EPA-650/1-75-003
June 1975
Environmental Health Effects Research Series
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EPA-650/1-75-003
LEAD:
ENVIRONMENTAL SOURCES
AND RED CELL TOXICITY
IN URBAN CHILDREN
by
Carol R. Angle and Matilda S. Mclntire
University of Nebraska
Omaha, Nebraska 68105
Grant No. 802043
Program Element No. 1AA005
EPA Project Officer: Gory J . Love
Human Studies Laboratory
National Environmental Research Center
Research Triangle Park, North Carolina 27711
Prepared for
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
WASHINGTON, D. C. 20460
June 1975
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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 docs 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
Thib 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/ J-75-003
11
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ABSTRACT
A comprehensive environmental study in Omaha for the correlation
of lead from multiple sources with the blood lead of children shows
a significant decrease in air and dustfall lead between 1970 and
1974. Despite this, urban children have higher blood leads than
their suburban counterparts in all three age groups, 2-5, 10-12,
and 14-18 years. The higher blood lead correlates with the urban
dustfall, yard soil and boot tray dirt and, in the preschool ages,
with lead in interior dust. Soil lead and blood lead both correlate
with residential proximity to traffic. There was no significant
urban-suburban difference in lead in the air, milk, or available
paint chips.
There is a significant linear decrease in red cell (rbc) membrane
Na/K ATPase as blood lead increases from 10 to 80_ug/dl. In
association with inhibition of this transport enzyme there is short-
ened rbc survival manifest in increased rbc G-6-PD and 6-PGD.
There is selective inhibition of rbc glutathione at a blood lead
above 20 jug/dl. Iron deficiency does not correlate with blood lead
and the administration of iron supplements to preschool children
does not decrease their blood lead.
This report was submitted in fulfillment of Grant Number R 802043
by the University of Nebraska College of Medicine, under the
sponsorship of the Environmental Protection Agency. Work was
completed as of October 31, 1974.
iii
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CONTENTS
Page
Abstract iii
List of Figures vi
List of Tables viii
Acknowledgments x
Sections
I Conclusions 1
II Recommendations 3
III Introduction 4
IV Design 8
A. Environmental Studies 14
B. Intrinsic Metabolic Differences 15
C. Health Effects 17
V Objectives 18
VI Development of Quantitative Methods 19
A. Environmental Studies 19
B. Lead and Iron Deficiency 2 1
C. Red Cell Metabolism 24
1. Enzymes of red cell glycolysis 24
2. Lead in young and old red cells 24
3. Na/K ATPase 24
iv
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CONTENTS (continued)
Page
VII Demonstration of Quantitative Methods (Results) 27
A. Environmental 27
1. Blood Lead - Residence, Age, 27
Season
2 . Air Lead 32
3. Dustfall Lead 35
4. Correlation of Environmental 37
Lead and Blood Lead
B. Lead and Iron Deficiency 50
C. Red Cell Metabolism 61
1. Enzymes of Red Cell Glycolysis 61
2. Red Cell Membrane Na/K ATPase 63
VIII Discussion 67
A. Environmental Studies 67
B. Red Cell Metabolism and Iron Deficiency 69
C. Red Cell Survival and Na/K ATPase 70
IX References 72
X Glossary 79
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FIGURES
No.
1 Map of Omaha 9
2 Close-up, North Central Omaha 11
3 Windrose Data, Omaha 12
4 Topographic Plot of Blood Leads 13
5 Iron Therapy Study 16
6 Seasonal Variations Placental Pb and Fe 23
7 Urban-Suburban Blood Lead and Age 28
8 Seasonal Variations in Blood Lead, Urban 31
and Suburban, Ages 14-18
9 Seasonal Change in Blood Lead, Ages 2-5 32
10 Air Lead - Omaha - 1970 through 1974 33
11 Air Lead at 3 Feet and 15 Feet Elevation 35
12 Dustfall Lead -Omaha - 1973 through 1974 36
13 Environmental Lead and Blood Lead, Ages 2-5 39
14 Environmental Lead and Blood Lead, Ages 10-12 41
15 Household Lead and Blood Lead, Ages 10-12 43
16 Lead in Yard Dirt and Residential Distance 50
From Traffic
17 Response of Serum Fe to Oral Iron 54
vi
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FIGURES (continued)
No. Page
18 Oral Iron vs Placebo: Change in Serum Iron 55
19 Oral Iron vs Placebo: Change in Hemoglobin 56
20 Oral Iron vs Placebo: Change in Blood Lead 57
21 Correlation of Blood Lead with Na/K ATPase 66
vii
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TABLES
No. Page
1 Age and Urban-Suburban Differences in Blood Lead 27
2 Monthly Blood Lead, Urban and Suburban Males, 29
14-18 Years
3 Blood Lead, Urban and Suburban, Ages 2-5 Years 30
4 Composite Air Lead in Omaha, 1970-1974 34
5 Environmental and Blood Lead, Ages 2-5, 1974 38
6 Environmental and Blood Lead, Ages 10-12 40
7 Household Lead and Blood Lead, Ages 10-12 42
8 Household Lead - Children, ages 10-12, with High 44
and Low Blood Lead
9 Household Lead, Correlation with Blood Lead, 45
Ages 10-12
10 Environmental and Blood Lead, Males, Ages 46
14-18
11 Household Lead and Blood Lead, Ages 14-18 47
12 Household Lead, Correlation with Blood Lead, 48
Ages 14-18
13 Proximity to Traffic and Blood Lead, Urban 49
Students
14 Blood Lead and Hematologic Indices 51
15 2,3 DPG Intercorrelations, Ages 2-12 52
viii
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TABLES (continued)
No. Page
16 Correlations of Blood Lead, Serum Iron and 53
Hematologic Indices
17 Iron Therapy vs Placebo: Effect on Serum Iron 58
and Blood Lead
18 Blood Lead and Iron Therapy 59
19 Blood Lead and Rbc Enzymes Reflecting Cell Age 61
20 Blood Lead and Rbc Enzymes Reflecting Cell Age: 62
G-6-PD Deficient Males
21 Lead in Young and Old Rbc 63
22 Blood Lead and Rbc Membrane Na/K ATPase 64
23 Na/K ATPase of Rbc Membrane: Blood Lead <20 65
vs 20-40 ug/dl
24 Blood Lead in Omaha School Children, 1971-1975 68
ix
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ACKNOWLEDGMENTS
The cooperation of the Board of Education, Omaha Public Schools,
Dr. Rene E. Hlavac, Assistant Superintendent; and of School
District 66, Dr. Vaughn Phelps, Superintendent, is gratefully
acknowledged. We are particularly indebted to Mrs. Betty Rundlett,
Supervisor of Health Services, Omaha Public Schools, to the nurses
and teachers at the participating schools for their enthusiastic
cooperation, active support, and sustained interest in the project.
Mr. Ronald Crampton, Science Department, Westside High School
actively participated in the environmental and student studies.
Our appreciation is also expressed to the directors of the seven
day care centers who participated in our study: Mrs. Betty Burton,
Mrs. Karen Ihrig, Mrs. Dorothy Jenkins, Mrs. Ardella Jones, Mrs.
Cleo Phillips, Mrs. Teresa Ruffin, and Mrs. Bobbie Young.
The Omaha-Douglas County Health Department participated in the
design of the project and conducted the environmental sampling
with major contributions by Mr. Donald Olson, Chief of the Environ-
mental Health Division; Mr. Clarence Monich, Supervisor of the
Sanitation Engineering Section; and Mr. Joe Palensky, Sanitarian.
Assays of environmental samples in 1974 were conducted by the
health department laboratories directed by Mr. John Wiley.
Gory Love, Ph.D., Project Officer, and Anthony Colucci, M.D.,
Environmental Research Center, EPA, provided invaluable guidance.
The analyses of air and dustfall lead in 1973-74 were directed by the
EPA and carried out by Dr. E.R.Williams, North Carolina Department
of Health.
Mrs. Marilyn Schlicht served as project coordinator and directed
all of the school sampling teams. Mrs. Kathryn Stelmak, M.S.,
Chief of the Laboratory, was ably assisted by Mr. Warren Hill.
x
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SECTION I
CONCLUSIONS
Lead in the air and dustfall in Omaha markedly declined from
1970 to 1974, coincident with the antipollution measures en-
acted by the EPA. Lead in both urban and suburban air fell to
neglible amounts in 1973, the time of introduction of unleaded
gasoline. Studies subsequent to the time of this report show
a concommitant and significant drop in the blood lead of urban
school children from 1971 to 1973 to 1975.
The decrease represents a prompt and gratifying response to the
Clean Air Act but stimulates an even closer scrutiny of the threshold
for health effects and the investigation of previously unrecognized
toxicity to lead.
Despite the decreases in environmental lead, the blood lead of
urban children and adolescents continues to be higher than that
of their suburban counterparts in all three age groups: 2-5, 10-12,
and 14-18 years. The urban-suburban difference is highest in pre-
school children and decreases with age: the younger the child, the
greater the exposure.
The increase in blood lead in urban children of school age correlates
with the increased lead in urban dustfall, yard soil and dirt brought
into the house. Lead in yard dirt and blood lead correlate with
residential proximity to traffic and industry. In preschool urban
children, ages 2-5, blood lead also correlates with the lead con-
centration in interior dust as well as with lead in playground soil.
In this study, correlation of blood lead with dustfall and dirt is
much more evident than with the lead in air, milk, water, or in avail-
able paint chips.
General environmental exposure relative to age appears to be a more
significant factor in the elevation of the blood lead of urban children
than intrinsic differences in red cell metabolism. Blood lead is not
increased in children with iron deficiency. Although urban black
children have both a higher lead and lower hemoglobin than suburban
white children, within any age group of environmentally homogenous
children, blood lead increases with the serum iron. The administra-
tion of ferrous sulfate, 200 mg/day, to preschool children with iron
1
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deficiency does not decrease the blood lead. To the contrary, data
from this study suggests that an increase in iron intake may actually
increase the blood lead. This might relate to the effect of ferritin
binding of lead or to facilitation of a common mucosal transport mech-
anism for heavy metals.
Lead toxicity is not an "all or none" phenomenon and the consideration
of a blood lead of 40 jjg/dl as the threshold for adverse effects denies
evidence for an actual continuum of toxicity. This continuum is shown
by the fact that children with blood leads increased to 20-40 >ig/dl
have significant depression of red cell membrane Na/K ATPase, the
enzyme considered critical to membrane stability, cation transport,
and cell size. They have evidence of a shortened red cell survival as
reflected in increased G-6-PD, enzymes that indicate a young red cell
population. They also have a decrease in red cell glutathione. Gluta-
thion, an index of red cell resistance to oxidation, normally increases
in a young red cell population, and its decrease in young cells suggests
a selective inhibition by lead.
This study demonstrates that an adverse health effect, decreased red
cell survival, occurs at 'hon-toxic"blood lead levels found even at the
reduced environmental exposure attributed to the successful antipollution
measures of the EPA.
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SECTION II
RECOMMENDATIONS
We have evidence that the urban child is exposed to significantly
greater amounts of lead from general environmental sources than
is the suburban child. Since this "sub-toxic" increase in blood
lead of urban children is associated with the adverse health effect
of decreased red cell survival, we strongly recommend continuing
efforts to decrease the high fallout of lead particles in the urban
dustfall, soil, and house dust.
We recommend investigation of the health effects of an inhibition
of red cell membrane Na/K ATPase by a standard protocol in several
areas of the United States to demonstrate its significance in ethnic
groups of differing environmental exposure and intrinsic differences
in red cell metabolism.
Decrease in the Na/K ATPase activity of the red cell membrane at
blood lead levels as low as 20 pg/dl also leads us to recommend
the study of the effect of lead on Na/K ATPase activity in leucocytes
and platelets. We recommend a study of leucocytic membrane trans-
port as a factor in the altered immune response of lead exposure.
Since a decrease in platelet Na/K ATPase is associated with a de-
creased uptake of serotonin, and may parallel a similar decrease in
cerebral uptake of serotonin1, we recommend investigation of the
effect of lead on the platelet enzyme. This could provide an available
peripheral index of neurotoxicity from lead.
A blood lead threshold for children of 40 pg/dl is no longer physio-
logically tenable. We propose that the Environmental Protection
Agency review and evaluate all current physiologic and biochemical
barometers of adverse health effects and make recommendations re-
garding the standards for acceptable levels of environmental lead
exposure.
The demonstrated success of the EPA in reducing environmental lead
and investigating its threshold effects is a model that should be
applied to other toxic metals such as arsenic, cadmium, and mercury.
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SECTION III
INTRODUCTION
There is much evidence that the urban population has a higher blood
lead and a higher total body burden of lead than the rural or even
the suburban population2. In any population, children are considered
particularly at risk for an increase in the body burden of lead and its
health effects, a fact attributed to the higher load per unit of weight,
the higher metabolic rate, and the smaller reservoir of cancellous
bone for lead storage"*^. There is evidence that the current level of
lead in the general urban environment accounts for an increased blood
lead in urban children, and may result in low level toxicity2,3. This
study focuses on three questions basic to the lead exposure of urban
children:
A. Environmental sources
B. Intrinsic differences in metabolism
C. Health effects.
A. ENVIRONMENTAL SOURCES
The report is first concerned with definition of the role of general
environmental exposure as distinct from unique factors such as de-
teriorated housing, food contamination and pica. Definition of the
contribution of the general sources of lead - air, dustfall, soil,
water and milk - is basic to the projection of the effects of a contin-
ued increase in environmental lead.
Lead in urban air is a major concern since the absorption of inhaled
lead ranges from 30% to 50%3. Under controlled conditions, the
elevation of air lead above 2 pg/M^ produces a discernable increase
in blood lead^. Our study investigates urban-suburban differences
in air lead as related to blood lead, and includes an assessment of
seasonal changes in air lead as related to seasonal changes in blood
lead.
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"he contribution of particulate fallout of air lead to the total ingested
lead is important. Although the usual intestinal absorption of lead
is only 10%, Delves has evidence that it may be as high as 50% in
young children^. Since an oral lead intake of only 0.6 - 1.0 mg/d
causes an increased blood lead and cumulative increases in body
burden^'^, the current levels of general urban exposure are significant.
Street dirt, dustfall, yard dirt, milk, water, and food are all general
environmental sources of lead with concentrations that often exceed
0.5 mg/gm7'8. interior sources of lead, even in the absence of flaking
paint and plaster of older housing, are reflected in the lead content of
household dust. Lead in house dust may exceed 5 mg/gm and signifi-
cantly contribute to the body burden of lead in children without pica^.
Within any environmentally homogenous population, individual variat-
ions in the lead load may result from increased mouthing behavior plus
exposure to sources that may be unique to the individual or to the
household such as water of low pH, cooking utensils, leaded pottery,
lead painted pencils and toys, food selection, ink and paper ingestion,
and even toothpaste^/ H.
B. INTRINSIC METABOLIC DIFFERENCES
Within culturally similar populations of the same age group, there also
are variations in the blood lead and in the susceptibility to lead toxicity
that support intrinsic differences in the absorption or metabolism of lead.
Black children in New York City, for example, have higher blood leads
than do Puerto Rican children living in apparently identical circumstan-
ces1^. Children with sickle cell disease seem particularly susceptible
to lead neuropathy 13,14. -y/Ve previously investigated the contribution
of a deficiency of red cell G-6-PD, a common metabolic phenomenon
found in 10-12% of black males. We did find some suggestion that red
cell lead, but not whole blood lead, was increased in children with this
genetic difference in molecular chemistrylS. This suggested the possi-
bility that other disorders of red cell metabolism common in the urban
population, such as iron deficiency anemia and sickle cell trait, might
alter red cell binding and, by virtue of the increased concentration
gradient, enhance the rate of transport of lead from red cell to tissuelS.
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Iron deficiency, the most common nutritional deficiency of urban
children, has a provocative epidemiologic association with lead
poisoning. Six and Coyer*7 showed marked enhancement of tissue
lead in iron deficient rats. Ferritin and transferrin have both been
shown to bind lead and this binding appears to relate to the increased
tissue lead of iron deficient animalsIS/19. The second focus of the
study is the evaluation, within a homogenous population, of the
correlation of serum iron and blood lead. It includes a clinical trial
of the effect of oral iron supplements on the blood lead of iron de-
ficient children to test the experimental evidence that iron supple-
mentation may prevent lead toxicity.
C. HEALTH EFFECTS
The third area of concern is the threshold for health effects of the
increased body burden of lead resulting from current urban levels of
exposure. Of all the indices of body burden available, blood lead
correlates best with tissue lead, although not with bone lead, and
is used throughout as evidence of lead load. Multiple clinical and
biochemical correlations, as reviewed by de Bruin^, support the
concept that lead toxicity is a continuum and not an all-or-none
phenomenon occurring at a specific blood level such as 40 or 80/Kj/dl.
The effects of lead on the red cell survival have long been known and
71
are well reviewed by Hernberg , but have been less used than the
suppression of erythropoiesis as clinical and biologic evidence of
lead toxicity. In vivo and in vitro, lead exposure results in rapid
loss of potassium from the red cell, leaving a shrunken cell with a
decreased capacity for survival.
Although changes in osmotic resistance and decreased red cell survival
99 -9 ^
are well documented in acute lead poisoning' , the complexities
of measurement of red cell survival have impeded broad scale clinical
studies at low levels of blood lead. Red cell enzyme activity is a
reliable indirect measurement of cell age26. Hexokinase, aldolase,
GOT, G-6-PD, 6-PGD are all significantly higher in young red cells
than old27'28. A young red cell population is evidence of decreased
survival; its correlation with minimal increases of blood lead is evi-
dence of a health effect at levels far below 40;ug/dl.
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The integrity of the red cell membrane relates to the cationic
transport system of membrane Na/K ATPase^/30. Recj ce\\ mem-
brane Na/K ATPase has been found to be depressed in lead workers
and even in urban adults with blood leads above 30 ;ig/dl31~33>
Although some aspects of the experimental suppression of K transport
into the red cell are all or none, as discussed by Passow™, the
usual effect of enzyme inhibition is linear. Linear inhibition of red
cell membrane Na/K ATPase at subtoxic levels of lead would be a
significant health effect and would also support the significance of
similar effects of lead on cation transport across cellular and mito-
chondrial membranes of tissue other than the red cell, and as shown
by Hexum in neuronal mitochrondria35,and suggested by Goyer in renal
tubular cells36.
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SECTION IV
DESIGN
LOCATION
The entire study was carried out in Omaha, Nebraska, a city of
350,000 population. Omaha's air has long been known to have a
high particulate concentration^7. Back in 1967, dustfall analysis
by the National Air Pollution Control Network had shown downtown
Omaha to have the highest dustfall lead of 22 midwestern cities^.
Since traffic density in Omaha should be commensurate with the low
population density, it is assumed that the high air lead and dustfall
lead in central Omaha relate to industrial sources. Figure 1 is an
outline map of Omaha, showing the urban and suburban areas of study.
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• Elementary School U' urban
• High School S: wfaurban
OMAHA, NEBRASKA
Figure 1. Map of Omaha reproduced at a scale of '—» =1.5 miles. The
west suburban area is a 3 mile radius centering on the two suburban
schools (S). The urban area is in the northeast quadrant and is a rectang-
ular area bordered by Ames to the north, Dodge to the south, 16th Street
on the east, and 42nd Street on the west. The urban grade school (U,
solid dot) has a battery manufacturer as its immediate north neighbor. Air
and dustfall were sampled at the two urban sites and at the common sub-
urban site, 1971-1974.
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Studies of blood lead (Pb-B) in school children as related to environ-
mental lead were conducted in two general areas, 1) a suburban area
in southwest Omaha predominantly white and middle class in population
where studies were conducted at 1 high school, 1 grade school and 2
day care centers, 2) a northcentral urban industrial area with a predom-
inantly black population with studies conducted at 1 high school, 1
junior high, 9 grade schools and 5 day care centers.
The northcentral area (Figure 2) has a significant amount of substandard
housing and has 3 known lead emission sources. There is a small
battery manufacturer in the north, immediately adjacent to a grade school.
Immediately southwest of the area there is a major lead smelter and a
battery reclamation plant. The windrose data for Omaha (Figure 3) shows
the worst residential area to be downwind from both lead emission areas.
School children, ages 6-12, living in this high lead area were studied
in 1970-71 for the interaction of G-6-PD deficiency and Pb-B15. Topo-
graphic analysis of the Pb-B of each of 107 school children studied in
1970 in the northcentral area is shown in Figure 440. This shows a
sharp peak in the blood lead of children living in the proximity of a
small battery paint. A significantly increased Pb-B was also demon-
strated in those children living within 500 feet of a major trafficway.
The census tract data on housing in the area was analysed but the blood
lead of these children, ages 6-12 years, did not show a significant
correlation with the percent of deteriorated housing within the census
tract. The same geographic areas are used in our present study for the
correlation of environmental lead with the Pb-B in high school students
14-18, grade school students, 10-12, and preschool children, 2-5 years
old.
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OMAHA,NEBRASKA
HOUSING
'/„ SUBSTANDARD
I I
10-19.9
20-29.9
30-50
Figure 2; Close up of the northcentral area of Figure 1 showing the
percent of substandard housing according to census tracts^ and the
location of 3 lead emission sources. Air sampling was conducted at
3 sites: M (mixed commercial) at the urban high school, 1970-1974;
C (commercial) in 1970; and at the urban grade school just south of the
battery plant in the center of the area, 1971-1974.
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OMAHA. NEBRASKA
WINDROSE DATA
WEATHER BUREAU AIRPORT STATION
OMAHA MUNICIPAL AIRPORT
OMAHA tO. NEBRASKA
NORTH
.i :D
WEST
0 5
I , . . . I . .
EAST
SOUTH
o =
= 3 MPM or l
= 4-15MPH
=a Or. IS MPH
Figure 3: Wind rose data from Omaha showing the prevailing NW and
SE winds which tend to focus emissions from the lead sources on the
north central urban area of Figure 2.
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TOPOGRAPHIC MAP OF BIOOD LEAD,SUBSTANDARD HOUSING AND LEAD EMISSION SOURCE
107 URBAN RUCK SCHOOl CHILDREN AGES 612
(MISSION SOURCE
HOUSING
SUBSTANDARD
figure 4; Topographic plot of the blood lead of 107 children living in
the northcentral area outlined in Figure 2 . The thickest black lines
define streets carrying more than 3500 vehicles per lane per day. There
is a sharp peak of blood leads (Pb-B) of children living near the central
battery plant and a ridge of increased values parallelling the major
north-south street. Little correlation of Pb-B with housing is seen
40
13
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Extremely important to this environmental assessment are the milk and
water supplies. In the Omaha area all the milk sold derives from a
common pool and is subsequently bottled at individual plants. All water
is from the 2 municipal processing plants, both yielding water at pH
8.0 - 9.0 and therefore unlikely to leach lead from pipes in the absence
of a water softener. The same grocery chains supply both urban and
suburban Omaha, and although general food supplies are similar,
variances in selection are assumed.
A. ENVIRONMENTAL STUDIES
The design of the environmental studies was based on the correlation
of the following measurements with the blood lead for group and indivi-
dual analysis. The specific details concerning dates, numbers of
samples, and numbers of subjects are given in Section VI.
Air Lead:
Dustfall Lead;
Soil Lead:
Interior Dust:
Paint Chips:
Water:
Milk:
2 urban, 1 suburban site, 1970-1974,
for group correlation.
2 urban, 1 suburban site, 1973-1974,
for group correlation.
7 urban and 4 suburban schools for group
analysis. Homes of 14 urban and 14 sub-
urban students for correlation with indivi-
dual Pb-B and with residential distance
from traffic.
7 preschools for group analysis.
As available.
10 urban and 10 suburban homes for group
and individual correlation.
10 suburban and 10 urban homes for group
and individual correlation.
14
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The correlations of the environmental lead were as follows:
1. Urban vs suburban, ages 2-5, 10-12, 14-18 years.
2. Group environmental studies for correlation of lead in
air, dustfall, milk, and water vs Pb-B of:
a) urban high schools
b) suburban high schools
c) urban grade schools
d) suburban grade school.
3. Exterior and interior dirt at day care centers vs mean
Pb-b at each center.
4. Individual correlations of Pb-B with lead in yard dirt,
boot tray dirt, housedust, residential distance from
traffic.
B. INTRINSIC METABOLIC DIFFERENCES
Iron Deficiency
The studies on iron deficiency involved screening in January, 1974 of
154 preschool children at 5 urban and 2 suburban day care centers for
comparison of Pb-B, serum iron (Fe), iron binding capacity (TIBC),
transferrin saturation (% Fe Sat), complete blood count (CBC), red cell
indices, and 2,3 DPG. Within each group, correlation was made of
blood lead with serum iron and hemoglobin.
After the January screening, 3 groups were chosen for the response to
oral ferrous sulfate or placebo. All subjects had a low serum Fe (below
80 ;ag/dl) but no anemia (Hgb> 10.5 gm%). The general design of the
study is diagrammed in Figure 5:
15
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Fe <80 pg/dl; Hgb >10.5 gm%
A (14)
Pb-B ^ 17
B(9)
Pb-B 17-40
Fe SO4 200 mg
5 day/week
Mar - June
Figure 5: Iron Therapy Study: The response of serum Fe and Pb-B to
oral iron was studied in 3 groups; A: 14 low lead children receiving
ferrous sulfate, B: 9 high lead children receiving ferrous sulfate,
C: 7 high lead children receiving a placebo. Groups B and C were
paired for comparable levels of blood lead and serum iron and a double
blind system was used for administration of iron.
16
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C. HEALTH EFFECTS
Total and Na/K ATPase were correlated with blood lead in 5 groups:
1. Ages 2-5 years
2. Ages 10-18 years
3. G-6-PD deficient, ages 10-18
4. Normal adults
5. Lead workers.
Evidence of hemolysis or other effect of lead was obtained by corre-
lation of G-6-PD, 6-PGD, 2,3 DPG, GSH with blood lead in the above
5 groups.
17
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SECTION V
OBJECTIVES
Definition of the contribution of general environmental sources of
lead: air, dustfall, dirt, milk, and water to the higher Pb-B of
urban children and assessment of the relative significance of gen-
eral environmental increases of lead and of sources unique to a
household.
2. Investigation of the role of intrinsic differences in metabolism on
Pb-B: the effects of iron deficiency and the possible beneficial
effect of oral iron supplements to correct the iron deficiency.
3. Investigation of the hemolytic effect of lead, even at Pb-B levels
below 40;ug/dl,as manifested by red cell membrane Na/K ATPase
and by enzymes indicating decreased red cell age.
18
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SECTION VI
DEVELOPMENT OF QUANTITATIVE METHODS
MATERIALS AND METHODS
A. ENVIRONMENTAL STUDIES
The urban and suburban areas of this study are shown in Figure 1.
Students live and attend school within their urban or suburban area. At
ages 1-12, urban children attend school within 0.25 miles of home; sub-
urban children live within a 5 mile radius. All high school students
studied live within a 3 mile radius of their respective schools, but
greater mobility is assumed in the high school age group. Comparison
is made of the blood lead and environmental lead of 4 groups:
1. Urban vs suburban high school students, ages 14-18
(1973).
2. Urban children, ages 10-12, vs suburban children,
ages 10-12 (1973).
3. Urban children, ages 10-12 years, with blood lead over
20 ug/dl vs those with blood lead below 20;jg/dl (1973).
4. Urban children, ages 2-5, attending 5 day care centers
vs preschool children at suburban centers (1974).
Blood Lead
Blood lead (Pb-B) studies in the 4 paired populations were as follows:
1. High School, Urban vs Suburban -
Venous samples in February and April, 1973 for micro Pb-B by the Delves
micro method41, and macro Pb-B by the Farrelly & Pybus method42j pius
3 fingerstick samples for micro lead in January, March, and May, 1973.
The lead content of fingerstick samples were corrected for the ratio of
venous to capillary hemoglobin.
19
-------
2. Grade School, Urban vs Suburban -
Fingerstick samples for micro assay in 35 urban black children in
December, 1972 and in 35 suburban children in early January, 1973.
3. Urban Grade School, High Lead vs Low Lead -
Of the 35 urban black children, 26 had a second fingerstick lead in
February, 1973, and a venous sample in April, 1973, for both a micro
and a macro assay of Pb-B. Fingerstick Pb-B leads of December and
February were corrected to the venous hemoglobin of April.
4. Preschool Children -
154 venous samples for micro Pb-B were collected from 92 urban children,
predominately black, and 31 suburban children, predominately white, all
ages 2-5. Fifty of the urban children and all of the suburban children
were sampled in January, 1974, and 45 children were sampled in April,
1974. Repeat Pb-B were obtained in July, 1974 from 30 of the children.
Environmental Lead
Air lead was collected as random 24-hour Hi Vol Samples at 3 sites:
1) the urban high school at 15' elevation, 2) the urban grade school
(located next to a small battery plant) with samples at both 3' and 15',
3) the suburban high school at 15'. The monthly values are the mean of
6 to 20 24-hour samples per month collected during 7 months of 1970,
8 months of 1972-1973, and 10 months of 1974.
Dustfall lead was collected in plastic buckets in 30 day samples at the
same 3 sites as the air lead for 6 months of 1973 and 4 months of 1974.
Soil lead was collected at the 2 high schools, the 2 grade schools, the
7 preschool centers and at 37 homes (7 urban high school, 7 suburban
high school, 16 urban grade school and 7 suburban grade school). At
each site, 4 samples were obtained, each consisting of a 2" core
collected at a distance halfway between the building and the lot margin
on all 4 sides of the building.
20
-------
Interior dust was sampled in two ways: dust as emptied from the
vacuum cleaner or, if no vacuum cleaner was in the home, as a floor
sweeping. Dirt brought into the house was collected over 30 days in
boot trays fitted with a plastic grid and placed in the entry way.
Paint chips were sampled as availabe from flaking interior or exterior
paint.
Air, dustfall, and soil lead were measured by AA spectrometry after
nitric acid extraction by E.R. Williams, D.P.H., South Carolina
Department of Health and Environmental Control in 1973. Additional
dustfall samples from 1973 and all environmental samples from 1974
were similarly assayed by the Omaha-Douglas County Health Depart-
ment.
Water was collected at 20 homes in 1973 as one month random collect-
ions. Each householder was given a 1 liter plastic jug and asked to
add approximately 15 ml every morning (before running water for other
purposes) and 15 ml every evening after dinner. Water was analysed
by the Delves cup assay after nitric acid extraction (20 samples) by
Dr. Williams.
All of the study homes used whole milk only. At the time of the soil
sampling, purchase was made of the same brand of whole milk as used
by each household. Lead assay was by the Delves cup assay (20
samples) of Haelen, Cooper and Pampel^ in our laboratory.
B. LEAD AND IRON DEFICIENCY
Initial screening of venous blood for blood lead (Pb-B) by the Delves
micro method^*, serum iron (Fe^ and total iron binding capacity (TIBC),
2,3 DPG^, CBC and hematologic indices was carried out in January,
1974, on 154 preschool children, ages 2-5 years. Of these, 31 white
children attended 2 suburban day care centers and 122 children, predom-
inantly black, attended 5 urban day care centers. All had been enrolled
for at least 4 months. The standard analyses, _t.test and Pearson's £
were used to evaluate the urban-suburban differences and the correlation
of blood lead with serum iron and other indices.
21
-------
From the initial group of 154 children, 3 groups of iron deficient
children without anemia were selected for evaluation of 1) the effects
of increased blood lead on response to iron therapy and, 2) the effects
of iron supplementation on blood lead. All children had a serum iron
below 80^ig/dl and a hemoglobin above 10.5 gm%.
Group A; Low Fe, low Pb with fe supplementation:
14 children, blood lead below 17.5 ug/dl.
Treated with oral ferrous sulfate, 200 mg/dl
(40 mg elemental Fe or 1.15 - 1.75 mgAgK
given as a noon time dose at their school, 5
days per week for 4 months, March through
June, 1974.
Group B: Low Fe, high Pb with Fe supplementation:
9 children, blood lead 17.5 - 40jug/dl, iron
supplementation as for Group A.
Group C: Low Fe, high Pb, no Fe supplementation:
7 children, blood lead 17.5 - 40>ig/dl, given
a "placebo" solution containing 2 mg of ferrous
sulfate, 5 days per week for 4 months.
Matched pairs from Groups B and C:
Groups B and C initially consisted of 11 pairs of children
matched for serum Fe + 20 >ig/dl and Pb-B + 8 jug/dl. They
received, in a double blind study, either 200 mg or 2 mg of
ferrous sulfate in an oral solution, 5 days per week for 4
months. Two children were lost from the treated group and
4 from the matched group. Seven matched pairs, including
3 sets of sibs, completed the study and analysis of the
response employed the Wilcoxen signed ranks test.
The 30 children in Groups A, B, and C had a repeat of the initial studies
within 4 weeks of completion of the oral iron supplement.
22
-------
Seasonal Sampling
The January to July sampling was partly convenience and partly an
dttrmpt lo modify Lho known seasonal changes in both serum iron and
blood lead. Serum iron is highest in the first half of the year with a
peak in Marches. Blood lead is highest in the second half of the year
with a peak in August. This relationship is shown in a graph of data
from Dawson et aP° of the seasonal changes in placental Pb, Ca, and
Te (Figure 6).
10
J FMAMJJASOND
Tigure 6: Seasonal variations in the placental concentrations of Pb
46
(uM/g x 10) and Fe (>iM/g) adapted from data by Dawson et aPD.
23
-------
C . RED CELL METABOLISM
1. Enzymes of Red Cell Glycolysis and Cell Age
Samples of venous blood from 79 urban and suburban grade school and
high school students, ages 10-18 (as cited in Section VI -A) and from
29 black males, ages 10-12, with G-6-PD deficiency, were assayed
for 2,3 DPG by the method of Maeda4^, glucose-6-phosphate dehydro-
genase (G-6-PD), 6-phosphogluonic dehydrogenase (6-PGD), and total
glutathione, all by the methods of Beutler47. The G-6-PD deficient
subjects all had less than 2.5 lU/gm Hgb and as black males were
probably, although not proven, to have the A~ enzyme. Enzyme
activity, hemoglobin, hematocrit, red cell indices, and reticulocytes
were compared by J^ test for those with Pb-B below and above 20 ug/dl.
2. Lead Concentration in Young and Old Red Cells
Heparinized venous blood obtained from 4 healthy adults was incubated
with lead at 100 jug/dl for one hour at room temperature. Differential
centrifugation was carried out by the method of Murphy^S in an angle-
head rotor at 30° and 15,000 rpm for one hour. The top and bottom 10%
of the red cells were separated and reconstituted with the subjects' own
plasma to the original hematocrit and assayed for lead content by the
micro method of Delves.
3. Assay of Na/K ATPase, Materials and Methods
Subjects -
Assays of red cell membrane Na/K ATPase were done on 65 samples
obtained from 44 males and 3 females. While no differences in enzyme
activity had previously been found in relation to sex, predominately
male subjects were chosen, with the composition of the five groups as
follows:
24
-------
Preschool:
School Age:
G-6-PD:
Adults:
Lead
Workers:
3 girls, 2 boys, all black, ages 2-4 years,
attending an urban day care center. The
children had no history of pica, no iron de-
ficiency anemia, and normal G-6-PD activity.
36 assays were done on the following 22 sub-
jects: 7 urban black males, ages 9-12, all
attending one school adjacent to a battery
plant; 8 males, ages 14-18 at one urban high
school (14 samples); 8 white males, ages 14-
18, attending a suburban high school (15 sam-
ples).
14 black males,ages 6-12, attending 3 urban
schools, all with G-6-PD deficiency, (less
than 2.5 lU/gm Hgb^7), but no anemia or
reticulocytosis. All were presumed to have
the A~ variant of the G-6-PD enzyme, but this
was not definitively established.
9 white males, ages 20-35, either medical
students or laboratory staff.
2 white males employed in the lead refinery,
no overt symptoms of lead poisoning.
Blood Lead -
All samples were drawn by venipuncture into lead free heparinized
vacuum tubes. Whole blood samples were assayed for blood lead by
the macro method of rarrelly and Pybus4 . Blood lead (Pb-B) is
reported as micrograms/deciliter (jag/dl).
25
-------
Red Cell Membrane Na/K ATPase, Membrane Preparation -
Membrane fragments were prepared and assayed as described by
except that no EDTA was used. All glassware was acid washed and
rinsed with water that was first distilled and then deionized. The red
cells (RBC) from 5 ml of heparinized blood were washed 3 times with 6
volumes of 0.15 M NaCl, were centrifuged at 400 x g for 4 minutes and
resuspended in NaCl. All subsequent procedures were done at 2° C.
The RBC suspension was separated for duplicate determinations. One
volume of cells was hemolyzed with 8 volumes of 2.2 mM imidazole,
buffered with HC1 to pH 6.8 + 0.1, and was centrifuged at 20,000 x g
for 10 minutes. This procedure was then repeated on the sediment. The
sediment was then washed and centrifuged 4 times with 8.6 mM NaCl
and 3.68 mM imidazole, pH 7.8 +_0.1. The fluffy sediment, consisting
of red cell ghosts, was refrigerated and incubated overnight in 1.3 mM
urea and then rewashed with the saline-imidazole solution at which time
it would be verified that the ghosts were broken into membrane fragments
These membrane fragments were suspended in the saline-imidazole sol-
ution to the original volume and the enzyme was assayed immediately.
In Vitro Studies of ATPase Activity -
In vitro studies of ATPase activity were done by the same method employ-
ing blood samples from 4 subjects for the effect of preincubation for
1 hour at 37° of whole blood with Pb of 100 jjg/dl before preparation of
the membrane fragment and of the incubation of membrane fragments with
lead under the same conditions.
26
-------
SECTION VII
DEMONSTRATION OF QUANTITATIVE METHODS
A. ENVIRONMENTAL STUDIES
1. Urban-Suburban and Age Related Differences in Blood Lead
As defined in the following table, the urban-suburban differences in
Pb-B were most extreme in preschool children and decreased with age:
Table 1. AGE AND URBAN-SUBURBAN DIFFERENCES
IN BLOOD LEAD
Age
1-5 years
1974
10-12 years
1973
14-18 years
1973
14-18 years
males only
1973
Urban
22.9 + 0.6
(122)
21.7 + 0.5
(35)
22.3 + 1.2
(14)
25.6 + 2.8
(8)
Suburban
14.4 + 0.6
(31)
17.1 + 0.7
(35)
20.2 + 0.7
(23)
20.7 + 0.9
(17)
P_
<.001
-C.01
n.s.
<.05
Blood lead of urban-suburban children. Each pair of groups was tested
in same month and same methodology (micro or macro). Pb-B given as
mean + SE with number of subjects in parentheses. Students _t_ used to
test for significant differences.
The decrease in Pb-B with age and the lessening urban-suburban differ-
ence is diagrammed in Figure 7.
27
-------
25
£ 20
IS
I
URBAN
SUBURBAN
I
B
a
BB
I
B
10-12
AGE-YEARS
14-18
Figure 7. Pb-B (mean + SE) of representative samplings of urban and
suburban children as listed in Table 1.
The decrease in Pb-B in urban children from ages 2 to 5 years is given
in Table 2. A comparable decrease in blood lead from ages 2 to 5 years
was not noted in the relatively small sample of suburban children:
28
-------
Table 2. BLOOD LEAD, URBAN-SUBURBAN,
AGES 2-5 YEARS
Age, years
Urban
Suburban
27.0
+ 1.9
(35)
25.9
± I-2
(70)
25.1
+ 0.9
(71)
23.1
± !-4
(34)
**
14.2*
+ 0.9
(7)
14.3*
+ 0.9
(21)
14.4*
+ 0.5
(8)
Blood lead, jag/dl, mean + SE, in urban and suburban pre-
school children in Omaha, 1974.
* p < . 05 by t test for urban-suburban difference.
** p < .05 for urban children age 2 vs age 5.
29
-------
In the age group, 14-18 years, the mean Pb-B of the total group (male
plus female) did not differ, but monthly measurements, over 3 to 5 months,
showed a significantly higher mean blood lead in urban than suburban
boys (25.0 vs 20.7; p < .05), as listed in Table 3:
Table 3. MONTHLY BLOOD LEAD URBAN-SUB URBAN
MALES, 14-18 YEARS
Urban
Suburban
1972
Dec.
1973
Jan.
Mar.
Apr.
May
Mean
27.0 + 4.7
(8)
22.7 + 3.6
(6)
27.1 +3.1
(8)
31.4 +4.2
(8)
21.1 + 1.7
(8)
25.0 + 2.8
(8)
22.7 +2.3
(15)
17.0 + 0.6
(16)
20.1 +1.9
(14)
22.1 +1.5
(17)
21.5 + 0.9
(17)
20.7 +0.9
(17)
n.s.
n.s.
.05
.01
n.s.
Monthly Pb-B, AJg/dl, mean + SE, of 8 urban and 17 suburban high school
boys. Macro assay used in January and May, micro assay in other months,
F ratio used for determination of significant difference.
Seasonal Changes in Blood Lead
The seasonal changes in Pb-B in males ages 14-18 have been given in
Table 3. The seasonal changes from December, 1972 - May, 1973 in
male and female students, 14-18 years old, are shown in Figure 8:
30
-------
60
50-
5 40
o
m
I
8 30-
.0
o.
c
S 20'
a.
1
s
k
S
10-
Suburban—N«23
Urban. xN=!5
Dec Jon Feb Mar Apr May
Months
Figure 8. Monthly variations in Pb-B (mean +_ SD) in 23 suburban and
15 urban students, ages 14-18. No on-line or lag-time correlation with
air lead or dustfall lead could be demonstrated.
In the age group 2-5 years, repeat venous sampling for blood lead was
done in 30 preschool children. The seasonal increase, given in Figure
9, is of similar magnitude in urban and suburban children. An increase
from March to April, similar to the high school students, is also found
in a small sample of urban children.
31
-------
BLOOD LEAD-AGES 1-5
o
o
o
_l
CD
UJ uj
^W
s*.
x
2
O
40
30
20
10
SUBURBAN-6
URBAN I-17
URBAN n- 6
DEC JAN FEB
MAR APR
MONTHS
MAY JUN JUL
Figure 9. Seasonal change in Pb-B from December to July in urban
and suburban children, ages 2-5, each with at least two serial venous
samples. Blood lead is given jig/dl, mean^SE.
2. Air Lead
The decline in air lead in Omaha over 5 years as monitored at 3 sites is
shown in Figure 10. The highest values each year were found at the
urban grade school which is located adjacent to a battery fabrication
plant. In 1973, the year of the introduction of unleaded gasoline, there
is a significant drop in suburban as well as urban air lead. City wide,
industrial installation of antipollution devices began in 1971-72.
32
-------
2.5r
2.0
AIR LEAD - OMAHA - 1970 -1974
m
x
k
V.J
f •>
•-r
111
.1
CC
1.0
05
M J J A S O N
1970
URBAN GRADE SCHOOL|
URBAN HIGH SCHOOL^
SUBURBAN HIGH SCHOOL Q
NDJ F MAMJ
1972-1973
MONTHS
Figure 10. Monthly mean air lead in Omaha 1970-1974 at 3 sites. The
urban grade school site was at 24th and Pinkney in 1970 and 30th and
Sprague thereafter. The urban high school site (30th and Cuming) was
constant. The suburban site was at 48th and Q in 1970 and 88th and
Pncifjc thereafter.
The annual mean composite air lead at the 3 sites is given in Table 4:
33
-------
Table 4. COMPOSITE AIR LEAD IN OMAHA 1970-1974
Suburban
48th & Q, 1970
88th & Pacific, 1972-74
Urban high school
30th & Cuming
Urban
24th & Pinkney, 1970
30th & Sprague, 1972-74
1970
May-Nov
0.79
1.48
1.69
1972-73
Nov-June
0.29
0.43
0.63
1974
Jan-Dec
0.12
-
0.10
Air lead jug/M3, as the 7 to 10 months mean of the monthly means of 24-
hour Hi Vol collections at 3 sites.
Because of the possibility of higher air leads at the nearground level of
air intake of the young child, air lead was monitored at both 3' and 15'
at the urban grade school. The correlation of monthly values was sig-
nificant at r =0.94 (p <.05), but no significant difference was found
between air lead at the two elevations, as shown in Figure 11:
34
-------
AIR LEAD-URBAN GRADE SCHOOL
<5
D
N D J F MAM
1972 -1973
J F MA MJ J
1974
O N D
M ONTHS
Figure 11. Air lead at 3' and 15' elevation. Air lead in;ug/M3, as
the monthly mean of 6 to 20 random 24-hour collections per month taken
in Hi-Vol samplers at 3' and 15' feet elevation at the urban grade school
(30th & Sprague). There is no significant difference in the 16 months
mean at 3' (0.25 >ig/M3) and at 15' (0.32 jug/M3).
3. Dustfall Lead
A significant decline was noted in dustfall lead as collected in 30 day
samples at the same 3 sites as the air lead from March, 1973 through
April, 1974 (Figure 12). Unlike air lead, urban-suburban differences
in the dustfall lead were still significant in 1974 with maximum levels
at the school adjacent to the battery plant.
35
-------
£ 40
o
o
<•>
x.
-------
Lead in Exterior Soil and Housedust
Hxterior soil samples (4 at each site) were obtained from 4 schools and
25 homes in 1973, and from 7 day care centers in 1974 for a total of
154 assays. Exterior soil from urban areas had a lead content ranging
from 100-2400 ug/£jm: all suburban samples had less than 400 ug/gm.
Housedust was obtained from 34 homes and 7 day care centers. The
urban values for lead ranged from 50 to 1200 jig/gm, suburban samples
contained 110 to 610pg/gm.
Boot tray dust collections, representing a composite of exterior and
interior soil, had a lead content between 90 and 2400jjg/gm in 14 urban
samples and 50 to 500 ug/gm in 9 suburban samples.
Lead in Milk and Water
Water, collected as a composite 1 month sample from 20 homes in 1973,
never had a lead content above .01 ug/ml. The lead content of 20 milk
samples was between .02 and .03 jug/ml.
4 . Correlation of Environmental Lead and Blood Lead
The higher Pb-B of urban than suburban preschool children was assoc-
iated with a significantly (p < .001) higher dustfall lead and interior
dust (p < .05), as shown in Table 5. The relation of the general environ-
mental sources to Pb-B is shown graphically in Figure 13.
37
-------
Table 5. ENVIRONMENTAL: BLOOD LEAD, AGES 2-5, 1974
Pb-B
Air Pb pg/M3
Dustfall Pb,
mg/M /mo
Interior dust Pb,
ug/gm
Yard dirt Pb,
;jg/gm
Suburban
14.4 + 0.6
(31)
0.14 + .02
2.94 + 0.91
175 + 60
(2)
97 + 84
(7)
Urban
22.3 + 0.6
(122)
0.12 + .05
20.47 +2.72
7.05 +1.19
550 + 155
(5)
219 + 105
(20)
P
< .001
n.s
*. .001
< .01
-d .05
n.s.
Blood lead of 31 suburban and 122 urban children in 1974 with compari-
son of the lead content of general environmental sources. All values
given as mean +_SE. Numbers in parentheses are number of subjects or
of samples. Urban dustfall at each of the 2 urban sites of Figure 1, is
significantly higher than the suburban dustfall.
38
-------
22.3
Environmental Lead
Blood Lead Ages 2—5
SSO
SUBURBAN
(31)
URBAN
(122)
Pb-B
Dustfall
A
mg/m /mo
Int
Dust
/•g/grn
Yard Dirt
>«g/gm
Figure 13. Environmental Pb and Pb-B, ages 2-5, of 122 children at 5
urban day care centers and 31 children at 2 suburban centers. The
children at the urban centers have significantly higher mean Pb-B,
22 . 3 +_ 0.6 vs 14.4 + 0.6 jug/dl (p < .001), associated with a higher
dustfall lead, 20.5 + 2.7 vs 2.9 + 0.9 (p •£ .001), and interior dust lead
550 + 155 vs 175 + 60 pg/gm (p < .05). All values as mean + SE.
In the grade school students, ages 10-12, studied in 1973, the higher
level of Pb-B in the urban group was associated with a higher dustfall
lead as shown in Table 6, and plotted in Figure 14.
39
-------
Table 6. ENVIRONMENTAL AND BLOOD LEAD, AGES 10-12
Number of subjects
Pb-B ( micro) ,/ag/dl
Air Pb, ;ag/M3
Dustfall, mg/M2/mo
Milk. , ;ig/ml
Water, ^ig/ml
Suburban
35
17.1
+ 0.7
0.29
3.05
<.04
*.oi
Urban
35
21.7
+ 0.5
0.63
32.96
<.04
-c.Ol
Blood lead (mean + SE) of 35 urban and 35 suburban studies by
micro assay, fingerstick sample of January, 1973.
Air lead is the mean for January, 1973 and dustfall lead is the
mean for April - May, 1973. Values for milk and water were the
means of a single milk and a one month composite water sample
collected in April and May, 1973 at the homes of 5 urban and 5
suburban children of each group.
40
-------
32.9'
General Environmental Lead
and Blood Lead Ages 10-12
21.7
|| Suburban (35)
Urban (35)
* p < .05
Pb-B
Dustfall-Pb
Air Lead
Figure 14. Environmental Pb and Pb-B, ages 10-12. There is a signifi-
cant (p <.05 by 1 test) difference in Pb-B, dustfall and soil lead with no
significant difference in lead in milk or water.
In the individual homes, there was a significant difference in the lead
content of soil and boot tray dirt as shown in Table 7 and Figure 15.
41
-------
Table 7. HOUSEHOLD LEAD AND BLOOD LEAD,
AGES 10-12
Suburban
Urban
Number
Pb-B, micro
Soil, mean, ppm
Boot tray, ppm
Housedust, ppm
Paint chips, > 1000 ppm
17.7
+ 2.3
123
+ 28
283
+157
335
+ 68
1/9
16
22.4
+ 2.4
444
+ 62 p<.005*
1001
+ 472 p<.05**
572
+ 147
1/16
*_t test.
** z ratio for uncorrelated proportions of values greater than the
group median of 370 ppm.
42
-------
ELEMENTARY SCHOOL
SUBURBAN vs URBAN
1500
o
<
5
a.
Q.
500
S U
SOIL
S U
TRAY
S U
DUST
Figure 15. Household lead, urban (U) and suburban (S). Lead content,
as mean j^SE, as measured at the homes of 9 suburban and 16
urban children, in yard dirt, boot tray collections, and house dust.
as given in Table 7.
Within the total group of 16 urban students, ages 10-12, those with Pb-B
above 20;jg/dl had significantly higher levels of lead in yard dirt and
boot tray dirt as shown in Table 8.
43
-------
Table 8. HOUSEHOLD LEAD - CHILDREN 10-12 WITH
HIGH AND LOW BLOOD LEAD
Number of subjects
Pb-B, ;jg/dl
Soil, mean, ppm
Boot tray, ppm
Housedust, ppm
Paint chips
Pb-B< 20
7
15.5 + 1.4
346 + 94
470 + 141
449 + 108
0
Pb-B > 20
9
27.4 +1.4**
519 + 79 **
1442 +847*
692 +278
1/9 (93,636
ppm)
All values given as the mean + SE.
** p<.005, * p <.02 by t test.
There was a significant correlation (r = .49; p< .05) of Pb-B with lead
in yard dirt in this age group (Table 9). Correlation Pb-B with lead in
boot tray dirt was positive (0.23) but not significant for 16 subjects.
There was no correlation of Pb-B with lead in house dust, milk or
household water for ages 10-12 years:
44
-------
Table 9. HOUSEHOLD LEAD, CORRELATION WITH
BLOOD LEAD, AGES 10-12
Urban, ages 10-12
Soil
Boot tray
Housedust
Milk
Water
.49 (p^.05)
.23
.01
.00
.00
In urban males of high school age, the higher blood lead (mean of serial
measurements over 5 months) was associated with an urban dustfall lead
that was almost 3 times the suburban value (Table 10). Air lead in the
two areas was not significantly different. Lead in milk and water was
low in all samples in both areas.
45
-------
Table 10. ENVIRONMENTAL AND BLOOD LEAD,
MALES, AGES 14-18
December, 1972 - May, 1973
Number of subjects
Pb-B x 5 , ug/Il
Air Pb, ug/M3,
Nov - June
2
Dustfall, mg/M /mo
Apr - May
Milk, pg/ml
Water, ;ag/nl
Suburban
17
20.7
± 0.9
0.29
3.0
<.04
<.01
Urban
8
25.0*
± 2.8
0.43
11.4**
<.04
< .01
*p<.05;** p <.01 by t test.
In the study of 15 individual homes, the homes of 8 urban high school
students (Pb-B 16.3), had significantly higher lead in yard dirt than
did the homes of 7 suburban students (Pb-B 13.1), as listed in Table 11,
46
-------
Table 11. HOUSEHOLD LEAD AND BLOOD LEAD,
AGES 14-18, 1973
Number of subjects
Pb-B, ug/dl
Soil, mean, ppm
Boot tray, ppm
Housedust, ppm
Suburban
7
13.1
89.6
+ 10.8
196.0
+ 80.9
339.6
+ 77.5
Urban
8
16.3
863.5
+ 243 *
254.2
+ 104
361.3
+ 36.4
* p < . 02 by _t test. Values given as mean + SE.
In the high school age, correlation of Pb-B with lead in soil and house
dust was positive although not statistically significant for the small
size of the sample as shown in Table 12. Correlation of Pb-B with lead
in yard dirt (r = 0.31) and with lead in house dust (r = 0.29) was similar.
47
-------
Table 12. HOUSEHOLD LEAD, CORRELATION WITH
BLOOD LEAD, AGES 14-18
Soil
Housedust
Boot tray
Milk
Water
£
.31
.29
-.04
.00
.00
Proximity to Traffic as Related to Blood Lead and Soil Lead -
The home address of each of the urban students, ages 10-18, was
plotted on a metropolitan map and the distance from a major trafficway
was plotted. In the northcentral area of Omaha, 6 trafficways are de-
fined by the City Traffic Department as carrying more than 3500 vehicles
per lane per day. As shown in Table 13, the 9 students living within
750' of a trafficway had a significantly higher mean Pb-B than the 18
students living at or beyond 750' (27.3 vs 21.0; p <.02).
48
-------
Table 13. PROXIMITY TO TRAFFIC AND BLOOD LEAD,
URBAN STUDENTS, AGES 10-18
Number of subjects
Pb-B, ,ug/dl
< 750'
9
27.3 +2.6
"5-7501
18
21.04+1.3*
The households of 27 urban students, ages 10-18, were categorized as
within or beyond 750 feet from a street carrying more than 3500 cars/
lane/day. The difference in Pb-B is significant.
* p<.02, Student's t test.
In the group of 24 urban students, ages 10-12 and 14-18, the best
correlation of Pb-B with an environmental source was with lead in
yard dirt. The lead content of yard dirt, in turn, was linearly related
(r =-.42) to the residential distance from traffic as shown in Figure 16.
49
-------
10000..
o
en
2
o
tf
2000
1000
500
100
--,. 0
1000 2000
DISTANCE FROM TRAFFIC IN FEET
3000
4000
Figure 16. Lead in yard dirt and residential distance from traffic. There
is a significant correlation (r =-42; p <.05) of the lead content of yard
dirt (mean of 4 samples) with residential proximity to traffic for the houses
of 24 urban students, ages 10-18, studied in 1973. Traffic is defined as
any of the streets in northcentral Omaha with over 3500 vehicles/lane/
day.
B. LEAD AND IRON DEFICIENCY
Urban-Suburban Differences in Blood Lead, Hematologic Indices and
2,3 PPG:
As shown in Table 14, the urban black preschool children had significant-
ly higher Pb-B (22.3 vs 14.4 jig/dll and lower hemoglobins (12.3 vs 12.7
gm%) than their suburban counterparts. There was no difference in serum
iron (Fe), iron binding capacity (TIBC), or percent saturation of iron
binding capacity (% Fe Sat).
50
-------
Table 14. BLOOD LEAD AND HEMATOLOGIC INDICES
Urban-Suburban Differences
Ages 2-5 Years
Pb-B,
ug/dl
Hgb,
gm%
Hct,
%
Fe
TIBC,
ug/dl
% Fe Sat.
2 , 3 DPG ,
uM/L
Urban
22.3
+ 0.6
(122)
12.3
+ 0.07
(122)
36.7
+ 0.2
(122)
93
+ 6
(107)
363
+ 6
(102)
25.3
+ 0.8
(101)
4.70
+ 0.11
(103)
Suburban
14.4
+ 0.6
(31)
12.7
+ 0.13
(31)
37.4
+ 0.3
(31)
94
+ 3
(31)
366
+ 8
(31)
25.4
+ 1.3
(31)
3.95
+ 0.16
(31)
P
< .001
<.01
n.s.
n.s.
n.s.
n .s.
<.001
All values given as mean +_ SE: numbers of subjects in parentheses;
p derived from Student's t test.
51
-------
The urban children also had significantly higher concentrations of red
cell 2,3 DPG (4.70 vs 3.95/tM/L), an observation not completely
explained by the differences in hemoglobin. Correlation of 2,3 DPG
with hemoglobin was somewhat higher, r = 0.36; p
-------
Table 16. CORRELATIONS OF BLOOD LEAD, SERUM IRON,
AND HEMATOLOGIC INDICES
Urban Children, Ages 2-5 Years
Pb-B
Fe
% re Sat.
Hgb
Hct
Fe
+
% Fe Sat.
+
+
Hgb
+*
+
+
Hct
+
+
+
+
2 , 3 DPG
0
0
0
-
-
Significant (p < 0.05) correlations are labelled as positive (+) or nega-
tive (-). Correlations not significant at the 95th percentile are labelled
0. * The correlation of Pb-B with Hgb was positive, r = 0.19 , although
significant at only p< 0.1.
Hematologic Indices in Fe Deficiency with High and Low Pb-B
The possible augmentation of Fe deficiency anemia by moderate eleva-
tion of the Pb-B was investigated by comparison of the hematologic
indices of iron deficient children (Fe< 80) with Pb-B below and above
20 >ig/dl. There was no significant difference in the mean Hgb, Hct,
MCV, MCH, MCHC, or 2.3 DPG of the 29 Fe deficient children who
had Pb-B < 20 jjg/dl and that of the 17 Fe deficient children with Pb-B
20-40>ig/dl.
53
-------
Serum Fe Response with High and Low Pb-B
The therapeutic response of serum Fe of 14 low lead children in
Group A (Fe< 80, Pb-B < 17. 5) to 4 months of oral FeSo. is compared
with that of the 9 in the high lead Group B (Fe< 80, Pb-B 17.5 - 40)
in Figure 17. The increase of the serum iron was almost identical in
Group A where Pb-B increased from 13.9 + 0.5 to 20.9 + l.Sjig/dl
and in Group B, where Pb-B increased from 18. 4^1. 6 to 25. 7 + 1. 8
jug/dl.
3. 100-
E
a
& 50
Pb-B Level and Response to FeSo4
• Jan. x July
10
15 20
Pb-B pg/dl
25
Figure 17. Response of serum Fe of Group A (N = 14, Fe <80, Pb-B< 17.5)
and Group B (N =9, Fe <80, Pb-B< 17.5) after 4 months of oral Fe therapy.
54
-------
Serum Iron and Hemoglobin: Response to Therapy vs Placebo
The response to oral iron supplementation was compared for the 23
children who received 200 mg FeSo^ 5 days per week, March through
June (Groups A & B) and for the 7 children who received only 2 mg FeSo4
for the same time (Group C). Although the iron treated children had
slightly higher levels of iron and hemoglobin, neither of these differen-
ces was significant (Figures 18 and 19).
120
100
O
o
cr
D
cr
UJ
80
60
40
PLACEBO
FES04
JAN
JUL
MONTHS
Figure 18. Oral iron vs placebo: January to July change in serum Fe
(mean + SE) in 23 children receiving FeSo^, 200 mg/d, 5 days per week,
March through June, and in 7 children receiving placebo.
55
-------
13
12
\
5
O
z
QQ
O
O II
UJ
I
10
PLACEBO •
FES0 •
I-
JAN
JUL
MONTHS
Figure 19. Oral iron vs placebo: January to July change in Hgb (mean +
SE) in FeSo4 treated (N =23) and placebo (N =7).
Effect of Iron Supplementation on Blood Lead
Comparison of the seasonal change in Pb-B of the 14 low lead (Group A)
and 9 high lead children (Group B) given 200 mg FeSo4 with that of the 7
receiving only a 2 mg dose (Group C) for 4 months, as graphed in Figure
20, showed no difference in the seasonal increase of the blood lead.
56
-------
o
LJ
O
o
m
LJ
o
40
30
10
PLACEBO
FES04
JAN
JUL
MONTHS
Figure 20. Oral iron vs placebo: change in blood lead. January to
July change in Pb-B in 9 high lead and 14 low lead children treated
with FeSo^ March through June, and in 7 children receiving placebo.
The data comparing the entire treated group (A and B) with the placebo
Group C is given in Table 17:
57
-------
Table 17. Fe THERAPY VS PLACEBO: EFFECT ON SERUM Fe
AND Pb-B IN IRON DEFICIENCY
Group
A & B
FeS04, 200 mg
x 4 months
N = 23
C
FeSo4, 2 mg/d
x 4 months
N = 7
Significant
Difference
p < .05*
Serum Fe
Jan July ^
57.7
+ 3.5
59.4
+12.2
No
101.1
+ 6.5
91.6
+11.6
No
42.6
+ 6.7
33.9
+13.1
No
Pb-B
Jan July £^
18.4
+ 1.6
26.4
+ 3.0
Yes
25.1
+ 1.8
30.7
+ 3.4
No
6.7
+1.1
4.2
+4.2
No
Serum iron (Fe), blood lead (Pb-B), and the January to July difference
(&), all as jjg/dl, mean +_ SE in preschool children given oral ferrous
sulfate, 200 mg or 2 mg/day, March through June.
* A+B vs C.
Table 18 details the response of 7 pairs of children matched for initial
levels of serum Fe (+^ 20) and Pb-B (+ 8) to the double blind administrat-
ion of either 200 mg or 2 mg of FeSo4 given at noon, 5 days per week for
the four months, March through June. In 6 of 7 pairs, the seasonal in-
crease in lead was greater in the treated group (p = .06 by sign test for
matched pairs). Iron therapy was associated with a greater decrease in
serum transferrin in 5 of 6 pairs and with a greater increase in hemoglo-
bin in 5 of 5 pairs.
58
-------
Table 18. BLOOD LEAD AND IRON THERAPY
Seasonal Response in 7 Pairs with Double Blind
Administration of Fe or Placebo (P)
1. Fe
P
A Fe - A P
2. Fe
P
A Fe - A P
3. Fe
P
A Fe - A P
4. Fe
P
A Fe - A P
Pb-B ;ag/
Jan
39.1
41.5
+ 1.4
20.6
22.3
+ 1.6
22.7
28.8
+ 2.0
21.3
30.8
+ 3.6
ai
June
38.2
39.2
23.3
23.4
24.9
29.0
24.3
30.2
Fe jug/dl
Jan June
89 90
119 88
-20
38 132
58 123
+29
66 86
68 146
-58
23 51
23 72
-21
TIBC jjg/dl
Jan June
316 286
326 296
-62
284 338
276 292
+32
398 320
342 324
-50
312 256
274 320
-102
Hgb gm%
Jan June
11.2 10.7
11.3 10.4
+0.4
12.4
11.8 13.2
11.9 13.7
11.6 12.5
+0.9
11.1 11.9
12.1 12.5
+0.4
en
ID
-------
Table 18 (continued). BLOOD LEAD AND IRON THERAPY
5. Fe
P
A Fe - & P
6. Fe
P
& Fe - A P
7. Fe
P
A Fe - b. P
& Fe> & P
P
Pb-Bjjg/dl
Jan June
29.6 37.5
21.6 26.5
+ 3.3
24.5 29.4
23.0 20.4
+ 7.5
17.5 20.1
17.1 46.1
-26.4
6/7
0.062
Fe jjg/dl
Jan June
59 63
50 74
-20
72 151
71 74
+76
27 66
36
2/6
n.s.
TIBC >ig/dl
Jan June
346 278
272 250
-36
336 258
328 222
-174
196
336 254
1/6
0.109
Hgb gm%
Jan June
12.9 13.3
12.0 11.7
+0.7
11.6 12.8
11.2 12.0
+0.4
12.9
9.5 11.6
5/5
0.031
CT)
O
-------
C. RED CELL METABOLISM
1. Enzymes of Red Cell Glycolysis
G-6-PD, 6-PGD, and GSH are of higher concentration in young red cells
than in older cells and are recognized as indices of cell age 26,27^
2,3 DPG is not a reliable index of cell age and varies with multiple
factors such as hemoglobin, oxygen saturation and serum phosphorus.
The data in Table 19, is from 79 students, ages 10-18, without G-6-
PD deficiency with Pb-B above and below 20^ig/dl. There is a signifi-
cant increase of G-6-PD and 6-PGD and a significant decrease in GSH
in those subjects with a Pb-B_£: 20 jag/dl. There was no significant
difference in 2,3 DPG, hematocrit, hemoglobin, red cell indices, reti-
culocytes, or haptoglobins between the high and low lead group.
Table 19. BLOOD LEAD AND RBC ENZYMES REFLECTING
CELL AGE, AGES 10-18
G-6-PD
6-PGD
GSH
HCT
2 , 3 DPG
Pb-B < 20
9.68 + 0.20
(67)
7.57 + 0.16
(67)
6.11 + 0.13
(68)
42.2 + 0.36
(68)
5.08 +0.16
Pb-B;>20
11.08 + 0.68
(11)
8.50 + 0.43
(11)
5.56 + 0.28
(ID
41.8 +0.61
(11)
5.30 +0.37
P
< .05
<; .05
<.05
n.s.
0.1
The values in this and the next two tables are expressed as 2,3 DPG
>aM/ml rbc44, G-6-PD, and 6-PGD TU/gm Hgb47, and GSH uM/gm Hgb,
mean + SE.
61
-------
Since GSH is normally increased in the young red cell, the decrease
in these subjects suggests a specific depression of GSH by lead, as
noted previously in lead workers with increased Pb-B^ ^ .
In 29 black males, ages 10-12, with G-6-PD deficiency, the difference
in the enzyme activity between those with Pb-B below and above 20jag/il,
as shown in Table 20, was not significant. The levels of G-6-PD,
6-PGD and GSH are intrinsically lower in G-6-PD deficiency and were
not stimulated by modest increases in Pb-B. There was no significant
difference in hemoglobin, hematocrit, red cell indices, reticulocytes
or haptoglobins between the low lead and high lead G-6-PD deficient
boys.
Table 20. BLOOD LEAD AND RBC ENZYMES REFLECTING
CELL AGE
G-6-PD Deficient Black Males,
Ages 10-12
G-6-PD
6-PGD
GSH
2,3 DPG
Pb-B -i 20
0.89 + 0.14
(13)
8.63 + 0.49
(13)
4.39 + 0.19
(13)
5.26 + 0.36
(12)
Pb-B -^20
0.69 +0.12
(16)
8.72 + 0.46
(16)
4.26 + 0.18
(16)
5.48 +0.25
(15)
All values given as mean + SE in the same units as Table 19. No signi-
ficant difference in mean of the two groups on 1 tailed _t test. Number
of subjects in parentheses.
Affinity of Lead for young and Old Red Cells
The affinity of lead, on in vitro incubation of whole blood with lead at
100 jug/fcl, for the top 10% fyoui
ized red cells is given in Table
100 pg/dl, lor the top 10%_(xqung cells) and bottom 10% (old) of heparin-
£• A .
62
-------
Table 21. LEAD IN YOUNG AND OLD RBC
Subject
A
B
C
D
Hct%
49
41
37
40
Pb-B
top 10% rbc
(young cells^
105.2
76.5
82.0
78.0
Pb-B
bottom 10% rbc
'old cells^
89.2
60.7
67.2
78.7
There was a greater concentration of lead in the top layer of red cells
in 3 of the 4 assays. The assay was done after reconstitution to the
original hematocrit of the whole blood. Normally, the bottom layer of
cells is denser with a lower hematocrit than the mean. Reconstitution
to the mean or higher hematocrit should thus result in a higher concen-
tration of lead unless there were differences in affinity for lead between
the young cells of larger volume and larger surface area at the top and
the shrunken older cells at the bottom.
2. Red Cell Membrane Na/K ATPase
A total of 65 blood samples were assayed for total and sodium-potass-
ium adenosine triphosphatase (Na/K ATPase) activity of the rbc mem-
brane. The subjects had a mean Pb-B of 22.7^g/dl with a range of
4.9 - 92; all but 4 were below 40^g/dl. The values in Table 22 are
those of the 3 age groups plus values obtained in 2 lead workers. Total
ATPase activity for the entire group was 1.57 + 0.05 mM phosphate
liberated/hour/mg protein 'mean + SE). Na/K ATPase or oubain inhibited
enzyme activity was 0.93 mM + 0.30.
Within the total group there was no consistent correlation of total or
Na/K ATPase activity with age, race, or hematocrit. No sex correlation
had been noted, but the subjects were predominantly male. Serial
assays of enzyme activity of individuals were relatively constant over
63
-------
several months time. Although enzyme activity has been said to be
increased in the young cell, the enzyme activity of cell membranes
from 13 G-6-PD deficient boys, ages 9-14, did not differ significantly
from that of the 7 urban black males of the same age, and these values
are grouped with ages 10-18 in Table 22.
Table 22. BLOOD LEAD AND RED CELL MEMBRANE
Na/K ATPase
10-18 yi
N =49
2-5 yr
N = 5
Adult
N =9
Lead
workers
Total
N =65
Pb-B
>jg/dl
18.5 +1.2
37.0 + 1.8
27.3 + 2.6
(1)92.0
(2)81.0
22.7 + 1.7
Hct
%
42.0 + 0.5
36.5 + 0.8
45.1 +0.6
50.0
46.0
42.1 + 0.3
ATPase
Total Na/K
1.69 +0.04
1.32 +0.06
1.24 + 0.06
0.80
0.87
1.57 + 0.05
1.03 + 0.04
0.73 + 0.06
0.70 +0.06
0.13
0.31
0.93 + 0.04
All values given as mean + SE. ATPase is measured as ^aMoles phosphate
liberated per mg protein per hour.
Tn the 34 samples with Pb-B below 20 jig/dl, total ATPase activity was
1.67 + 0.39, Na/K activity was 1.04 + 0.25. In 27 samples with the
Pb-B of 20-40jug/dl, there was no significant depression of total ATPase
activity, but Na/K ATPase (oubain inhibited activity) was significantly
decreased to 0.89 + 0.28 mM, p < . 05 (Table 23).
64
-------
Table 23. Na/K ATPase OF RED CELL MEMBRANE
BLOOD LEAD< 20 VS 20-40
Blood Leadjjg/dl
< 20 20-40
N = 34 N=27
N =4
Na/K ATPase
Total ATPase
1.04*
+ 0.04
1.67
+ 0.07
0.89*
+ 0.05
1.53
+ 0.07
0.40
+ 0.10
1.02
+ 0.09
*p -s . 05 by _t for Na/K ATPase of Pb-B < 20 |ig/dl vs Pb-B 20-40
The correlation of Na/K ATPase activity with blood lead for the 5 popu-
lations is plotted in Figure 21. There is an inverse linear correlation,
r = -.56, p< .05, with the regression coefficient of Na/K ATPase =
1.21 - 0.01 Pb-B. Elimination of the data from the 2 lead workers does
not significantly alter the correlation or the regression equation.
65
-------
1.8
IS Z 1.2
4
X.
<
Z
NA/K ATPASC -121 — OOI Pb
N -65
• -ADULTS (9)
Q- PRESCHOOL CHILDREN (5)
O- LEAD WORKERS (2)
O-SCHOOL CHILDREN
G6PD NORMAL (30)
C-SCHOOL CHILDREN
G6PD DEFICIENT CI4>
20
30 40 50 60 70 80
JJGM Pb/100 ML WHOLE BLOOD
90 100
110
120
Figure 21. Correlation of blood lead with red cell membrane Na/K ATPase
as assayed in 65 samples. The number of G-6-PD normal school children
actually totals 35, not 30, as indicated on the label. The correlation
of enzyme activity and blood lead, r = -0.56 is significant (p <.05).
66
-------
SECTION VIII
DISCUSSION
A. ENVIRONMENTAL STUDIES
One of the most striking aspects of the study is the sharp drop in air
lead in urban Omaha. By 1974, the lead in urban air was lower than
the suburban values of 1970-1972 (Figure 10, p. 33). Although indust-
rial air filters might be expected to remove relatively more of the large
particles, there was less of a decrease in dustfall lead than in air lead
(Figure 12, p. 36). This suggests that recirculation of the large part-
icles of lead in street dirt and soil may be a factor in persistence of
high dustfall lead even after a reduction in lead emission.
The fallout from air lead to dustfall and dirt shows a consistently better
correlation with the urban-suburban differences in blood lead in all
ages of children than does air lead itself37 ,40,53^ jn Omaha, lead in
water and milk are insignificant. Lead in housedust, in this study,
relates to the blood lead only in the preschool age group where mouthing
behavior and long hours of playing on the floor might be expected to
increase the ingestion of house dust to the levels of a significant load
per unit of body weight.
Because of the drop in environmental lead we reached ahead to blood
lead studies done in 1975 (ES 00939) and examined Pb-B as comparably
assayed (macro method, spring season) in students at an urban junior
high school in 1971, 1973, and 1975 (Table 2). There is a significant
decrease in Pb-B from 1971 to 1975 and from 1973 to 1975 that parallels
the decrease in air and dustfall leads:
67
-------
Table 24. BLOOD LEAD, 1971-1975,
URBAN JUNIOR HIGH SCHOOL, AGES 12-14
May, 1971
25.1**
+ 1.1
(32)
March, 1973
21.0*
+ 2.4
(13)
March, 1975
16.1
+ 0.9
(17)
** p ^.001, 1971 vs 1975
* p<.05, 1973 vs 1975
All blood leads done by macro method of Farrelly & Pybus on hepar-
inized venous samples. Values are the mean + SE, with the num-
bers of subjects given in parentheses. Data from this school was
selected for analysis because of the consistency of methodology
(macro assay) and the comparable season of sampling.
By _t test there is a significant difference between 1971 and 1975
(p <.001) and 1973 and 1975 (p <.05).
Although the annual trend in Pb-B and its correlation with general en-
vironmental lead in air and dustfall can be seen, we were unable to
relate the monthly and seasonal fluctuations in Pb-B to the monthly
variations in air lead or dustfall lead. Soil lead was obtained in the
standard two-inch core to avoid rapid fluctuations. To better evaluate
a seasonal correlation of Pb-B with environmental lead, monthly samples
of the top layer of soil or of street dirt would be more appropriate.
Despite the critical role of deteriorated housing as a cause of clinical
lead poisoning in young children, the urban-suburban difference in
housedust lead was much less obvious than the urban-suburban differ-
ences in dustfall and soil lead. High levels of dustfall and soil lead
clearly related to industrial sources as shown by the comparison of the
2 urban schools. Dustfall lead at the urban high school averaged 9.4
mg/M /30 day collection. The urban grade school is in a comparable
mixed residential-commercial area but is immediately adjacent to a
battery plant - an industrial source that presumably accounts for a mean
of dustfall lead of 22.7 mg/M2/30 d. All soil lead samples adjacent
68
-------
to this plant were below 2000/jg/gm, and far below the levels of soil
lead in smelter studies such as that in El Paso7.
Traffic as well as industry can be incriminated as a significant lead
source even though the Omaha criterion of 3500 cars/lane/day is far,
far below the traffic density in New Jersey as studied by Caprio et al54.
Children living within 750 feet of an Omaha trafficway had a mean Pb-B
of 27.3 vs 21.0 for those living at a greater distance and proximity to
traffic related linearly to soil lead.
This environmental study in Omaha supports multiple surveys^'7~^
conclusively documenting the higher lead load of the urban child. By
school age, this increase in lead burden relates directly to the general
environmental exposure to lead in proximity to traffic and industry.
B. Red Cell Metabolism and Iron Deficiency
This study of the effect of oral iron on the Pb-B was designed as a
clinical trial of the experimental evidence that iron deficiency enhances
the absorption and transport of lead . The study can be criticized
on the grounds that, 1) the subjects had only a low serum iron but no
real anemia, 2) the daily dose of 200 mg ferrous sulfate is less than the
therapeutic dose of 600 mg/day and was given only 5 days per week,
3) the response is obscured by the known seasonal fluctuations in both
iron and lead. Despite these valid criticisms, the conditions of the
study are those that might be accomplished in any broad scale test of the
hypothesis. The fact that absolutely no decrease in Pb-B occurs on
giving oral ferrous sulfate, 200 mg/d, 5 days a week, for 4 months
supports previous studies showing no effect of iron on pica or on blood
lead", and allows two other inferences. First, since Pb-B increases
as the serum iron increases and as the hemoglobin increases, the corr-
ection of Pb-B for anemia remains physiologically valid. Second, the
binding of lead by ferritin and transferrin may affect the kinetics of lead
absorption in different ways at different concentrations and could explain
the failure of physiologic levels of iron supplementation to interfere with
lead absorption. Although saturation of ferritin and transferrin with iron
may displace lead from a common transport system; iron supplementation
short of saturation might facilitate lead transport by enzymatic induction
or stimulation of another common carrier mechanisms-SB ^
69
-------
C. Red Cell Survival and Na/K ATPase
This is the first report of a linear inhibition of Na/K ATPase of the red
cell membrane. As the blood lead increases within the normal range
from 0 to 40/ig/100 ml59-61t ALA dehydratase of the red cell is also
linearly depressed; the health effects of this are unknown since ALA-D
has no known metabolic activity in the mature red cells. Membrane
Na/K ATPase, in contrast, is a critical determinant in cation permea-
bility and red cell surviva^S-SO. Almost 50 years ago, Aub documented
that in vivo and in vitro exposure to lead causes a rapid loss of inter-
cellular cation resulting in small, shrunken, hyperosmotic red cells^ ,
an effect similar to the inhibition of Na/K ATPase. In vitro exposure of
rbc to lead evokes a rapid efflux of K+ that occurs at rates so much faster
than the maximum pumping capacity of the cell that it is considered a
passive leak of potassium^. Since the potassium leak is reversed by
EDTA, it is postulated that it represents the binding by lead of surface
reactors on the membrane^, in vitro exposure to lead also causes a
decrease in ability to pump sodium out of the red cell; unlike the potass-
ium leak, this inhibition of the sodium pump is not reversed by treatment
with EDTA and may represent lead binding to Na/K ATPase, or at least
to a site other than the outer membrane.
Hasan and Hernberg were the first to show a decrease in Na/K ATPase,
but not total ATPase, of industrial lead workers at Pb-B of 60-80>ig/dl
31i32_ Secchi and Alessio also found a lower Na/K ATPase in lead
workers and subsequently reported decreases in membrane Na/K ATPase
in urban men with increased occupational exposure to lead such as
traffic policemen^. We modestly attribute our demonstration of a linear
inhibition to improved methodology of the enzyme assay, since we were
unable to demonstrate inhibition, even at high Pb-B, employing these
previously reported methods of enzyme assay.
Since Na/K ATPase is a critical enzyme in integrity of the red cell mem-
brane, we then investigated the evidence for decreased red cell survival
in the same range of values. Red cell enzymes are well accepted indices
of red cell age and of red cell survival. Although G-6-PD and 6-PGD are
less sensitive indicators of red cell age than hexokinase or aldolase,
demonstration of an increased value in children with a Pb-B above 20 jug/
dl may be evidence of a decreased rbc survival in association with alter-
ed cation transport of the rbc membrane. Under varying experimental
circumstances, lead may inhibit or enhance G-6-PD activity. Because
G-6-PD activity did not change with Pb-B in G-6-PD deficient subjects,
an enzyme increase relating to increased rbc turnover might be postulated.
Since inhibition of GSH at low levels of Pb-B, similar to that recently
70
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reported by Taniguchi°^, might be expected to depress G-6-PD activity,
the intrepretation is proposed that increased G-6-PD represents acceler-
ated turnover. Further studies of the effects of lead on Na/K ATPase and
also on Ca++ ATPase of white cells and platelets would seem indicated.
71
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SECTION IX
REFERENCES
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5. Rabinowitz, M., G. Wetherill, and JKopple. Absorption,
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of Missouri - Columbia (Presented at the 9th Annual Conference
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7. Landrigan, P.J., S.H. Gehlbach, B.F.Rosenblum, J.M.
Shoults, R.M.Candelaria, W.F.Barthel, J.A.Liddle, A.L.
Smrek, N.W. Staehling, and J.F.Sanders. Epidemic Lead
Absorption Near an Ore Smelter. New Eng J Med 292; 123-
129, January, 1975.
8. Pinkerton, C., D.I.Hammer, T.A.Hinners, J.L.Kent, V.
Hasselbad, J.V.Lagerwerff, and E.S.Ferrand. Trace Metals
in Urban Soils and Housedust. Environment, November 16,
1972.
72
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REFERENCES (continued)
9. Sayre, J.W., E. Charney, J. Vostal, I.E. Press. House
and Hand Dust as a Potential Source of Childhood Lead
Expsoure. Am J Dis Child 127; 167-170.
10. Joselow, M.D., and J.D.Bogden. Lead Content of Printed
Media. Am J Public Health 64_: 238-240, 1974.
11. Shapiro, I.M., G.H.Cohen, H.L. Needleman, and O.C.
Tuncay. The Presence of Lead in Toothpaste. J A D A
86; 394-395, 1973.
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1973.) p 763-772.
13. Erenberg, G., S. Rinsler, and 8. Fish. Lead Neuropathy and
Sickle Cell Disease. Pediatrics J54; 438-441, October, 1974.
14. Anku, V.D., and J.W. Harris. Peripheral Neuropathy and
Lead Poisoning in a Child with Sickle Cell Anemia. J Pediat
8^: 337-340, 1974.
15. Mclntire, M.S., and C.R. Angle. Air Lead: Relation to Lead
in Blood of Black School Children Deficient in Glucose-6-
Phosphatase Dehydrogenase. Science 177: 520-522, August,
1972.
16. Angle, C.R. and M.S.Mclntire. More on the Relevance of the
Concentration of Lead in Plasma vs that in Blood. J Pediat
85; 286-287, 1974.
17. Six, K.M., and R.A. Goyer. The Influence of Iron Deficiency
on Tissue Content and Toxicity of Ingested Lead in the Rat.
J Lab Clin Med 79: 128-135, 1972.
73
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REFERENCES (continued)
18. Kochen, J., and Y. Greener. Interaction of Ferritin with
Lead and Cadmium (abst). Pediat Res 9_: 323, 1975.
19. Kochen, J., and Y. Greener. Lead Binding by Transfer-Tin
(abst). Pediat Res 9_: 323, 1975.
20. DeBruin, A. Certain Biological Effects of Lead Upon the
Animal Organism. Arch Environ Health 23: 249-264, October,
1971.
21. Hasan, J., and S. Hernberg. Interactions of Inorganic Lead
with Human Red Blood Cells. Work - Environ Health 2\ 26-
44, 1966.
22. Qazi, Q.H., and D.P. Madahar. A Simple Rapid Test for
Lead Poisoning. J Pediat 79; 805-808, 1971.
23. Riordan, J.R., and H. Passow. Effects of Calcium and Lead
on Potassium Permeability of Human Erythrocytes. Biochim
et Biophys Acta 249; 601-605, 1971.
24. Hernberg, S., M. Nurminen, and J. Hasan. Nonrandom
Shortening of Red Cell Survival Times in Man Exposed to
Lead. Environ Res J_: 247-261, 1961.
25. Vincent, P.C., and C.R. Blackburn. The Effects of Heavy
Metal Ions on the Human Erythrocyte. I. Comparison of the
Action of Several Heavy Metals. Austral J Exp Biol 36; 471-
478, 1958.
26. Komazawa, M., and F.A.Oski. Biochemical Characteristics
of "Young" and "Old" Erythrocytes of the Newborn Infant.
J Pediat 8J7_: 102-106, 1975.
27. Chapman, R.G., and Schaumberg. Glycolysis and Glycolytic
Enzyme Activity of Aging Red Cells in Man. Brit I Haemat 13:
663, 1967.
74
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REFERENCES (continued)
28. Macdougall, L.G., I.MJudisch, and S.B.Mistry. Red Cell
Metabolism in Iron Deficiency Anemia. II. The Relationship
Between Red Cell Survival and Alterations in Red Cell
Metabolsim. J Pediat 76; 660-670, 1970.
29. Dahl, J.L. , and L.E.Hokin. The Sodium Potassium Adenosine
Triphosphatase. Ann Rev Biochem £3: 327-356, 1974.
30. Glader, B.E., N. Portier, M.Albala, and D. Nathan.
Congenital Hemolytic Anemia Associated with Dehydrated
Erythrocytes and Increased Potassium Loss. New Engl I Med
291; 491-496, 1974.
31. Hasan, J., V. Vihko, and S. Hernberg. Deficient Red Cell
Membrane Na/K ATPase in Lead Poisoning. Arch Environ
Health 14_: 313-318, 1967.
32. Hernberg, S., V. Vihko, and J. Hasan. Red Cell Membrane
ATPase in Workers Exposed to Inorganic Lead. Arch Environ
Health 14_: 319-324, 1967.
33. Secchi, G.C., L. Alessio, and G. Cambiaghi. Na/K ATPase
Activity of Erythrocyte Membranes. Arch Environ Health 27;
399, 1973.
34. Passow, H. Passive Ion Permeability of the Erythrocyte
Membrane. Progr Biophys and Molec Biol 19; 423-467, 1969.
35. Hexum, T.D. Studies on the Reaction Catalyzed by Transport
(Na, K) Adenosine Triphosphatase. I. Effects of Heavy Metals.
Biochem Pharmacol_23: 3441-3447, 1974.
36. Goyer, R.A. Lead and the Kidney. Current Topics Pathol 55:
147-176, 1971.
75
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REFERENCES (continued)
37. Mclntire, M.S. and C.R.Angle. Increased Red Cell Lead in
G-6-PD Deficient Urban Black School Children. Paediatri-
cian 1,: 114-124, 1973.
38. Mid United States City Dustfall Results, R. Horton, NAPCA,
April, 1968.
39. Data from the Metropolitan Area Planning Agency, University
of Nebraska at Omaha.
40. Angle, C.R., M.S. Mclntire, and G. Vest. Blood Lead of
of Omaha School Children - Topographic Correlation with
Industry, Traffic, and Housing. Neb St Med J 60; 97-102,
1975.
41. Delves, H.T. The Determination of Trace Metals and Their
Significance in Clinical Chemistry. Atomic Absorption
Newsletter l£: 50-54, 1973.
42. Farrelly, R.O, and J. Pybus. Measurement of Lead in Blood
and Urine by Atomic Absorption Spectrometry. Clin Chem
15; 566-574, 1969.
43. Haelen, P., G. Cooper, andC. Pampel. The Determination
of Lead in Evaporated Milk by Delves Cup Atomic Absorption
Spectrometry. Atomic Absorption Newsletter I: 1-3, 1974.
44. Maeda, N., H. Chang, R. Benesch, and R.E. Benesch.
A Simple Enzymatic Method for the Determination of 2,3
diphosphoglycerate in Small Amounts of Blood. New Eng J
Med.284: 1239-1242, 1971.
45. Vellar, O.D., F.O.Winther, K. Horthe, and A. Lystad.
Upper Respiratory Tract Infections in Relation to Iron Medi-
cation in Healthy Students. J Laryngol Otol 88; 773-783.
76
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REFERENCES (continued)
46. Dawson, E.B., H.A.Croft, R.R.Clark, andW.J. McGarity.
Study of Seasonal Variations in Nine Cations of Normal
Placentas. Am J Obst Gynec 102; 354-361, 1968.
47. Beutler, E. R. Red Cell Metabolism. Grune and Stratton,
New York, 1971.
48. Murphy, J.R. Influence of Temperature and Method of Centri-
fugation on the Separation of Erythrocytes. J Lab Clin Med
81: 334-341, 1973.
49. Sen, A.K., and R.L. Post. Stochiometry and Localization
of Adenosine Triphosphate Dependent Sodium and Potassium
Transport in the Erythrocyte. J Biol Chem 239: 345, 1964.
50. Post, R.L., C.R. Merritt, C.R. Kinsolving, and C.D.
Albright. Membrane Adenosine Triphosphatase as a Partici-
pant in the Active Transport of Sodium and Potassium in the
Human Erythrocyte. J Biol Chem 235; 1796, 1960.
51. Batolska, A., and H. Marinova. Glutathione Changes in an
Employee of an Ore Smelting Works. Arch des Malad Prof 31;
117-122, 1970.
52. Bonsignore, D., C. Cartasegna, V. Ardoino, and C. Vergnano.
Reduced Glutathione in Saturnisme. Lavoro Umano 19; 97,
1967.
53. Angle, C.R., and M.S. Mclntire. Lead in Air, Dustfall,
Soil, Housedust, Milk, and Water: Correlation with Blood
Lead of Urban and Suburban School Children (Proceedings
of the 8th Annual Conference on Trace Substances in Environ-
mental Health, Columbia, Missouri, 1974.) p 23-30.
54. Caprio, R.J., H.L. Margulis, and M.M.Joselow. Lead
Absorption in Children and its Relationship to Urban Traffic
Densities. Arch Environ Health 28_; 193, 1974.
77
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REFERENCES (continued)
55. Gutelius, M.F., F.K.Millican, E.K. Layman, G.J. Cohen,
and C.C.Dublin. Nutritional Status of Children with Pica.
Pediatrics ^9: 1012-1023, 1962.
56. Ciket, M. The Uptake of Hg 203, Cu 64, Mn 54, Pb 212 by
the Intestinal Wall of the Duodenal and Heal Segment In Vitro.
Int J Clin Pharmacol 3-4; 351-357, 1970.
57. Pollak, S., J.N. George, R.C.Reba, R.M. Kaufman, and
W.H. Crosby. The Absorption of Non-Ferrous Metals in Iron
Deficiency. J Clin Investor 1470-1473, 1965.
58. Angle, C.R., and M.S.McIntire. Blood Lead of Iron Deficient
Children - Increase Following Iron Supplementation, (abst)
Ped Res^: 257, 1975.
59. Angle, C.R., and M.S.McIntire. Red Cell Lead, Whole Blood
Lead, and Red Cell Enzymes. Environ Health Perspect 1\ 133-
137, May, 1974.
60. Angle, C.R., and M.S. Mclntire. Low Level Lead and
Shortened Rbc Survival (abst). Ped Res J: 257, 1975.
61. Angle, C.R., and M.S. Mclntire. Normal Urban Increases
in Red Cell Lead and Depression of Red Cell Membrane Na/K
ATPase (abst). Ped ResjJ: 397, 1974.
62. Aub, J.C., L.T.Fairhall, A.S.Minot, and P. Reznikoff. Lead
Poisoning. Medicine 4j 1-250, 1925.
63. Taniguchi, N., T.Sato, T.Kondo, H. Tamachi, K.Saito, and
E.Takakuwa. Carbonic Anhydrase Isozymes, Hemoglobin F and
Glutathion in Lead-Exposed Workers. Clin Chim Acta 59- 29-
34, 1974.
78
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SECTION X
GLOSSARY
ATPase - adenosine triphosphatase.
2 , 3 PPG -2,3 diphosphoglycerate.
Serum iron
Fe Saturation -
Total iron binding capacity x 100%.
G-6-PD - Glucose-6-phosphate dehydrogenase.
GOT - Glutamic oxalacetic transaminase.
6-PGD - 6-phosphogluconic dehydrogenase.
GSH - Reduced glutathione.
Hct - Hematocrit.
Hgb - Hemoglobin.
MCV - Mean corpuscular volume of red cells.
Na/K ATPase - sodium-potassium activated or oubain inhibited adenosine
triphosphatase.
Pb-B - Blood lead injjg/dl.
Rbc - Red blood cell.
Serum Fe - serum iron.
TIBC - Total iron binding capacity of serum or serum transferrin.
79
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
l REPORT NO
EPA-650/1-75-003
4 TITLE AND SUBTITLE
Lead: Environmental Sources and Red Cell Toxicity
in Urban Children
3 RECIPIENT'S ACCESSION-NO
S REPORT DATE
June 1975
6 PERFORMING ORGANIZATION CODE
7 AUTHOR(S)
8 PERFORMING ORGANIZATION REPORT NO
Carol R. Angle, Matilda S. Mclntire
9 PERFORMING ORGANIZATION NAME AND ADDRESS
University of Nebraska
Omaha, Nebraska 68105
10 PROGRAM ELEMENT NO
1AAQQ5
11 CONTRACT/GRANT NO
802043
12 SPONSORING AGENCY NAME AND ADDRESS
Human Studies Laboratory
Environmental Research Center
U.S. Environmental Protection Agency
Rocoarrh Triannlp Park. North C.an'Mna ?7711
13 TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-ORD
CULL
CfTE
15 SUPPLEMENTARY NOTES
16 ABSTRACT
A comprehensive environmental study was carried out for correlation of lead
in multiple sources with the increased blood lead of urban children. In the three
age groups, 2-5, 10-12, and 14-18 years, urban children had higher blood leads
than their suburban counterparts, although the difference decreased with age. The
increased blood lead correlated with increased lead in the urban dustfall, yard soil
and boot tray lead. There was no significant urban-suburban difference in air lead,
housedust lead, available paint chips or lead in milk and water. Lead in yard dirt
and blood lead both correlated with residential proximity to traffic.
Although all blood lead were below 40 pg/dl, there was a significant linear
decrease in red cell (rbc) membrane Na/K ATPase as blood lead increased; children
with a blood lead above 20 ;ug/dl had decreased activity of rbc glutathione and
increased rbc G-6-PD and 6-PGD. The increase in the latter two enzymes supports
decreased rbc survival at the level of lead exposure of urban children. Iron
deficiency anemia showed no correlation with blood lead and the administration of
iron supplements to preschool children resulted in higher blood leads than their
matched controls.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Air Pollution, Dust, Enzymes, Phosphatase
Hemolysis, Erythrocytes, Iron deficiency
anemia, Lead (metal), Lead poisoning,
milk, soil analysis, water analysis
b IDENTIFIERS/OPEN ENDED TERMS
Environmental Lead and
Blood Lead; Urban Black
Children; (erythrocyte)
Na/K ATPase, gluthathiom
G-6-PD, 6 PGD and 2, 3
DPG; iron therapy
c COSATI I icId/Group
06P
:, 06T
3 DISTRIBUTION STATEMENT
Release Unlimited
19 SECURITY CLASS (This Report)
Unclassified
21 NO OF PAGES
90
20 SECURITY CLASS (Thispage)
llnrl acc ifioA
22 PRICE
EPA Form 2220-1 (9-73)
80
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