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Lead and δ-aminolevulinic acid dehydratase in RBC's of urban mothers and fetuses
ENVlRONMENl
AL
14, 73 -80 (1977)
RESEARCH
Lead and SAminolevulinic Acid Dehydratase of Urban Mothers and Fetuses P. M. KUHNERT,
P. ERHARD,
in RBC’s
AND B. R. KUHNERT
Depurtment of Obstetrics und Gynecology. Cleveland Metropolitan General Hospital and the Department of Reproductive Biology, Case Western Reserve University Cleveland, Ohio 44109
Received July 19. 1976 Environmental lead pollution may pose a health hazard to the mother and her fetus, but limited information concerning this problem is available. In this study. we examined erythrocyte &aminolevulinic acid dehydratase (ALAD) activity and erythrocyte lead levels in urban pregnant women and fetuses. The data show that the ratio of activatedinonactivated ALAD activity and erythrocyte lead are positively correlated in both the mothers and fetuses. The mean level of ALAD inhibition was found to be 28% in the mothers and 12%~in the fetuses. The data also show that fetal erythrocytes have significantly higher levels of activated ALAD activity than maternal erythrocytes. and that a positive correlation exists between maternal and fetal erythrocyte lead levels. These results indicate that “normal” urban blood lead levels inhibit erythrocyte ALAD activity in the pregnant woman and fetus.
INTRODUCTION
The adverse effects of excessive exposure to lead are well known. For many years they have been the subject of investigations ranging from purely clinical descriptions to studies of interactions with discrete enzymes. Less well known are the toxic effects of exposure to relatively low concentrations of lead. In recent years, this area of research has received much attention because of the widespread dissemination of lead by man into the general environment. The earliest manifestation of lead toxicity at low concentrations appears to be the inhibition of the enzyme aminolevulinic acid dehydratase (ALAD), an enzyme involved in the biosynthesis of heme (Secchi et nl., 1974). ALAD is so sensitive to lead that it is partially inhibited long before many of the other biological effects of lead are measurable (e.g., ALA in urine). Thus, many studies utilizing the activity of this enzyme have assessed the level of lead exposure of certain groups of the general population, e.g., people living near lead smelters (Secchi et al., 1972), traffic policemen (Secchi et al., 1972), and even the urban population of highly industrialized cities (Secchi et a/., 1972; Hernberg et al., 1970; Hernberg and Nikkanen, 1970). The degree of ALAD inhibition in these studies and in others, has been found to correlate closely with the concomitant level of lead in blood. One group of the general population which may be particularly vulnerable to the toxic effects of lead is the pregnant woman and fetus (Environmental Protection Agency, 1972). Recognizing their vulnerability, the Environmental Protection Agency has suggested that the upper acceptable level of lead in the pregnant woman and fetus should be 30 pg Pb/lOO ml of whole blood, 10 pg Pb/lOO ml lower than the general population. Likewise, Barltrop (1969) has concluded that until further information becomes available, it is reasonable to limit the exposure of the 73 Copyright All
rights
t0 of
1977 reproduction
by
Academic m
any
Press.
Inc.
form
reerved.
74
KUHNERT,
ERHARD,
AND
KUHNERT
pregnant woman, and hence the fetus, to lead. These studies and others (National Research Council, 1972), suggest that lead may be a health hazard to these subjects and that further research in this area is necessary. Accordingly, we investigated the levels of erythrocyte lead and ALAD activity in the urban pregnant woman and fetus. Our primary purpose was to determine whether or not the “normal” urban blood lead levels inhibit the activity of the enzyme ALAD. We report herein the results of our investigation. MATERIALS
AND METHODS
Forty-seven pregnant women and their fetuses (matched pairs) were included in this study. All of them resided in or very near Cleveland, Ohio and delivered in Cleveland Metropolitan General Hospital. Urban pregnant women and fetuses were chosen for study because they are, most likely, exposed to higher levels of lead than those living in a suburban environment. They were not specially selected in regard to prenatal or intrapartum medical problems, although indepth clinical information was recorded. Informed consent was obtained from the subjects in the appropriate manner. Blood from pregnant women and from the cord of their infants was obtained in polyethylene heparinized tubes. For maternal blood analysis. 4 ml of venous blood was drawn when the mother was admitted to the hospital in labor. For fetal blood analysis, 4 ml of venous umbilical cord blood was obtained immediately following delivery of the infant. A small amount of blood from each sample was used to determine the hematocrit, the red cell count, and the erythrocyte lead concentration; the remainder of the sample was placed on ice until analyzed for enzyme activity. Erythrocyte lead concentrations were determined by means of atomic absorption spectrophotometry. The analytical procedure was similar to the method described by Fernandez (1975). An atomic absorption spectrophotometer (Model 303; Perkin Elmer Corp., Norwalk, Conn.) equipped with a Deuterium Background Corrector and a graphite furnace (Model HGA-2100; Perkin Elmer) was used. Peak signals were registered on a strip-chart recorder (Model 165; Perkin Elmer). The HGA2100 was operated with an internal flow of argon purge gas (a setting of ten divisions on the HGA controller). The temperature program followed was that recommended by Fernandez (1975): dry at 100°C for 25s, ash at 525°C for 5Os, atomize at 2300°C for 9s. The within-run precision of the lead analysis was determined for a blood sample (38 pg Pb/lOO ml RBC) and an aqueous standard (25 pg PbllOO ml). The blood sample standard deviation for ten determinations was 1.87 and the coefficient of variation was 4.9%. For the aqueous standard, the standard deviation was 1.25 and the coefficient of variation was 4.1%. All of the samples were run in duplicate. ALAD activity was assayed using the microprocedure developed by Granick et al. (1973) with these modifications: (1) 50 ~1 of blood was assayed using appropriate dilutions of the assay media; (2) lysing of the red blood cells was accomplished by freezing and thawing; and (3) absorbance of the sample was read at 553 nm. Both activated and nonactivated levels of ALAD activity were determined since this procedure permits a better correlation to be made between lead poisoning and decreased ALAD activity by partially eliminating genetic variations (Granick et al.,
LEAD
AND
ALAD
75
ACTIVITY
1973). Activated ALAD activity is defined as the level of ALAD activity measured with dithiothreitol present in the assay medium; nonactivated ALAD activity is the level measured without dithiothreitol in the assay medium (dithiothreitol provides sulfhydryl groups that readily bind lead and thus remove it from the enzyme). All of the samples were stored at 4°C until analyzed for enzyme activity within 2 hours. The assay procedure is based on the conversion of aminolevulinic acid by ALAD to porphobilinogen, which is then measured spectrophotometrically. Enzyme activity is expressed as nanomoles of PBG formed per hour per milliter of erythrocytes at 37°C.
Red blood cell counts were determined using a D, Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.) and the methodology described in the instrument instruction manual. Statistical analyses were performed in the manner described by Sokal and Rohlf (1969) using a Hewlett Packard Model 25 programmable calculator. RESULTS
Figure 1 shows a scatter plot between the activated/nonactivated ALAD ratio and lead in fetal cord erythrocytes. The solid line is a least-squares best lit regression line with a correlation coefficient of 0.58; this represents a significant correlation at P < 0.01. Lead levels in fetal erythrocytes were found to vary from 16.3 to 67 pg Pb/lOO ml RBC with a mean value of 32.9 & 10.2 pg Pb/lOO ml RBC. The ALAD ratios ranged from 1.027 to 1.35 with a mean value of 1.138 ? 0.068. A scatter plot between the activated/nonactivated ALAD ratio and lead in maternal erythrocytes is presented in Fig. 2. Again, a significant correlation (r = 0.43; P < 0.01) was observed. The erythrocyte lead levels in the mothers ranged from 25 to 78.8 pg Pb/lOO ml RBC with a mean value of 49.1 ? 11.9 pg Pb/lOO ml RBC. The maternal ALAD ratios ranged from 1.102 to 1.988 with a mean ratio of 1.406 ? 0.156. It is of interest to note that the correlation coefficient in the maternal samples was less than that observed in the fetal samples and that the variance of the maternal ALAD ratios was significantly greater than the variance of the fetal ALAD ratios.
z
y = O.W38x
BLOOD
FIG. 1. Correlation erythrocytes.
between
the ratio
LEAD
flg/lOOml
of activated’nonactivated
+ I0134
RBC
ALAD
activity
and lead in fetal cord
76
KUHNERT,
z
FIG. 2. Correlation erythrocytes.
ERHARD,
‘111111”1”##“’ IO
between
AND
KUHNERT
20 30 40 50 60 BLOOD LEAD pg/lOO ml RBC
the ratio
of activatedinonactivated
70
ALAD
80
activity
and lead in maternal
Correlation analysis of the maternal and fetal erythrocyte lead levels also showed a significant correlation (r = 0.79: P < 0.01). This data is shown in Fig. 3. Other investigators have also observed this relationship and have reported correlation coefficients of 0.54 (Haas ef al., 1972) and 0.84 (Barltrop, 1968). The interception of the regression line on the vertical axis at 19 &lOO ml RBC is in agreement with the generally higher levels of lead found in the maternal erythrocytes. Because of the size difference between maternal and fetal erythrocytes, it is difficult to directly compare the absolute levels of nonactivated ALAD activity and activated ALAD activity in maternal and fetal erythrocytes. In Fig. 4, we have
r= 0.79 y = 09230x
+/8.4632
, IO FETAL
FIG 3. Correlation
between
20 BLOOD
maternal
30 LEAD
40 50 60 .ug/lOOml RBC
and fetal erythrocyte
lead concentrations
LEAD
AND
ALAD
NON-ACTIVATED
FIG 4. Activated
and nonactivated
ALAD
77
ACTIVITY
ACTIVATED
activity
in maternal
and fetal
erythrocytes.
expressed ALAD activity per LO9 RBC’s in order to compare ALAD activity per unit cell number. Looking at the data in this manner, significant differences are observed between maternal and fetal nonactivated ALAD activity and the maternal and fetal activated ALAD activity. However, if the difference in mean corpuscular volume is taken into consideration (approximately 16%), the activated levels of ALAD activity are no longer significantly different. Apparently, the greater cell volume of the fetal erythrocyte enables a correspondingly greater amount of the cytoplasmic enzyme ALAD. The same situation mentioned above is also encountered when comparing the absolute lead levels in the maternal and fetal erythrocytes. Therefore, in Table 1 we have expressed the lead levels as pg I%/ 109 RBC’s. The data indicate that despite the increased size of the fetal erythrocyte. the maternal erythrocytes contained significantly more lead (t test; P < 0.01). The effect of lead on the maternal and fetal erythrocyte ALAD activity is shown in Table 2. In both the mothers and fetuses, significant inhibition of ALAD activity was observed. However, the percent inhibition reported in Table 2 is quite likely below the actual level of ALAD inhibition. According to Sassaet al. (1975) another assay procedure is required in order to obtain maximal activation of the ALAD enzyme. DISCUSSION
The principal purpose of this study was to determine whether or not the “normal” levels of erythrocyte lead found in the urban pregnant woman and fetus affect the activity of the enzyme ALAD. Our data indicate that the erythrocyte lead levels TABLE Lt.&r)
COSCLNTRAI.IONS
Number’ Maternal Fetal (’ Fewer subjects on all of them. b Mean 5 SD.
37 35 are included
1 AND
Fural.
Kg Pb/lO’
RBC”
IN M.ATERNAL
0.044 0.037 in this
table
because
FRYTHRO(
Range
t 0.011 -t 0.012 red
blood
Y.I~S
0.024-0.072 0.02 l-0.076 cell
counts
were
not performed
78
KUHNERT,
ERHARD,
AND
TABLE EFFECT
OF LEAD
ON MATERNAI.
ALAD Nonactivated Maternal Fetal
2 225 1200 i 190
1035
AND
FETAL
activity
2 ERYTHROCYTE
ALAD
ACTIVITY
Level of
Activated 1436 1358
KUHNERT
2 ‘57 -c 188
significance
9%Inhibition
P i 0.001 P < 0.001
28.1 11.9
o Activity is expressed in nmoles porphobilinogen formed/ml RBCihour at 37°C (mean 2 SD) Quadruplicate enzyme determinations were made for each sample from 47 paired maternal and fetal blood samples.
in both mothers and fetuses are high enough to inhibit erythrocyte ALAD activity, and that the degree of inhibition is related linearly to the erythrocyte lead concentration. The results obtained also confirm that the degree of lead exposure of the fetus depends directly on the level of exposure of the mother. Manyrstudies performed to evaluate the effect of lead on ALAD activity determine whole blood lead levels rather than erythrocyte lead levels as was done in this study. Our reasons for this approach are similar to those of Evenson and Pendergast (1974), i.e., we feel that determining erythrocyte lead enables a greater precision and accuracy than the determination of whole blood lead. The lead levels we obtained can, of course, be converted to whole blood lead levels by calculation (Rosen et al., 1974). Assuming 3 pg PbllOO ml in the plasma (Rosen et al., 1974) and calculating the mean whole blood lead levels for the mothers and fetuses, we found their respective lead levels to be 20 and 17 pg Pb/ 100 ml whole blood. These values are within the range of values previously reported for fetal (Rajegowdaet al., 1972; Scanlon, 1971) and maternal (Barltrop, 1968) whole blood lead. The ALAD assay procedure utilized in this study was chosen because it provides a maximal difference between lead-poisoned blood and normal blood, and partially eliminates variations in activity due to genetic differences (Sassa et al., 1975). It is based on the fact that erythrocyte ALAD activity is inhibited by lead in the circulation and that its inhibition may be reversed by the addition of sulfhydryl groups. However, the activated levels of enzyme activity which it measures are not the maximum levels of activity attainable. Further activation may be achieved by increasing the concentration of dithiothreitol and increasing the incubation volume (Sassa et al., 1975). Consequently, the levels of activated ALAD activity and the percent inhibition of ALAD activity reported here are lower than the actual levels occurring in the maternal and fetal erythrocytes. How much lower these values are cannot be estimated from other studies; another investigation using the assay procedure designed to measure the maximally activated ALAD activity is necessary. Nonetheless, significant inhibition of ALAD activity was observed in both the mothers and fetuses in the present study. The mean corpuscular volume of the maternal erythrocyte is approximately 88 fl. whereas the mean corpuscular volume of the fetal erythrocyte is approximately 107 fl. (Wintrobe et al., 1974). These facts make it very difticuit to compare in a meaningful way the ALAD activity and lead levels of the mothers and fetuses. We feel that it is more useful comparing lead and ALAD levels when they are expressed
LEAD
AND
ALAD
79
ACTIVITY
per lo9 RBC’s rather than per 100 ml RBC’s. Thus, we have done so in this report when comparisons were made. However, to avoid this problem in regard to lead affecting the activity of the ALAD enzyme, we treated the data on the pregnant women and fetuses separately. A significantly greater variance of the maternal ALAD ratios than the fetal ALAD ratios was observed in the present study. In contrast, the variance of the maternal and fetal lead levels were similar. These findings suggest that the maternal ALAD ratio may have been influenced by another factor or factors besides lead. Assuming that this is the case would also explain the lower coefficient of correlation between lead and the ALAD ratio in the mothers. Although only speculative, we hypothesize that the maternal ALAD ratios were influenced in some manner by the metabolic changes that occur during pregnancy. We plan to investigate these differences further in our laboratory. The medical significance of maternal and fetal erythrocyte ALAD inhibiton is difficult to assess. Its inhibition in the red blood cell probably represents functional loss of a reserve enzyme since heme for hemoglobin is synthesized in nucleated precursors of red blood cells in the bone marrow (Secchi et al., 1974). However, this same enzyme does play an important metabolic role in the synthesis of other heme containing enzymes, among them catalases and cytochromes (Secchi et al., 1974). These hemoproteins are found in many tissues of the body and are thought to be present in the cytoplasm of all cells possessing aerobic metabolism. Thus, the inhibition of ALAD in erythrocytes is one of the many sites where inhibition may occur and be significant. Several studies in animals and in humans have confirmed that ALAD inhibition in the red blood cell correlates well with inhibition of the same enzyme in other tissues. In animal studies, Mouw et al. (1975) observed that blood and kidney ALAD activity are inhibited in rats living in an urban environment, but not brain or liver ALAD activity. Also, Millaret al. (1970) have reported that liver, brain, and kidney ALAD activity are inhibited in lead-poisoned suckling rats, and that agood correlation exists between erythrocyte ALAD activity and brain ALAD activity. In the human, Secchi et al. (1974) found a significant correlation between erythrocyte ALAD activity and liver ALAD activity in individuals not occupationally exposed to lead. They concluded that the inhibition of ALAD activity in hepatic tissue cannot be devoid of biological significance. However, additional studies need to be carried out in order to clarify the effect of lead on ALAD activity in other tissues besides blood, particularly with reference to what dosage level results in ALAD inhibition. The partial inhibition of ALAD activity that has been observed in erythrocytes and in other cells and tissues may have no health significance, since the availability of the enzyme appears to be largely in excess of the real requirement. On the other hand, this inhibition may be detrimental to certain groups of the population who are particularly vulnerable to the adverse effects of lead, e.g., the fetus. Whichever the case may be, the results of this study clearly indicate that erythrocyte ALAD activity is inhibited by lead in the urban pregnant woman and her fetus. ACKNOWLEDGMENTS This study
was supported
in part by NIHUSPHS
Grant
No.
5MO1-RROO:!lO-11.
80
KUHNERT,
ERHARD,
AND
KUHNERT
REFERENCES I. Secchi, G. C., Erba, L.. and Cambiaghi, G. (1974). Delta-aminolevulinic acid dehydratase activity of erythrocytes and liver tissue in man. Arch. Environ. Health 28, 13@132. 2. Secchi, G. C., Alessio, L., Cambiaghi, G. (1972). ALA-dehydratase activity of erythrocytes and blood lead levels in “critical” population groups. Itl “Proceedings of the International Symposium on Environmental Health Aspects of Lead.” pp. 595-602. Amsterdam. 3. Hernberg, S., Nikkanen. .I.. Mellin. G.. and Lilius, H. (1970). &aminolevuhnic acid dehydratase as a measure of lead exposure. Arch. Environ. Healrh 21, 140-145. 4. Hernberg, S., and Nikkanen, J. (1970). Enzyme inhibition by lead under normal urban conditions. Lancer 1, 63-64. 5. Environmental Protection Agency (1972). “EPA’s Position on the Health Effects of Airborne Lead.” Washington. D.C. 6. Barltrop, D. (1969). Environmental lead and its paediatric significance. Postgrad. Med. J. 45, 129-134. 7. National Research Council (1972). Committee on biological effects of atmospheric pollutants. 1,~ Natl. Acad. Sci., Washington, D.C. “Lead: Airborne Lead in Perspective.” 8. Fernandez, F. J. (1975). Micromethod for lead determination in whole blood by atomic absorption, with use of the graphite furnace. Clin. Che. 21, 55&561. 9. Granick, J. L., Sassa. S.. Granick, S.. Levere. R. D., and Kappas. A. (1973). Studies in lead poisoning 11. Correlation between the ratio of activated to nonactivated &aminolevulinic acid dehydratase of whole blood and the blood lead level. Biochem. Med. 8, 149-159. 10. Sokal. R. R.. Rohlf. F. J. (1969). “Biometry.” Freeman, San Francisco. 11. Haas, T., Wieck, A. G., Staller, K. H., Mache, K.. and Valentine, H. (1972). Die usuelle Bleibelastung bei Neugeborenen und ihren Muttern. Zbl. B&t. Hyg. I. Abt. Orig. B 155, 341-349. 12. Barltrop. D. (1968). The transfer of lead to the human foetus. In “Mineral Metabolism in Paediatrics.” Blackwell Scientific Publications. Oxford. 13. Sassa, S., Granick, S., and Kappas. A. (1975). Effect of lead and genetic factors on heme biosynthesis in the human red cell. Ann. N. Y. Acad. Sci. 244, 419-140. 14. Evenson, M. A., and Pendergast, D. D. (1974). Rapid ultramicro direct determination of erythrocyte lead concentration by atomic absorption spectrophotometry. with use of agraphite tube furnace. C/in. Chem. 20, (2). 163-171. 15. Rosen, J. F., Zarate-Salvadore, C.. and Trinidad, E. E. (1974). Plasma lead levels in normal and lead-intoxicated children. J. Ped. 84 (I), 4548. 16. Rajegowda. B. K., Glass. L.. and Evans, H. E. (1972). Lead concentrations in the newborn infant. /. Ped. 80, 116-117. 17. Scanlon. J. (1971). Umbilical cord blood lead concentration. Amer. J. Dis. Child. 121, 325-326. 18. Wintrobe, M. M., Lee, R. G.. Boggs, D. R., Bithell. T. C., Athens, J. W.. and Foerster. J. ( 1974). “Clinical Hematology.” Lea & Febiger, Philadelphia. 19. Mouw, D., Kalitis. K., Anver. M.. Schwartz, J., Constan. A., Hargung, R.. Cohen, B., and Ringler. D. (1975). Lead-possible toxicity in urban vs. rural rats. Arch. Emiron. Health 30, 276280. 20. Millar, J. A., Battistini, V., Cumming, R. L. C.. Carswell. F.. and Goldberg, A. (1970). Lead and b-aminolevulinic acid dehydratase levels in mentally retarded children and in lead poisoned suckling rats. Lnwet 2, 695-698.
AL
14, 73 -80 (1977)
RESEARCH
Lead and SAminolevulinic Acid Dehydratase of Urban Mothers and Fetuses P. M. KUHNERT,
P. ERHARD,
in RBC’s
AND B. R. KUHNERT
Depurtment of Obstetrics und Gynecology. Cleveland Metropolitan General Hospital and the Department of Reproductive Biology, Case Western Reserve University Cleveland, Ohio 44109
Received July 19. 1976 Environmental lead pollution may pose a health hazard to the mother and her fetus, but limited information concerning this problem is available. In this study. we examined erythrocyte &aminolevulinic acid dehydratase (ALAD) activity and erythrocyte lead levels in urban pregnant women and fetuses. The data show that the ratio of activatedinonactivated ALAD activity and erythrocyte lead are positively correlated in both the mothers and fetuses. The mean level of ALAD inhibition was found to be 28% in the mothers and 12%~in the fetuses. The data also show that fetal erythrocytes have significantly higher levels of activated ALAD activity than maternal erythrocytes. and that a positive correlation exists between maternal and fetal erythrocyte lead levels. These results indicate that “normal” urban blood lead levels inhibit erythrocyte ALAD activity in the pregnant woman and fetus.
INTRODUCTION
The adverse effects of excessive exposure to lead are well known. For many years they have been the subject of investigations ranging from purely clinical descriptions to studies of interactions with discrete enzymes. Less well known are the toxic effects of exposure to relatively low concentrations of lead. In recent years, this area of research has received much attention because of the widespread dissemination of lead by man into the general environment. The earliest manifestation of lead toxicity at low concentrations appears to be the inhibition of the enzyme aminolevulinic acid dehydratase (ALAD), an enzyme involved in the biosynthesis of heme (Secchi et nl., 1974). ALAD is so sensitive to lead that it is partially inhibited long before many of the other biological effects of lead are measurable (e.g., ALA in urine). Thus, many studies utilizing the activity of this enzyme have assessed the level of lead exposure of certain groups of the general population, e.g., people living near lead smelters (Secchi et al., 1972), traffic policemen (Secchi et al., 1972), and even the urban population of highly industrialized cities (Secchi et a/., 1972; Hernberg et al., 1970; Hernberg and Nikkanen, 1970). The degree of ALAD inhibition in these studies and in others, has been found to correlate closely with the concomitant level of lead in blood. One group of the general population which may be particularly vulnerable to the toxic effects of lead is the pregnant woman and fetus (Environmental Protection Agency, 1972). Recognizing their vulnerability, the Environmental Protection Agency has suggested that the upper acceptable level of lead in the pregnant woman and fetus should be 30 pg Pb/lOO ml of whole blood, 10 pg Pb/lOO ml lower than the general population. Likewise, Barltrop (1969) has concluded that until further information becomes available, it is reasonable to limit the exposure of the 73 Copyright All
rights
t0 of
1977 reproduction
by
Academic m
any
Press.
Inc.
form
reerved.
74
KUHNERT,
ERHARD,
AND
KUHNERT
pregnant woman, and hence the fetus, to lead. These studies and others (National Research Council, 1972), suggest that lead may be a health hazard to these subjects and that further research in this area is necessary. Accordingly, we investigated the levels of erythrocyte lead and ALAD activity in the urban pregnant woman and fetus. Our primary purpose was to determine whether or not the “normal” urban blood lead levels inhibit the activity of the enzyme ALAD. We report herein the results of our investigation. MATERIALS
AND METHODS
Forty-seven pregnant women and their fetuses (matched pairs) were included in this study. All of them resided in or very near Cleveland, Ohio and delivered in Cleveland Metropolitan General Hospital. Urban pregnant women and fetuses were chosen for study because they are, most likely, exposed to higher levels of lead than those living in a suburban environment. They were not specially selected in regard to prenatal or intrapartum medical problems, although indepth clinical information was recorded. Informed consent was obtained from the subjects in the appropriate manner. Blood from pregnant women and from the cord of their infants was obtained in polyethylene heparinized tubes. For maternal blood analysis. 4 ml of venous blood was drawn when the mother was admitted to the hospital in labor. For fetal blood analysis, 4 ml of venous umbilical cord blood was obtained immediately following delivery of the infant. A small amount of blood from each sample was used to determine the hematocrit, the red cell count, and the erythrocyte lead concentration; the remainder of the sample was placed on ice until analyzed for enzyme activity. Erythrocyte lead concentrations were determined by means of atomic absorption spectrophotometry. The analytical procedure was similar to the method described by Fernandez (1975). An atomic absorption spectrophotometer (Model 303; Perkin Elmer Corp., Norwalk, Conn.) equipped with a Deuterium Background Corrector and a graphite furnace (Model HGA-2100; Perkin Elmer) was used. Peak signals were registered on a strip-chart recorder (Model 165; Perkin Elmer). The HGA2100 was operated with an internal flow of argon purge gas (a setting of ten divisions on the HGA controller). The temperature program followed was that recommended by Fernandez (1975): dry at 100°C for 25s, ash at 525°C for 5Os, atomize at 2300°C for 9s. The within-run precision of the lead analysis was determined for a blood sample (38 pg Pb/lOO ml RBC) and an aqueous standard (25 pg PbllOO ml). The blood sample standard deviation for ten determinations was 1.87 and the coefficient of variation was 4.9%. For the aqueous standard, the standard deviation was 1.25 and the coefficient of variation was 4.1%. All of the samples were run in duplicate. ALAD activity was assayed using the microprocedure developed by Granick et al. (1973) with these modifications: (1) 50 ~1 of blood was assayed using appropriate dilutions of the assay media; (2) lysing of the red blood cells was accomplished by freezing and thawing; and (3) absorbance of the sample was read at 553 nm. Both activated and nonactivated levels of ALAD activity were determined since this procedure permits a better correlation to be made between lead poisoning and decreased ALAD activity by partially eliminating genetic variations (Granick et al.,
LEAD
AND
ALAD
75
ACTIVITY
1973). Activated ALAD activity is defined as the level of ALAD activity measured with dithiothreitol present in the assay medium; nonactivated ALAD activity is the level measured without dithiothreitol in the assay medium (dithiothreitol provides sulfhydryl groups that readily bind lead and thus remove it from the enzyme). All of the samples were stored at 4°C until analyzed for enzyme activity within 2 hours. The assay procedure is based on the conversion of aminolevulinic acid by ALAD to porphobilinogen, which is then measured spectrophotometrically. Enzyme activity is expressed as nanomoles of PBG formed per hour per milliter of erythrocytes at 37°C.
Red blood cell counts were determined using a D, Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.) and the methodology described in the instrument instruction manual. Statistical analyses were performed in the manner described by Sokal and Rohlf (1969) using a Hewlett Packard Model 25 programmable calculator. RESULTS
Figure 1 shows a scatter plot between the activated/nonactivated ALAD ratio and lead in fetal cord erythrocytes. The solid line is a least-squares best lit regression line with a correlation coefficient of 0.58; this represents a significant correlation at P < 0.01. Lead levels in fetal erythrocytes were found to vary from 16.3 to 67 pg Pb/lOO ml RBC with a mean value of 32.9 & 10.2 pg Pb/lOO ml RBC. The ALAD ratios ranged from 1.027 to 1.35 with a mean value of 1.138 ? 0.068. A scatter plot between the activated/nonactivated ALAD ratio and lead in maternal erythrocytes is presented in Fig. 2. Again, a significant correlation (r = 0.43; P < 0.01) was observed. The erythrocyte lead levels in the mothers ranged from 25 to 78.8 pg Pb/lOO ml RBC with a mean value of 49.1 ? 11.9 pg Pb/lOO ml RBC. The maternal ALAD ratios ranged from 1.102 to 1.988 with a mean ratio of 1.406 ? 0.156. It is of interest to note that the correlation coefficient in the maternal samples was less than that observed in the fetal samples and that the variance of the maternal ALAD ratios was significantly greater than the variance of the fetal ALAD ratios.
z
y = O.W38x
BLOOD
FIG. 1. Correlation erythrocytes.
between
the ratio
LEAD
flg/lOOml
of activated’nonactivated
+ I0134
RBC
ALAD
activity
and lead in fetal cord
76
KUHNERT,
z
FIG. 2. Correlation erythrocytes.
ERHARD,
‘111111”1”##“’ IO
between
AND
KUHNERT
20 30 40 50 60 BLOOD LEAD pg/lOO ml RBC
the ratio
of activatedinonactivated
70
ALAD
80
activity
and lead in maternal
Correlation analysis of the maternal and fetal erythrocyte lead levels also showed a significant correlation (r = 0.79: P < 0.01). This data is shown in Fig. 3. Other investigators have also observed this relationship and have reported correlation coefficients of 0.54 (Haas ef al., 1972) and 0.84 (Barltrop, 1968). The interception of the regression line on the vertical axis at 19 &lOO ml RBC is in agreement with the generally higher levels of lead found in the maternal erythrocytes. Because of the size difference between maternal and fetal erythrocytes, it is difficult to directly compare the absolute levels of nonactivated ALAD activity and activated ALAD activity in maternal and fetal erythrocytes. In Fig. 4, we have
r= 0.79 y = 09230x
+/8.4632
, IO FETAL
FIG 3. Correlation
between
20 BLOOD
maternal
30 LEAD
40 50 60 .ug/lOOml RBC
and fetal erythrocyte
lead concentrations
LEAD
AND
ALAD
NON-ACTIVATED
FIG 4. Activated
and nonactivated
ALAD
77
ACTIVITY
ACTIVATED
activity
in maternal
and fetal
erythrocytes.
expressed ALAD activity per LO9 RBC’s in order to compare ALAD activity per unit cell number. Looking at the data in this manner, significant differences are observed between maternal and fetal nonactivated ALAD activity and the maternal and fetal activated ALAD activity. However, if the difference in mean corpuscular volume is taken into consideration (approximately 16%), the activated levels of ALAD activity are no longer significantly different. Apparently, the greater cell volume of the fetal erythrocyte enables a correspondingly greater amount of the cytoplasmic enzyme ALAD. The same situation mentioned above is also encountered when comparing the absolute lead levels in the maternal and fetal erythrocytes. Therefore, in Table 1 we have expressed the lead levels as pg I%/ 109 RBC’s. The data indicate that despite the increased size of the fetal erythrocyte. the maternal erythrocytes contained significantly more lead (t test; P < 0.01). The effect of lead on the maternal and fetal erythrocyte ALAD activity is shown in Table 2. In both the mothers and fetuses, significant inhibition of ALAD activity was observed. However, the percent inhibition reported in Table 2 is quite likely below the actual level of ALAD inhibition. According to Sassaet al. (1975) another assay procedure is required in order to obtain maximal activation of the ALAD enzyme. DISCUSSION
The principal purpose of this study was to determine whether or not the “normal” levels of erythrocyte lead found in the urban pregnant woman and fetus affect the activity of the enzyme ALAD. Our data indicate that the erythrocyte lead levels TABLE Lt.&r)
COSCLNTRAI.IONS
Number’ Maternal Fetal (’ Fewer subjects on all of them. b Mean 5 SD.
37 35 are included
1 AND
Fural.
Kg Pb/lO’
RBC”
IN M.ATERNAL
0.044 0.037 in this
table
because
FRYTHRO(
Range
t 0.011 -t 0.012 red
blood
Y.I~S
0.024-0.072 0.02 l-0.076 cell
counts
were
not performed
78
KUHNERT,
ERHARD,
AND
TABLE EFFECT
OF LEAD
ON MATERNAI.
ALAD Nonactivated Maternal Fetal
2 225 1200 i 190
1035
AND
FETAL
activity
2 ERYTHROCYTE
ALAD
ACTIVITY
Level of
Activated 1436 1358
KUHNERT
2 ‘57 -c 188
significance
9%Inhibition
P i 0.001 P < 0.001
28.1 11.9
o Activity is expressed in nmoles porphobilinogen formed/ml RBCihour at 37°C (mean 2 SD) Quadruplicate enzyme determinations were made for each sample from 47 paired maternal and fetal blood samples.
in both mothers and fetuses are high enough to inhibit erythrocyte ALAD activity, and that the degree of inhibition is related linearly to the erythrocyte lead concentration. The results obtained also confirm that the degree of lead exposure of the fetus depends directly on the level of exposure of the mother. Manyrstudies performed to evaluate the effect of lead on ALAD activity determine whole blood lead levels rather than erythrocyte lead levels as was done in this study. Our reasons for this approach are similar to those of Evenson and Pendergast (1974), i.e., we feel that determining erythrocyte lead enables a greater precision and accuracy than the determination of whole blood lead. The lead levels we obtained can, of course, be converted to whole blood lead levels by calculation (Rosen et al., 1974). Assuming 3 pg PbllOO ml in the plasma (Rosen et al., 1974) and calculating the mean whole blood lead levels for the mothers and fetuses, we found their respective lead levels to be 20 and 17 pg Pb/ 100 ml whole blood. These values are within the range of values previously reported for fetal (Rajegowdaet al., 1972; Scanlon, 1971) and maternal (Barltrop, 1968) whole blood lead. The ALAD assay procedure utilized in this study was chosen because it provides a maximal difference between lead-poisoned blood and normal blood, and partially eliminates variations in activity due to genetic differences (Sassa et al., 1975). It is based on the fact that erythrocyte ALAD activity is inhibited by lead in the circulation and that its inhibition may be reversed by the addition of sulfhydryl groups. However, the activated levels of enzyme activity which it measures are not the maximum levels of activity attainable. Further activation may be achieved by increasing the concentration of dithiothreitol and increasing the incubation volume (Sassa et al., 1975). Consequently, the levels of activated ALAD activity and the percent inhibition of ALAD activity reported here are lower than the actual levels occurring in the maternal and fetal erythrocytes. How much lower these values are cannot be estimated from other studies; another investigation using the assay procedure designed to measure the maximally activated ALAD activity is necessary. Nonetheless, significant inhibition of ALAD activity was observed in both the mothers and fetuses in the present study. The mean corpuscular volume of the maternal erythrocyte is approximately 88 fl. whereas the mean corpuscular volume of the fetal erythrocyte is approximately 107 fl. (Wintrobe et al., 1974). These facts make it very difticuit to compare in a meaningful way the ALAD activity and lead levels of the mothers and fetuses. We feel that it is more useful comparing lead and ALAD levels when they are expressed
LEAD
AND
ALAD
79
ACTIVITY
per lo9 RBC’s rather than per 100 ml RBC’s. Thus, we have done so in this report when comparisons were made. However, to avoid this problem in regard to lead affecting the activity of the ALAD enzyme, we treated the data on the pregnant women and fetuses separately. A significantly greater variance of the maternal ALAD ratios than the fetal ALAD ratios was observed in the present study. In contrast, the variance of the maternal and fetal lead levels were similar. These findings suggest that the maternal ALAD ratio may have been influenced by another factor or factors besides lead. Assuming that this is the case would also explain the lower coefficient of correlation between lead and the ALAD ratio in the mothers. Although only speculative, we hypothesize that the maternal ALAD ratios were influenced in some manner by the metabolic changes that occur during pregnancy. We plan to investigate these differences further in our laboratory. The medical significance of maternal and fetal erythrocyte ALAD inhibiton is difficult to assess. Its inhibition in the red blood cell probably represents functional loss of a reserve enzyme since heme for hemoglobin is synthesized in nucleated precursors of red blood cells in the bone marrow (Secchi et al., 1974). However, this same enzyme does play an important metabolic role in the synthesis of other heme containing enzymes, among them catalases and cytochromes (Secchi et al., 1974). These hemoproteins are found in many tissues of the body and are thought to be present in the cytoplasm of all cells possessing aerobic metabolism. Thus, the inhibition of ALAD in erythrocytes is one of the many sites where inhibition may occur and be significant. Several studies in animals and in humans have confirmed that ALAD inhibition in the red blood cell correlates well with inhibition of the same enzyme in other tissues. In animal studies, Mouw et al. (1975) observed that blood and kidney ALAD activity are inhibited in rats living in an urban environment, but not brain or liver ALAD activity. Also, Millaret al. (1970) have reported that liver, brain, and kidney ALAD activity are inhibited in lead-poisoned suckling rats, and that agood correlation exists between erythrocyte ALAD activity and brain ALAD activity. In the human, Secchi et al. (1974) found a significant correlation between erythrocyte ALAD activity and liver ALAD activity in individuals not occupationally exposed to lead. They concluded that the inhibition of ALAD activity in hepatic tissue cannot be devoid of biological significance. However, additional studies need to be carried out in order to clarify the effect of lead on ALAD activity in other tissues besides blood, particularly with reference to what dosage level results in ALAD inhibition. The partial inhibition of ALAD activity that has been observed in erythrocytes and in other cells and tissues may have no health significance, since the availability of the enzyme appears to be largely in excess of the real requirement. On the other hand, this inhibition may be detrimental to certain groups of the population who are particularly vulnerable to the adverse effects of lead, e.g., the fetus. Whichever the case may be, the results of this study clearly indicate that erythrocyte ALAD activity is inhibited by lead in the urban pregnant woman and her fetus. ACKNOWLEDGMENTS This study
was supported
in part by NIHUSPHS
Grant
No.
5MO1-RROO:!lO-11.
80
KUHNERT,
ERHARD,
AND
KUHNERT
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