exposure days.
i
Six biochemical blood paraiteters were measured pre and post exposure: glutathione,
lysozyme, glutathione reductase;, serum glutamic oxaloacetic acid transaminase, serum
vitamin E and 2,3-diphosphoglyceric acid. The results indicate no significant effect of
one four hour exposure of humans to sulfuric acid aerosol (100 ug/m3).
One significant effect did occur indicating an increase in glutathione reductase
post exposure for both the control group and acid group.
This report covers a period from October 23, 1978, to December 15, 1978, and work
was completed as of May 1, 1979.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Blood biochemistry
Human exposure
Sulfuric Acid Aerosol
Pollutant insult
screening
Reductive detoxification
06A
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report!
UNCLASSIFIED
21. NO. OF PAGES
24
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLET-
19
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