Influence of membrane sodium transport upon the relation between blood lead and blood pressure in a general male population.

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+,K+ 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+,K+ cotransport was taken into account; the same trend was observed with diastolic blood pressure. These findings suggest that a blood lead-related Na+,K+ 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)(4)(5), and other reports suggest that one or more RBCTS, including Na+,K+ pump, could be impaired in hypertensive subjects (6)(7)(8). * 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 of five RBCTS, including the Na+,K+ pump, and the role of red blood cell Na+ and K+ content in the blood lead-blood pressure relationship.

Population
The study population consisted 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 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 1000C, ash 7500C, atomized 2,4500C). 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 40C 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 Na -NaCOUNJIIATRANSPOAT in gmole/L.cells. 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/L cells*hr.

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 gg/L, 160.8 ± 5.2 ig/L, and 188.3 ± 8.8 ,ug/L. Age was weakly positively associated with blood lead (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 ig/L and 178.5 ± 9.9 pg/L, significantly higher than the mean in nonsmokers (146.8 ± 6.2 ,g/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 0.17 (p = 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 lowest blood lead classes (lower than 120 ,ug/L, between 120 to 160 ,ug/L, and those between 161 to 200 ig/L) and then, at > 200 ,ug/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.

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  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 ,umole/L.cells.hr Na+,K+ cotransport decrease for a 100 ,ug/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). bNumbers of subjects are in parentheses. 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.

Discussion
The overall blood lead mean observed in this study (165.8 gg Pb/L) is in agreement with blood lead levels found in other general populations (11)(12)(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. bRegression coefficient x 100 indicates the systolic blood pressure increase in mm Hg for a 100 pg Pb/L increase and for a 100 limole/L cells-hr Na+,K+ cotransport increase.
CNumbers of subjects are in parentheses. bRegression coefficient x 100 indicates the systolic blood pressure increase in mm Hg for a 100 pg Pb/L increase and for a 100 pmole/L cells-hr 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)(18)(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)(4)(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 far from significance (p = 0.21), while Na+,K+ cotransport is highly significantly negative (p < 0.01) ( Table 3). By comparison, when Na+,K+ cotransport is no longer taken into account, the blood lead regression coefficient is either nearly significant (p = 0.09) when adjusting for alcohol consumption, age, and body mass index, or significant (p = 0.05) without any adjustment. Put together, these results show that Na+,K+ cotransport impairment explains the relationship between blood lead and systolic blood pressure to a greater extent than do the confounder variables alcohol consumption and age. This trend is also observed, to a lesser 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.