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OAR Box 1128 

Prepped by Ollie Stewart 

Document Number: 

76) 11-1-254 

Docket Number: 

A-83-22 

Printed 4/21/2008 11:07:13 AM Sheet 76 of 198 
)n 

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Environmental Health Perspectives 

Vol. 78. pp. 47-51 1988 

influence of Membrane Sodium Transport 

upon the Relation Between Blood Lead and 

Blood Pressure in a General Male 

population 

by Thierry Moreau,* Patrick Hannaert, 

Genevieve Orssaud, Guy Hue.,* Ricardo P. Garay,* 

jean R. Claude/ Bernard Juguet,* Bernard Festy,8 

and Joseph Lellouch* 

Five red blood cell cation transport systems (RBCTS), together with blood lead level and blood 

pressure, were measured in 129 male adult subjects who were not occupationally exposed to lead or 

subsequent to a course of treatment for hypertension. Blood lead was positively related with systolic 

blood pressure, and to a lesser degree with diastolic blood pressure. Blood lead was found 

significantly negatively related to one of the RBCTS, Na*,K* cotransport, and in addition, Na'JC 

cotransport appeared negatively related to blood pressure. Final results showed that blood lead no 

longer accounts for an increase in systolic blood pressure when Na'JC cotransport was taken into 

account; the same trend was observed with diastolic blood pressure. These findings suggest that a 

blood lead-related Na'^C cotransport impairment could explain the blood pressure increase observed 

to parallel the blood lead increase. 

Introduction 

The relationships of blood lead to blood pressure have 

been investigated in several general populations, with 

somewhat divergent published results (1,2). 

None of these studies attempted to explain the mechanisms possibly involved in these relationships; however, several works have shown that blood lead could 

inhibit one of the red blood cell cation transport systems (RBCTS): the Na+

,K+pump (3-5), and other 

reports suggest that one or more RBCTS. including 

Na+,K+ pump, could be impaired in hypertensive 

subjects (6-8). 

SERM. Unite 169. 16 Ave P.V. Couturier. 94807. Villejuif Cedex. 

France. 

+

INSERM. Unite 7. 161 rue de Sevres. 75730. Paris Cedex 15. 

France. 

tLaboratoire DASES. 88 rue de La Jonquiere, 75017. Paris. France. 

§Laboratoire LHVp. l bis rue des Hospitalieeres St. Gervais. 7.5004. 

Paris. France. 

Therefore, it was logical to test the hypothesis that the 

increase of blood pressure with blood lead could be 

explained at least partly by RBCTS impairment due to 

an effect of lead exposure as reflected in blood lead concentration. It is the aim of the present study to ininvestigate in a sample of not occupationally lead-exposed 

men the possible role ol live RBCTS. including the 

Na+.K+ pump, and the role of red blood cell Na+ and 

K

+

 content in the blood lead-blood pressure relationship. 

Materials and Methods 

Population 

The study population consisied of 129 adult men belonging to the Paris Police Administration, who were 

not occupationally exposed to lead, who were not following a course of treatment for hypertension, and 

who were aged 23 to 57 years (mean, 36.2 years). Each 
P.3 

48' MOREAUETAL. 

subject was given a standardized questionnaire asking 

for detailed alcohol and tobacco consumption, among 

other information. Several physical parameters were 

recorded and a fasting blood sample was taken for 

biochemical measurements. 

Alcohol Consumption and Smoking 

Habits 

For each subject, the daily consumption of wine, beer, 

aperitif, and spirits was recorded and converted in 

grams of alcohol per day. The smoking status was also 

noted: nonsmokers, 45 subjects; exsmokers, 24 subjects; 

current smokers, 57 subjects. For current smokers, the 

quantity of tobacco smoked per day was expressed in 

grams. 

Physical Examination 

Systolic and diastolic blood pressures were measured 

using a mercury apparatus. Three measurements were 

performed: the first after 10 min resting, the second 10 

min later (both in supine position), and the third in 

the standing position. This protocol followed the 

recommendations for blood pressure measurements of 

the British Hypertension Society (9). Individual blood 

pressure values reported (systolic or diastolic) are the 

means of these three measurements. Height and 

weight were also recorded. 

Biochemical Measurements 

The following parameters were measured on each 

blood sample. 

Blood Lead. Whole heparinized blood was diluted 

(1/10) into an ammonium nitrate solution; the 

dilution was injected into a tantalum-coated graphite 

furnace of a Jarrel-Ash 850 AA spectrometer (dry 100°C, 

ash 750°C, atomized 2,450°C). Measurements were 

performed at 283.3 nm wavelength with background 

correction and calibration by standard additions. 

Special precautions were taken to avoid contamination 

by ambient lead, reagents, and materials (heparinized 

collection tubes, Vacutainer, BD 06527). 

RBCTS and Red Blood Cell Na+ and K+ Contents. Five RBCTS were considered in fresh red cells: 

Na+,K+ pump; Na+,K+ cotransport; Na+,Li+ countertransport; and Na+

 and K+ passive permeabilities. 

Their mechanisms of action are outlined in Figure 1. 

Methods of measurement of each RBCTS are described 

in detail elsewhere (10) and can be summarized as follows. Blood samples were stored at 4°C and used within 

3 hr. After removal of plasma and buffy coat, red cells 

were washed free of Na+. The activity of the different 

transport systems was assessed by spectrophotometrically measuring Na+

 efflux in four different Na+-

free media containing: a) K+; b) ouabain; c) ouabain 

and bumetanide; and d) ouabain, bumetanide, and 

Li+. Red blood cell Na+ and K+ contents were expressed 

»>• pn'coumn i««»Sf0«i 

NiMii*: 

PtKMIOIllIt 

Nl' IT C0t««NSP0IU 

FIGURE 1. Red blood cell sodium (Na+) and potassium (K+) transport 

systems (RBCTS). 

in nmole/Lcells. Na+ and K+ passive permeabilities 

were expressed in hr1. Activity of Na+,K+ pump, 

Na+,K+ cotransport,! and Na+,Li+ countertransport 

were expressed in umole/Lcellshr. 

Results 

Blood Lead and Blood Pressure 

Blood lead levels and systolic blood pressure values 

had log-normal distributions, so that log transformations were used in statistical analysis. 

Among information obtained from the questionnaires, daily alcohol consumption appeared to be 

strongly positively related to blood lead (correlation 

coefficient r = 0.29, p < 0.01); the blood lead means ± 

SEM in nondrinkers (19 subjects), in subjects who 

drank between 1 and 50 g/day (64 subjects), in subjects 

who drank more than 50 g/day (35 subjects) were, 

respectively: 142.1 ± 8.6 ng/L, 160.8 ± 5.2 jig/L, and 

188.3 ± 8.8 jig/L. Age was weakly positively associated 

with blood iead (r = 0.14, p = 0.13). Daily tobacco consumption was not related to blood lead in current 

smokers (r = 0.01); the blood lead means ± SEM in current smokers and in exsmokers were 175.4 ± 5.9 ug/L 

and 178.5 ± 9.9 |J.g/L, significantly higher than the 

mean in nonsmokers (146.8 ± 6.2 ug/L). 

Blood lead was found to be significantly positively 

related to systolic blood pressure (r = 0.19, p = 0.05) and 

to a lesser degree to diastolic blood pressure (r = 0.17, 

p = 0.07) Taking into account alcohol consumption and 

age yielded the partial correlation coefficients 0.1? 

ip = 0.09) and 0.14 (p = 0.15), respectively; taking further 

into account body mass index did not change these 

latter results. The means of systolic blood pressure 

according to blood lead levels are shown in Table 1; they 

appear to increase about 8 mm Hg through the three 
r 

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SODIUM TRANSPORT, BLOOD LEAD, AND BLOOD PRESSURE 49 

lowest blood lead classes (lower than 120 ug/L, between 

120 to 160 |J.g/L, and those-between 161 to 200 ug/L) 

and then, at > 200 (xg/L, to reach a plateau in blood 

pressure values. As shown in Table 1, similar results 

were obtained after adjusting systolic blood pressure 

values for alcohol consumption, age, and body mass 

index. Table 1 also shows that the same trend is 

observed concerning diastolic blood pressure. 

RBCTS, Red Blood Cell Na+ and K+ 

Contents, and Blood Pressure 

The distribution of Na+,K+ pump appeared log-normal, so that statistical tests were performed after log 

transformation. 

Among RBCTS and red blood cell Na+ and K+ 

contents, only Na+, K+ cotransport was significantly 

inversely related to either systolic or diastolic blood 

pressure (r = -0.20, p < 0.05; r = -0.19, p < 0.05, respectively)- Figure 2 shows the systolic and diastolic blood 

pressure means according to Na+,K+ cotransport 

activity values. The systolic or, respectively, diastolic 

blood pressure mean increase is about 6 mm Hg or 5 

mm Hg from the highest Na+,K+ cotransport class 

(271 nmole/Lcellshr and over) where the means are 

126.0 mm Hg for systolic (83.7 mm Hg for diastolic) 

until the two lowest classes (131-200 (imole/Lcellshr 

and lower than 130 nmole/Lcellshr), in which the 

mean pressures are nearly equal: 131.9 mm Hg and 

131.1 mm Hg (respectively, diastolic 88.6 mm Hg and 

88.1 mm Hg). 

After adjustment for alcohol consumption, age, and 

body mass index (whose correlations with Na+,K+ cotransport were: r = -0.20, p < 0.05; r = -0.21, p < 0.05; 

r = 0.13, p = 0.15; respectively), the partial correlation 

coefficients between Na+,K+ cotransport and either 

systolic or diastolic blood pressure were r = -0.28 

(/xO.Ol). 

RBCTS, Red Blood Cell Na+ and K+ 

Contents, and Blood Lead 

The correlation coefficients of RBCTS and red blood 

cell Na+ and K+ contents with blood lead are listed in 

Blood pressure (mmHg) 

150 r140 -

130 

120 

110 

100 

90 

80 

70 

i I , Systolic 

I _ Diastolic 

60 \-

-130 131-200 201-270 271* 

(23) (35) (30) (26) 

Na+,K+ Cotransport (iimole/l.cells hour) 

FIGURE 2. Means of systolic (•) arid diastolic (•) blood pressure 

according to Na+,K+ cotransport activity. Means are represented 

together with their 95% confidence limits. Numbers of subjects in 

each Na+,K+ cotransport class are in parentheses. 

Table 2. It appears that only a decrease in Na+,K+ cotransport is significantly related to an increase in 

blood lead (r = -0.23, p < 0.05). The "corresponding regression coefficient indicates a 39.5 nmole/Lcellshr 

Na+,K+ cotransport decrease for a 100 (xg/L blood lead 

increase. This negative relationship remains significant after allowance for alcohol consumption, age, and 

body mass index (partial correlation coefficient, 

r= 0.19, p< 0.05). 

Table 1. Systolic and diastolic blood pressure means according 

to blood lead values. 

Blood 

lead, 

K/l 

Systolic blood pressure, 

mmHg 

Adjusted 

Mean ± SEM mean3 

Diastolic blood pressure, 

mmHg 

Adjusted 

Mean ± SEM meana 

< 121 124.7 11.9 (18)b 

121-160 126.711.7 (44) 

161-200 133.4 ± 2.2 (31) 

-JgO 130.9 ±2.4 (20) 

124.6 (17) 82.9 ± 1.9 (18) 83.8 (17) 

126.3 (40) 85.7 ± 1.4 (44) 86.4 (40) 

132.0(27) 89.8 ±2.0 (31) 88.9(27) 

129.0 (20) 88.012.8 (20) 87.4 (20) 

'Systolic and diastolic blood pressure values were adjusted for 

alcohol consumption, age, and body mass index. 

Numbers of subjects are in parentheses. 

Table 2. Correlation coefficients of blood lead levels with 

RBCTS and red blood cell Na+ and K+ contents. 

Parameter Correlation coefficient Significance 

Na*,K* pump 

NafK* cotransport 

NafLi* countertransport 

Na* passive permeability 

K* passive permeability 

Red blood cell Na* content 

Red blood cell K* content 

0.05 (124)a 

-0.23 (124) 

-0.04 (124) 

0.01 (124) 

0.09 (119) 

0.11 (124) 

-0.09(124) 

NSb 

0.02 

NS 

NS 

NS 

NS 

NS 

aNumbers of subjects are in parentheses. 

bNS, not significant at the 10% level. 
50 MOREAUETAL. 

__•_•_-_-_._-• r 

Na+,K+ Cotransport, Blood Lead, and 

Blood Pressure 

The effect of blood lead upon blood pressure 

independent of Na+,K+ cotransport was studied by the 

means of multiple regressions with either systolic or 

diastolic blood pressure as the dependent variable. 

Results concerning systolic blood pressure are summarized in Table 3, showing the decrease of the blood 

lead regression coefficients as a) blood lead alone considered as an independent variable (regression coefficient, 3.7 mm Hg/100 ug Pb/L, p = 0.05); b) alcohol 

consumption, age, and body mass index added as 

independent variables (regression coefficient, 3.0 mm 

Hg/100 ag Pb/L, p = 0.09); c) Na+,K+ cotransport 

included further in the regression equation (regression coefficient, 2.1 mm Hg/100 ug Pb/L, p = 0.21). In 

this latter case, the Na+,K+ cotransport regression 

coefficient is -3.5 mm Hg/100 nmole/L-cells-hr. 

O<0.01). 

Table 4 shows the results of the regression analyses 

performed as described with diastolic blood pressure as 

a dependent variable. The blood lead regression coefficients are: 3.0 mm Hg/100 ng Pb/L, (p = 0.07); 2.1 mm 

Hg/100 ng Pb/L, (p = 0.15); 1.4 mm Hg/100 ng Pb/L, 

(p = 0.32). In the latter regression equation, the Na+,K+ 

cotransport regression coefficient is -3.2 mm Hg/100 

nmole/Lcellshr (p < 0.01). 

Discussion 

The overall blood lead mean observed in this study 

(165.8 ng Pb/L) is in agreement with blood lead levels 

found in other general populations (11-13). The 

factors contributing to variation of blood lead found in 

this study are the same as those found in other studies: 

alcohol consumption is positively related to blood lead; 

age and blood lead are weakly positively correlated. The 

association between blood lead and tobacco consumption is of less importance, since the amount of tobacco 

smoked by current smokers is not correlated with blood 

lead. 

Table 3. Multiple regressions of systolic blood pressure on 

blood lead, Na+,K+ cotransport, age, body mass index, and 

alcohol consumption. 

Table 4. Multiple regressions of diastolic blood pressure on 

blood lead, Na+,K+ cotransport, age, body mass index, and 

alcohol consumption. 

Independent variables3 

Blood lead alone (113)c 

Blood lead and A, B, C (104) 

Blood lead, Na+,K+ 

cotransport, and A, B, C 

(102) 

Regression coefficients x 100 ± SEMb 

Blood lead 

3.7 ± 0.22 

p = 0.05 

3.0 ± 0.22 

p - 0.09 

2.1 ±0.22 

p = 0.21 

Na+,K+ cotransport 

-3.5 ±0.13 

p < 0.01 

"Independent variables: A, age; B, body mass index; C alcohol 

consumption. 

••Regression coefficient x 100 indicates the systolic blood pressure 

increase in mm Hg for a 100 ug Pb/L increase and for a 100 

umole/Lcellshr Na+,K+ cotransport increase. 

cNumbers of subjects are in parentheses. 

Independent variables3 

Blood lead alone (113)c 

Blood lead and A, B,C (104) 

Blood lead, Na+,K+ 

cotransport, and A, B, C 

(102) 

Regression coefficients x 100 ± SEMb 

Blood lead 

' 3.0 ± 0.20 

/> = 0.07 

2.1 ± 0.20 

/> = 0.15 

'. 1.4 ± 0.20 

. /> = 0.32 

Na+,K+ cotransport 

-3.2 ± 0.12 

p < 0.01 

"Independent variables: A, age; B, body mass index; C, alcohol 

consumption. 

bRegression coefficient x 100 indicates the systolic blood pressure 

increase in mm Hg for a 100 ug Pb/L increase and for a 100 umole/L 

cellshr Na+,K+ cotransport increase. 

cNumbers of subjects are in parentheses. 

The association of blood lead to systolic blood pressure 

was found to be significant (r = 0.19, p = 0.05). The 

correlation of blood lead with diastolic blood pressure is 

slightly lower (r = 0.17, p = 0.07), as already noted in 

our earlier study (14,15). Taking into account alcohol 

consumption and age as confounding variables 

slightly decreases these correlations (systolic, r = 0.17, 

p = 0.09; diastolic, r = 0.14, p = 0.15); adjusting further 

for body mass index does not change these values. 

A significant negative relationship was observed 

between Na+,K+ cotransport and both systolic and 

diastolic blood pressure (r = -0.20, and r = -0.19 

respectively). Adjusting for alcohol consumption, age, 

and body mass index appears to strengthen these 

relationships (r = -0.28). This result confirms those 

obtained in a previous study where hypertensive and 

normotensive subjects were compared (16). Although 

the interpretation of Na+,K+ cotransport remains 

controversial as far as red blood cells are concerned, it 

must be stressed that the role of Na+,K+ cotransport in 

regulating Na+ cell content appears to be of greater 

importance in other cells, including vascular smooth 

muscle cells (17-19). Thus, an impaired Na+,K+ 

cotransport activity in these cells could increase Na+ 

content. This in turn may increase Ca2+ content and 

blood pressure, via a Na+, Ca2+ exchange mechanism. 

Results presented above show that Na+,K+ cotransport is significantly negatively related to blood 

lead. No other association was found between blood lead 

and either other RBCTS or red blood cell Na+ and K+ 

contents. In particular, the lack of correlation observed 

between Na+, K+ pump and blood lead level is not in 

agreement with other results (3-5). However, it must 

be noted that, in these latter studies, Na+,K+ ATPase 

activity was measured on red blood cell hemolysates; 

instead, we measured Na+,K+ pump activity. This 

particular point deserves further confirmation. 

Finally, considering the influence of both Na+,K+ 

cotransport and blood lead upon systolic blood pressure, 

after adjustment for alcohol consumption age and body 

mass index, the blood lead regression coefficient is very 

L 
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SODH M TRANSPORT. BLOOD LEAD. AND HLOOD PRESSURE 51 

from significance (p = 0.21), while Na+,K+ 

^'transport is highly significantly negative (p < 0.01) 

/Table 3)- By comparison, when Na+,K+ cotransport is 

a longer taken into account, the blood lead regression 

".efficient is either nearly significant (p = 0.09) when 

for alcohol consumption, age. and body mass 

far 

,diusting 

ndex. or significant (p = 0.05) without any adjustI

 ent.'Put together, these results show that Na+

.K+ coII

 nSport impairment explains the relationship be1

 ee n blood lead and systolic blood pressure to a greater 

extent than do the confounder variables alcohol conmption and age. This trend is also observed, to a 

?eSser degree, for diastolic blood pressure (Table 4). 

In conclusion, these results are in agreement with 

the hypothesis that blood lead could impair Na+.K+ 

cotransport activity, and this Na+,K+ cotransport alteration could increase blood pressure. Further studies 

are needed to confirm this finding. 

The authors gratefully acknowledge the work of Josianne Sahuquillo 

and Gisele Salomon in providing valuable technical assistance. This 

work was supported by the Programme Interdisciplinaire de Re-

•herche sur 1'Environment (PIREN) under contract number 034598. 

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