Release of lead from bone in pregnancy and lactation

Environmental Research 92 (2003) 139–151

Release of lead from bone in pregnancy and lactation$ W.I. Manton,a,
C.R. Angle,b K.L. Stanek,c D. Kuntzelman,b Y.R. Reese,d and T.J. Kuehnemannb a

Department of Geology, University of Texas at Dallas, Mail Drop FO21, P.O. Box 830688, Richardson, TX 75083-0688, USA b Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE, USA c Department of Nutrition Science, University of Nebraska–Omaha, Omaha, NE, USA d Department of Geology, University of Texas at Dallas, Richardson, TX, USA Received 22 February 2002; received in revised form 23 December 2002; accepted 17 January 2003

Abstract Concentrations and isotope ratios of lead in blood, urine, 24-h duplicate diets, and hand wipes were measured for 12 women from the second trimester of pregnancy until at least 8 months after delivery. Six bottle fed and six breast fed their infants. One bottle feeder fell pregnant for a second time, as did a breast feeder, and each was followed semicontinuously for totals of 44 and 54 months, respectively. Bone resorption rather than dietary absorption controls changes in blood lead, but in pregnancy the resorption of trabecular and cortical bone are decoupled. In early pregnancy, only trabecular bone (presumably of low lead content) is resorbed, causing blood leads to fall more than expected from hemodilution alone. In late pregnancy, the sites of resorption move to cortical bone of higher lead content and blood leads rise. In bottle feeders, the cortical bone contribution ceases immediately after delivery, but any tendency for blood leads to fall may be compensated by the effect of hemoconcentration produced by the postpartum loss of plasma volume. In lactation, the whole skeleton undergoes resorption and the blood leads of nursing mothers continue to rise, reaching a maximum 6–8 months after delivery. Blood leads fall from pregnancy to pregnancy, implying that the greatest risk of lead toxicity lies with first pregnancies. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Pregnancy; Lactation; Blood lead; Bone lead; Lead isotope ratios

1. Introduction Concern has been expressed that changes in physiology during pregnancy increase the turnover of bone, which could raise maternal blood lead concentrations to levels that would harm the fetus (Manton, 1985; Silbergeld, 1991). But even if maternal blood lead levels rise during pregnancy, as was observed by Rothenberg et al. (1994), one must still demonstrate that the increase stems from the release of lead from the skeleton. This could not be achieved by direct observation but for the fact that lead varies in isotope ratio and so behaves as a natural tracer. Even so, the problem remains difficult $ This work was funded by the National Institute for Environmental Health Sciences through grant ES 04762 to Carol Angle. The procedures and consent forms were reviewed and approved by the Institutional Review Board of the University of Nebraska Medical Center.
Corresponding author. E-mail address: [email protected] (W.I. Manton).

because the isotope ratio of bone lead must be known if changes in blood lead isotope ratio are to be uniquely attributed to the skeleton. With biopsies out of the question, investigators have had either to dose animals with enriched isotopes (Franklin et al., 1997) or to devise an indirect means of inferring the isotope ratios in the maternal skeleton, such as that accomplished by Gulson et al. (1997, 1998b) in their studies of women who grew up in Europe and then emigrated to Australia. Although the human and animal studies referred to above demonstrated an increased flux of lead from bone during late pregnancy, they both treated the skeleton as a single entity, whereas recent statistical analyses of blood lead and in vivo bone lead concentrations suggest that trabecular and cortical bone contribute differing amounts of lead to blood during pregnancy (Rothenberg et al., 2000). Isotopic techniques may provide a means of addressing this problem because many women of childbearing age in the United States grew up in household environments dominated by lead of one

0013-9351/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0013-9351(03)00020-3

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isotope ratio and then at early maturity moved to others characterized by lead of a different ratio. As trabecular bone turns over more rapidly than cortical bone these two portions of the skeletons will in time come to contain lead of different isotope ratios. We recently used mothers as controls in an isotopic study in which we followed children from birth for periods of at least 12 months (Manton et al., 2000). We have, therefore, observations over an extended period on a number of women, including both breast and bottle feeders. Some showed large changes in blood lead concentration; two, who also showed large changes in isotope ratio, we followed for a second pregnancy. Although the information was not obtained from a study designed specifically to examine the release of lead from the maternal skeleton, we believe that our findings are sufficient to allow speculation on the role of cortical and trabecular bone in pregnancy and lactation.

out the period of observation. For the second pregnancies, blood and diet were taken monthly until we ceased following the subjects 4–8 months after delivery. Hematocrits were obtained for all blood samples. The calcium contents of the diets were estimated from the mothers’ records by means of the Minnesota Nutrient Data System Version 2.3 software (Nutrition Coordinating Center, 1991), and 36 aliquots of the homogenized samples were sent to Midwest Laboratories (Omaha, NE, USA) for measurement of calcium content by inductively coupled plasma optical emission spectrography following acid digestion. Concentrations and isotope ratios of lead were measured simultaneously with a 205Pb spike, which yields precisions of better than 70.001 in 206Pb/207Pb and 71% in lead concentrations. The methods, both recruiting and analytical, have been described in Manton et al. (2000).

2. Subjects and methods

3. Results

Of the 22 mothers followed in the original study, the 12 that had been recruited earliest in pregnancy, i.e., during the second trimester, are the focus of this report. They were equally divided between breast feeders and bottle feeders. At delivery, the former, born in the 1950s and 1960s, averaged 34 years and the latter, born in the 1960s and 1970s, averaged 25 years (Table 1). All but three were multiparous. Six had up to three previous deliveries and one, the oldest in the study, had six. Four nursed between 4 and 8 months and two for 18. Two, one of whom was a breast feeder, fell pregnant for a second time during the study. During each month of pregnancy, a blood and a 24-h duplicate diet sample with a record of its contents were obtained from all subjects. For those electing to bottle feed, no diets were collected after delivery, but blood samples continued to be taken monthly for 4 months and thereafter every 6 months. Cord blood was collected, as its lead concentrations and isotope ratios provide a useful check of the accuracy of the maternal blood measurements at term. We used spot urines collected in the intervening months as approximate measures of blood lead isotope ratios. Our justification was the high correlation (r2 40:97) between the isotope ratios of lead in urine with those of whole blood (Angle et al., 1995; Gulson et al., 1998a). To monitor accuracy through the measurement of renal clearance, a 24-h urine was collected the day the 6th-month blood sample was drawn. Blood and duplicate diets were obtained monthly from the breast feeders throughout lactation. Once they stopped nursing, no more diets were collected and blood and urine were collected at the same frequency as that of the bottle feeders. Hand-wipe samples were taken monthly from all subjects through-

3.1. Single pregnancies Blood lead concentrations of the bottle feeders are shown in Fig. 1 and are grouped according to the pattern they exhibit. Those of the subjects in Fig. 1A changed little during pregnancy but rose after delivery, peaking 1–3 months later and then falling back to the levels of midpregnancy. In contrast, those of the subjects in Fig. 1B rose during late pregnancy and remained nearly constant for 4 months after delivery before rapidly declining to levels greater than those of early to midpregnancy. The blood lead concentrations of the four women who breast fed for 8 months or less are shown in Fig. 2. Blood lead concentrations that rose in late pregnancy continued to do so after delivery. Throughout lactation they remained elevated and declined once it ceased. The higher the concentration in pregnancy, the greater the rise during lactation. Two women, subjects 102–1 and 105–1 (Fig. 3), breast fed for 18 months. Both showed a continuous increase in blood lead concentration for 8 months and a slow decline thereafter. Subject 102-1 fell pregnant again at 18 months and was dropped from the study. A blood sample taken 6 months after subject 1051 stopped nursing showed that her blood lead concentration fell by half over that interval. The subjects’ dietary intakes of calcium and lead are shown in Table 2. Estimated calcium intakes of the bottle feeders averaged 62% of the recommended adequate intake of 1000 mg/day (Institute of Medicine, 1999), with only two subjects falling within the experimental error of that value. Individual mean lead intakes ranged from 2.4 to 14 mg/day, with an average for the group of 6.7 mg/day. In contrast, the breast feeders’

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Table 1 Race, year of birth, age at delivery, number of previous deliveries, and months nursed for the mothers followed in this study Subject no.

Race

Year born

Age at delivery

Previous deliveries

Months nursed

Bottle feeders 103-1 W 109-1 W 110-1 W 111-1 B 112-1a B 124-1 W

1969 1960 1964 1969 1974 1969

23 32 28 24 19 25

0 2 2 3 0 1

n/a n/a n/a n/a n/a n/a

Breast feeders 102-1 W 104-1 W 105-1 W 107-1a W 116-1 W 121-1 B

1952 1964 1963 1958 1960 1957

40 29 30 34 33 36

6 0 1 3 3 1

18 8 19 8 6 4

W, White; B, Black; n/a, not applicable. a These subjects were followed through a subsequent pregnancy.

Fig. 2. Blood lead concentrations vs. time from delivery for the four mothers who breast fed for 8 months or less. The dashed portions of the lines connect samples taken after weaning. The y-axis is drawn to the same scale as that of Fig. 1.

Fig. 3. Blood lead concentrations vs. time from delivery for the two mothers that breast fed for 18 months. The dashed portions of the lines connect samples taken after weaning. The y-axis is drawn to the same scale as those of Figs. 1 and 2. Fig. 1. Blood lead concentrations of bottle-feeding mothers vs. time from delivery. (A) Subjects whose blood lead concentrations changed little in late pregnancy but increased after delivery. (B) Subjects whose blood lead concentrations increased during late pregnancy but remained at a plateau for about 3 months after delivery.

average estimated calcium intakes (Table 2) were 102% of the adequate intake. They took in an average of 9.0 mg Pb/day, with individual mean values ranging from 6.0 to 12 mg/day.

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Table 2 Daily calcium intakes estimated from mothers’ records and daily lead intakes measured in duplicate diets Subject no.

Caa n

Pb b

% AI

c

DðnÞ

n

mgd

Bottle feeders 103-1

19

58715

3(2)

7

109-1

16

107721

16(1)

4

6.0

110-1

17

45712

+2(2)

5

2.4

111-1

18

90733

0(2)

7

3.4

112-1

29

68712

+33(4)

16

9.1

124-1

25

56715

+24(2)

14

5.7

Breast feeders 102-1 25

67717

20(5)

25

6.0

14

104-1

19

153720

+12(3)

15

8.8

105-1

30

113715

26(3)

29

7.9

107-1

24

80713

16(6)

29

12

116-1

15

115744

+4(5)

10

11

121-1

17

89728

14(2)

9

8.6

+9 6 +2.0 1.5 +2.6 1.2 +2.8 1.5 +4.6 3.0 +3.9 2.3

+1.6 1.2 +1.9 1.6 +1.7 1.4 +3 2 +5 3 +6. 3.8

a Expressed as percentage of the adequate intake (AI) of 1000 mg/ day for women 19–50 (Institute of Medicine, 1999). b Arithmetic means and 95% confidence limits. c D ¼ 100  ðCaestimated 2Cameasured Þ=Cameasured : Positive values imply dietary Ca was over-estimated. n is number of comparisons. d Geometric means and 95% confidence limits.

The success of a study of this kind depends on the recruitment of subjects who have been exposed to lead with isotope ratios falling far outside the range normally encountered in the US environment; the probability of doing so is, in our experience, about one in five. For the majority of the subjects, blood lead isotope ratios fell in the typical range of 1.185–1.215 for 206Pb/207Pb, and, although small changes occurred in some both before and after delivery, no consistent trends were observed. There were, however, two exceptions, both involving the subjects with the highest blood lead concentrations. As they were followed through a second pregnancy they are discussed in the next section. 3.2. Successive pregnancies The blood lead concentrations and isotope ratios of the two pregnancies of bottle-feeding subject 112-1 are plotted in Fig. 4. Her blood lead concentration declined

Fig. 4. Blood lead concentration and associated 206Pb/207Pb ratios of blood and urine for bottle-feeding subject 112-1, who was followed through two pregnancies. The interval P denotes pregnancy. The concentration and isotope ratio of lead in the umbilical cord of each infant are also shown. The latter fall close to the maternal value and attest to the overall precision of the measurements, which is better than 70.001 in 206Pb/207Pb.

from the 5th to the 7th month of her first pregnancy, but then rose rapidly until delivery, after which it remained constant for 3 months before falling to near constant values. During the first trimester of her second pregnancy, the blood lead concentration declined to one-half the value it had been 3 months before conception, but due to a lack of samples the exact nature of the change was undocumented. The change in concentration in the second pregnancy was small compared to the first, and 6 months after delivery the woman’s blood lead was 1 mg/dL, whereas 6 months after the first delivery it was twice that value. In her first pregnancy, the isotope ratios of the subject’s blood lead rose continuously from the 5th month to delivery, at which point they reversed and decreased for 6 months. Thereafter the woman’s blood lead isotope ratios, as reflected in urine samples, assumed a rising and falling pattern, ending with a steep decline once her second pregnancy began. During the second trimester the ratios remained constant, but they rose steeply during the third. After delivery the ratios began a decline that continued for the remaining 8 months of observation. The magnitudes of the changes in isotope ratio associated with each pregnancy were similar, as opposed

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4. Discussion 4.1. Blood lead concentrations

Fig. 5. Blood lead concentration and associated 206Pb/207Pb ratios of blood and urine for breast-feeding subject 107-1, who was followed through two pregnancies. The interval P denotes pregnancy and L lactation. The concentration and isotope ratio of lead in the umbilical cord of each infant are also shown. The latter fall close to the maternal value and attest to the overall precision of the measurements, which is better than 70.001 in 206Pb/207Pb.

to the changes in blood lead concentration, which were much diminished in the second pregnancy. The other mother who fell pregnant a second time during the course of the study was subject 107-1, the breast feeder with the highest blood lead concentration. Concentrations that were unchanging from the third to the 5th month of the first pregnancy rose until delivery and continued to do so into lactation, reaching a maximum after 6 months (Fig. 5). Two months later she stopped breast feeding and her blood leads continued to decline steadily over the next 14 months. A 5-month gap in sampling prevented us from observing precisely what changes occurred at the beginning of her next pregnancy, which was her fifth. By midpregnancy, however, her blood lead concentration was less than half the value at the equivalent point during her previous pregnancy, and the increase during the third trimester was even smaller, so that at delivery her blood lead was 1.2 mg/dL as opposed to 3.8 mg/dL at the delivery of her fourth child. During her pregnancies the isotope ratios of her blood first rose and then fell, but the steep declines of late pregnancy did not continue into lactation, which was characterized by a pattern of rising and falling ratios that were independent of changes in blood lead concentration.

The concentration of lead in blood during and after pregnancy is a function of plasma volume, red cell mass, and changes in the flux of lead from sources internal or external to the body. Plasma volume increases throughout pregnancy but more slowly during the third trimester, while red cell mass appears to increase linearly from the time of conception (Hytten, 1985). Together these changes bring about an increase in blood volume of about 37.5% at term. Immediately after delivery the plasma volume increases, but by the third day postpartum has decreased by 1 L and continues to decrease for another 6 weeks (Stock and Metcalfe, 1994). In contrast, the excess red cells produced during pregnancy persist over their normal lifespan of 120 days. These changes in volume are reflected by the hematocrit, which falls until the third trimester, then abruptly rises in the third trimester and remains above normal values until blood reattains the volume of the nonpregnant state (Fig. 6). If the flux of lead into blood during pregnancy remains constant a woman’s blood lead concentration will behave much like total calcium, 60% of which is bound to albumin, and will decline as blood volume increases (Dahlman et al., 1994; Pitkin and Gebhardt, 1977). If, however, her blood lead concentration is observed to remain constant (Fig. 1A) or increase during late pregnancy (Rothenberg et al., 1994; this paper, Fig. 1B and Fig. 2), an increased flux of lead into blood is indicated. Of the bottle-feeding subjects, the greatest change in blood lead concentration occurred during the first pregnancy of subject 112-1 (Fig. 1B), and with this was associated a rapid change in blood lead isotope ratio (Fig. 4), indicating the appearance of a new source of lead in her blood. It is reasonable to assume that the decline in isotope ratio after delivery reflects the disappearance of lead from this same source. Her blood lead concentration did not fall, however, because any reduction would be counteracted by a contraction of the plasma volume. The two effects cancelled each other for 3 months, after which her blood lead concentration abruptly fell. In the case of the women shown in Fig. 1A, the blood lead concentration changed little in late pregnancy, indicating that the amount of lead coming from this new source was small. After delivery the effect of plasma contraction was dominant, causing the blood lead concentrations to rise. Hematocrits and blood lead concentrations for two of the subjects are shown in Fig. 6. The breast feeder who showed the greatest change in blood lead concentration is subject 107-1 (Fig. 4), and this change was accompanied by a change in isotope ratio (Fig. 5), which again indicated the presence of a

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months. The decline in blood lead concentration that followed the cessation of lactation was associated with no change in isotope ratio and thus could not be attributed to lead from a particular source leaving blood. It is clear that the maintenance of elevated blood lead after delivery and its decline once lactation ceased were not caused by the same agent that caused the blood lead to rise in late pregnancy. 4.2. Source of lead in late pregnancy

Fig. 6. Relationships between blood lead concentration and hematocrit. (A) Blood lead concentration and percentage hematocrit for subject 109-1. Her blood lead changed little over her last two trimesters of pregnancy, indicating that lead was entering her blood in an amount that compensated for the effect of increasing blood volume, shown by her falling hematocrit. After delivery, plasma contraction caused her hematocrit to rise, which more than compensated any tendency for her blood lead concentration to decrease due to disappearance of lead mobilized in pregnancy from blood. (B) Blood lead concentration and percentage hematocrit for subject 112-1’s first pregnancy. Her blood lead rose over the last 2 months of pregnancy due to lead from a new source entering blood and not to the increase in hematocrit, which simply reflected the fact that her red cell mass was growing faster than her plasma volume. After delivery her blood lead concentration did not increase despite plasma contraction, because any tendency for this is compensated by the disappearance of the lead that has been mobilized in pregnancy. (C) Blood lead concentration and percentage hematocrit for subject 112-1’s second pregnancy. Over the last 2 months of pregnancy the influx of lead into her blood more than compensated for hemodilution and her blood lead concentration rose. After delivery, blood lead continued to rise for 2 months over which the effect of hemoconcentration proved somewhat greater than that of the disappearance of the lead mobilized in pregnancy. (For the isotope ratios of this subject, see Fig. 4.)

new source of lead in her blood. After delivery, however, the subject’s blood lead concentration continued to increase while no further change in isotope ratio occurred. As the new source of lead was not leaving blood, plasma contraction was in part responsible for the continued increase in blood lead concentration, but could not have maintained the elevated values for 6

The rise in blood lead in late pregnancy may be linked to increasing calcium demand, which could be satisfied by increased absorption through the gut, by resorption of bone mineral, or by both. Gulson et al. (1997) showed that the blood lead isotope ratios of immigrant European women moved away from the Australian diet as if mobilization of bone was the dominant agent, but in Franklin et al.’s (1997) dosed monkeys both dietary absorption and bone resorption increased, with the former being dominant. House dust, which can be ingested from contaminated hands, is an unlikely agent for changes in blood lead, not because it is negligible, but because it is ingested principally in the absence of dietary calcium and because it is probably under normal conditions absorbed to an extent that is already so high that it is not significantly affected by increasing calcium demand. We confirmed this for subject 112-1. Her hand wipe lead had, for 39 samples with a geometric mean content of 0.94 mg Pb, a mean 206Pb/207Pb ratio of 1.19970.007 in contrast to her mean blood ratio of 1.22970.003 (95% confidence). In late pregnancy her blood lead attained ratios greater than 1.235 (Fig. 4) and thus differed from the results of her hand wipes, indicating that exposure to house dust could not be the cause of the rise in her blood lead concentration. Subject 107-1’s hand wipe lead was unusable, however, having lead that was isotopically indistinguishable from her blood lead. With respect to dietary lead, it turned out that subject 112-1’s diet lead was isotopically indistinguishable from her blood lead, but that of subject 107-1, with a mean value of 1.18370.010 for 29 samples, was lower than her mean blood ratio of 1.19770.002. Thus, as her blood lead concentration increased in late pregnancy, its isotope ratio (Fig. 5) moved toward that of her diet. From this observation, one can argue that increased dietary absorption was the agent responsible for the rise of blood lead in late pregnancy. This is strengthened by the fact that the dietary lead of both subjects was high compared to that of the other subjects (Table 2), but without knowledge of the isotope ratios of bone lead the argument is not compelling because in each case the woman’s bone lead ratios may have been identical to those in her diet. If blood lead concentrations and isotope ratios are considered together, a much stronger

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case can be made that the source is internal. The amplitude of the changes in isotope ratio and the relative changes in blood lead concentration in both of the pregnancies of subject 112-1 are comparable (Fig. 4), but the absolute blood lead concentrations differed by a factor of two, as if the pools that interacted to bring about the changes in isotope ratio did this in exactly the same way in each pregnancy irrespective of their size. This is readily understandable if both pools were components of the skeleton reacting to a common physiological process, but less so if one pool was external (the diet) and the other internal (the skeleton).

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other hand, the initial blood lead of the younger group of 1.6 mg/dL fell by 13% to 1.4 mg/dL over the same period. If hemodilution was the only factor controlling the midpregnancy decline in blood lead concentrations, the percentage decreases would be independent of blood lead concentration and would be the same in each group. That they were not and that the women with the higher blood leads showed the greater decrease is consistent with our hypothesis that the midpregnancy decline is in part due to reduced flux of lead from bone. 4.4. Dietary calcium and bone resorption in late pregnancy

4.3. Decline of blood lead in midpregnancy In a sample of 105 women Rothenberg et al. (1994) observed a decline in blood lead concentration of 15% between the 12th and 20th weeks of pregnancy. This figure agrees exactly with Hytten’s (1985) calculation of a 15% increase in blood volume by the 20th week, most of which occurs after the 10th week. Decreases of similar size occurred in some of Gulson et al.’s (1997) subjects. It is, however, inescapable that a decline in blood lead of about 50% occurred over the first trimester of subject 112-1’s second pregnancy (Fig. 4) and that this cannot be accounted for by hemodilution. An accompanying change in isotope ratio indicates that the decline was due to the removal of a source that usually contributes lead to blood. Again the argument revolves around whether the source removed is dietary or skeletal and cannot be settled in the absence of the relevant isotope ratios. Franklin et al. (1997), however, observed in cynomolgous monkeys a decrease in the flux of lead from bone in midpregnancy, favoring the interpretation that the decline in subject 112-1 resulted from the same process. If this is true, it follows that changes in the flux of lead from the skeleton may cause both increases in blood lead at the end of pregnancy and declines in midpregnancy beyond those predicted by hemodilution. If the whole skeleton were involved the isotope ratios would not change; but the fact that they do change indicates that only part of the skeleton contributes lead at the end of pregnancy and that this same component is essentially shut down in midpregnancy. Gulson et al. (1997) also observed a 50% decrease in concentration in one well-documented case (their subject 1042). As she had entered Australia with a blood lead of 20 mg/dL, a level six times higher than that of any other of their subjects, it may be presumed that her bone lead was also the highest, so what we have described will probably not be infrequently encountered. In fact, Hertz-Picciotto et al. (2000) have published smoothed curves for the blood leads of 18- and 38-year-old women between the 5th and 40th weeks of pregnancy. At the 5th week the older group had an average blood lead of 2.8 mg/dL, which at the 20th week had declined by 29% to 2.0 mg/dL. On the

Gulson et al. (1998b), noting that in late pregnancy the blood of women who were on calcium supplements appeared to contain less lead derived from bone, concluded that bone resorption in late pregnancy was enhanced by insufficient calcium in the diet. HertzPicciotto et al. (2000) concluded likewise by observing that the blood lead concentration of women ingesting 600 mg Ca/day increased more rapidly in late pregnancy than that of ingesting 2000 mg Ca/day. We have argued above that the entry of a new source of lead into blood, now interpreted to be bone, can be inferred for bottlefeeding women from the pattern of blood lead concentrations in late pregnancy and after delivery. The subjects shown in Fig. 1A are interpreted as having experienced a smaller amount of bone resorption than those in Fig. 1B, and Table 2 shows that their average dietary calcium was 850 mg/day while the corresponding value for those in Fig. 1B was 560 mg/day. The breast feeders, who mostly had dietary calcium intakes close to or in excess of the recommended 1000 mg/day, tended not to show an increase in blood lead concentration in late pregnancy (Fig. 2), with subject 107-1 being a notable exception. Our observations, albeit based on a small sample, seem to confirm that an intake of 1000 mg Ca/day (Institute of Medicine, 1999) protects the skeleton from excessive resorption in late pregnancy. 4.5. Source of lead during and after lactation The blood lead concentrations of the breast feeders continued to increase after delivery (Figs. 2 and 3), but in the case of subject 107-1 cannot be attributed to increased dietary absorption because her isotope ratios moved away from those of her diet (which had an average ratio of 1.183) after each delivery (Fig. 5). The explanation may be that lead is coming from a part of the skeleton that was not resorbed during pregnancy, which does not necessarily mean that the part of the skeleton contributing lead during pregnancy shuts down once lactation begins. Isotope ratios are, like the hematocrit, relative measures and tell only the proportions of lead from each source, saying nothing of the

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absolute quantities contributed by each. Insofar as the blood lead concentrations were increasing, the changing ratios may mean that lead from another part of the skeleton was entering blood and overwhelming the source that formerly was dominant. Blood lead concentrations continued to rise for 6–8 months (Figs. 2 and 3), before beginning a slow decline, clearly seen in the case of the long-term breast feeders (Fig. 3). After weaning they continued their decline, from 3.8 to 1.3 mg/ dL over 16 months in the case of subject 107-1 (Fig. 5). What is clear from her record is that the lactation and postweaning isotopic patterns did not differ, indicating that changes in blood lead concentration were not controlled by the mobilization of different regions of the skeleton, as seemed to be the case in pregnancy, but instead by changes in the balance between bone resorption and bone formation. 4.6. Seasonal changes in blood lead isotope ratio Manton (1977) observed seasonally changing isotope ratios in a South African subject who had lived in the United States for 10 years and speculated that they resulted from differing amounts of lead in his blood derived from bone. Isotope ratios of lead in bone obtained by iliac crest biopsy were consistent with this hypothesis (Manton, 1985). Although it is not clear how the seasonal changes arise, they are nonetheless valuable because in winter the ratios moved toward those of cortical bone (see Manton, 1985, Fig. 3). In Figs. 7 and 8 we plot against calendar month the blood and urine lead isotope ratios of subjects 112-1 and 107-1 and superimpose sinusoids with 365-day periods and amplitudes that correspond as closely as possible to the observed range in ratios. With blood samples taken at 6-month intervals, the relationship is for the most part dependent on spot urines. Nonetheless, the match between the observed ratios and the sinusoid is not unconvincing for bottle-feeding subject 112-1 (Fig. 7). From the changes in her ratios, we infer that in midpregnancy the flux of lead from the cortical portion of her skeleton decreased by an amount far beyond that of its normal seasonal limits (Fig. 4), while in late pregnancy it similarly increased. The fall in blood lead concentration that accompanied the isotopic change of midpregnancy implies that the lead content of her trabecular bone, which dominated her blood lead at midpregnancy, was less than that of her cortical. This is not unreasonable, given that the subject was in her early 20s and that the high 206Pb/207Pb ratios of her blood imply exposure to an unusual source of lead in childhood. In the case of breast-feeding subject 107-1 (Fig. 8), the fit is excellent for the blood samples that were taken monthly while she was lactating. Thereafter, the every 6 months blood samples fall close to the sinusoid, but the spot urines at times fall consistently far from it. From

Fig. 7. Blood and urine 206Pb/207Pb ratios plotted against calendar month for bottle-feeding subject 112-1. She delivered her first child on April 12 of the first year of observation. When taken monthly, blood leads are joined by a solid line. When taken monthly, spot urines are joined by a dashed line. The heavy broken line is a sinusoid of the 365-day period and amplitude corresponding to the observed range in isotope ratios. Turning points in August and February appear to match the observations.

Fig. 8. Blood and urine 206Pb/207Pb ratios plotted against calendar month for breast-feeding subject 107-1. She delivered her fourth child on June 2 of the first year of observation. Blood leads, which were taken monthly until she stopped nursing, are joined by a solid line. Monthly spot urines are joined by a dashed line. The heavy broken line is a sinusoid of 365-day period and amplitude corresponding to the observed range in isotope ratios. Turning points in January and July appear to match the observations with the exception of the last 6 months of the second year, when spot urines fall far off the predicted curve.

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her winter minima, we again infer that as she approached midpregnancy her blood contained diminishing amounts of lead from cortical bone, but in late pregnancy the quantity began to increase. Although the frequency of the blood lead measurements is not ideal in these subjects and spot urines may at times be inaccurate measures of blood lead isotope ratio, the patterns lead to consistent and thus credible interpretations of what occurs in pregnancy. The isotopic pattern of subject 107-1’s blood was different the second time she lactated (Fig. 5). The reason may be the imposition of seasonal variations on her pattern of blood lead isotope ratios, which predict a minimum at month 42 and a maximum at month 48 (Fig. 5). It is important to note that the presence of seasonal variations in isotope ratios in lactation implies that the regions of the skeleton that are resorbed are the same as those in the nonpregnant, nonlactating state (or for that matter in the male) and that changes in blood lead concentration result solely from changes in the rate of resorption of bone. 4.7. Blood lead concentrations in multiparity It has been known for some years that blood leads of multiparous women tend to be lower than those of women delivering for the first time (Rabinowitz and Needleman, 1984; Rothenberg et al., 1994), leading to speculation that pregnancy removes lead from the skeleton. What we have observed in multiple pregnancies verifies this, and when Figs. 5 and 6 are taken together they give a dramatic illustration of the blood leads of a multiparous woman decreasing step-wise from pregnancy to pregnancy. 4.8. Calcium metabolism in pregnancy and lactation Up to this point we have restricted our discussion to observations on blood lead and have not considered to any extent what calcium kinetics, the calciotropic hormones, or markers of bone formation and resorption imply about the skeleton’s behavior in pregnancy and lactation. In this section we summarize the calcium literature; in the final section of the paper we examine what we have inferred from lead in the light of current knowledge of calcium metabolism in order to evaluate to what extent our interpretations might be universally applicable, bearing in mind that they were based essentially on two subjects whose behavior may well have been idiosyncratic. The literature on calcium, already vast, grows annually. Fortunately, it is periodically reviewed (Kalkwarf, 1999; King et al., 1992; Kovacs and Kronenberg, 1997; Pitkin, 1975, 1985, 1992; Prentice, 2000). These facts seem established. In pregnancy the mother delivers about 30 g of calcium to the fetus (Pitkin, 1985), most of

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which is supplied in the third trimester (Forbes, 1976). If she breast feeds for 6 months she will pass a further 47– 63 g to her child at a rate of 240–320 mg/day (King et al., 1992). The calcium demands of pregnancy are met by increased gastrointestinal absorption (Cross et al., 1995a; Kent et al., 1991a; Ritchie et al., 1998), which is promoted by increased synthesis of 1,25-dihydroxyvitamin D (1,25 (OH)2 D3) (Kumar et al., 1979; Seely et al., 1997; Specker et al., 1994). Absorption rises in the first trimester long before appreciable amounts of calcium are needed by the fetus. Pregnancy is a state of increasing bone turnover, shown both by kinetic studies (Heaney and Skillman, 1971) and by increases in the concentrations of markers of bone formation and resorption (Cross et al., 1995a; Naylor et al., 2000). Once lactation begins, 1,25 (OH)2 D3 levels almost immediately decline (Cross et al., 1995a; Reddy et al., 1983; Wilson et al., 1990) and gastrointestinal absorption of calcium returns to normal (Specker et al., 1994). Calcium is conserved at the kidney (Kent et al., 1991b; Ritchie et al., 1998) and the skeleton is resorbed, not under the action of the calciotropic hormones, but under falling estrogen levels (Cross et al., 1995b; Kalkwarf, 1999). The amount of bone loss may be as high as 10% in women who nurse for prolonged periods (Kolthoff et al., 1998). During pregnancy, markers of bone resorption progressively rise above normal levels, but markers of bone formation fall before rising (Cross et al., 1995a; Naylor et al., 2000). From similar observations Black et al. (2000) argued that in the first two trimesters of pregnancy bone formation and bone resorption are uncoupled. Bone mineral density measurements indicate that losses occur in trabecular bone (Black et al., 2000; Gambacciani et al., 1995; Naylor et al., 2000; Sowers et al., 2000; Yamaga et al., 1996). Bone biopsies made at clinically terminated human pregnancies (Purdie et al., 1988; Shahtaheri et al., 1999) and during nonhuman primate pregnancies (Ott et al., 1999) confirm both the reduction in bone formation and the loss of trabecular bone in early pregnancy. This loss is, however, temporary and at term trabecular bone volume is completely restored (Shahtaheri et al., 1999). Whole body scans of 47 women made between 0.5 and 5 months of lactation (Laskey et al., 1998) show the greatest loss in bone mineral content occurring at the lumbar and thoracic spine (3–4%), followed by the pelvis and ribs (1–2%) and the head (1%). Losses from the arms and legs were 0.1% or less. Polatti et al. (1999) in a study of 308 women who elected to breast feed for 6 months found the loss in bone mineral in the spine and radius greater at 6 months than at 3. Hopkinson et al. (2000) measured the change of bone mass at intervals over a period of 2 years for nonlactating and lactating women. They found that for those who nursed for an extended period, the maximum loss of bone mineral

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occurred at different times for different regions: earliest in the vertebrae (3–6 months), next in the pelvis (6 months), and lastly in the head (12 months). In contrast to the work of Laskey et al. (1998), they report the legs as never recovering from a 6% loss of bone mineral at 6 months. For 2–4 weeks postpartum, bone resorption as measured by deoxypyridinoline excretion rapidly increases while bone formation declines (Dahlman et al., 1994; Naylor et al., 2000; Prentice et al., 1998; Yamaga et al., 1996). Thereafter, deoxypyridinoline excretion falls to levels characteristic of midpregnancy and about twice those of the nonpregnant, nonlactating state (Black et al., 2000; Cross et al, 1995b; Prentice et al., 1998; Yamaga et al., 1996). Women who nurse for 6–9 months more than recover losses in bone mineral by 12 months postpartum (Polatti et al., 1999; Hopkinson et al., 2000), but those who do so for more than 9 months are still deficient 2 years after delivery (Hopkinson et al., 2000). 4.9. Inferences from lead vis-a`-vis calcium Lead, being, like calcium, a divalent ion, is absorbed through the gut by vitamin D-regulated mechanisms and is deposited in and removed from bone by the osteocytes. In blood, most calcium is bound to albumin and most lead to the red cells, so that the concentrations of both are affected by changes in blood volume. The principal difference between these two elements is that the concentration of ionized calcium in blood is homeostatically regulated while that of lead is not. Lead is slowly excreted by the kidney, with the amount lost being proportional to the concentration in blood plasma. Thus, if the flux of lead into blood increases, concentrations will rise along curves of the form 1  ekt ; if it decreases, blood lead will fall exponentially as ekt : The half-life of the change is long, however (about 3 weeks), because more than 99% of the lead in blood resides on the red cells. An increase in blood lead concentration attributable to resorption of bone may be interpreted in two ways: either resorption in a particular part of the skeleton has accelerated; or resorption has changed to a site of higher lead content without an increase in or even with a decline in resorption rate. The converse is also possible, and blood lead concentration may fall with increasing bone resorption if the site of resorption moves to one containing less lead. Isotope ratios detect shifts in the sites where bone is being resorbed. As such they are unique and can detect processes that are invisible to biomarkers and bone mineral density measurements. In normal circumstances they give no information pertaining to bone formation. If it is assumed that present day dietary lead levels are so low that even in late pregnancy negligible gastrointestinal absorption of lead occurs, then the interpretations put forward in this study are in accord

with current understanding of the behavior of the skeleton in that we have argued for an increase in bone resorption in late pregnancy which continues into lactation. There are, however, major differences, especially in pregnancy, where the blood lead concentrations indicate a decline in bone resorption in midpregnancy, while the biomarkers indicate an increase. This contradiction can be resolved if the site of resorption moves from one of higher to lower lead content, and we have argued from changing isotope ratios that the resorption of cortical bone diminishes in favor of trabecular bone in midpregnancy. Support for our interpretation comes from Shahtaheri et al.’s (1999) reexamination of Purdie et al.’s (1988) transiliac crest biopsies, which were taken in the 8th–10th week of pregnancy. They found a 27% reduction in trabecular bone mass compared to that of nonpregnant controls which had been brought about largely by thinning of the trabeculae and by a loss of connectivity. In biopsies taken at term, however, they found complete restoration (with subtle changes in architecture) of trabecular bone mass. This observation is of interest because it implies that demands made upon the skeleton in late pregnancy to supply calcium to the fetus can only be met by resorption of cortical bone, which is precisely what we have argued. Naylor et al.’s (2000) observation that cortical bone accumulates bone mineral over the course of pregnancy is not contradictory because they argued that such bone was periosteally deposited, whereas what we have isotopically identified as cortical must necessarily come from deep within the skeleton. In the case of the lactating women, especially subject 107-1 (Fig. 2), we observed blood lead concentrations increasing from late pregnancy into lactation without hiatus or change in slope. We expected markers of bone resorption to do likewise, but Cross et al. (1995a) report them to be lower in lactation than in the third trimester of pregnancy. The rise in blood lead over a period of 8 months that we observed in the women who breast fed for 6 months or more, subjects 107-1 (Fig. 2) and 102-1 and 105-1 (Fig 3), corresponds to the 6-month decline in the bone mineral content of the subjects that Hopkinson et al. (2000) studied. Their measurement intervals were such that they were unable to define the point at which maximum loss occurred, but some time after 6 months the bone mineral content of their subjects began to increase and continued to do so for up to 24 months after delivery. Since the amount of lead in blood measures the activity of the whole skeleton, it is not surprising that it is in comparison with whole body measurements of bone mineral that we find the closest agreement with inferences made from blood lead concentrations. It is possible that the continuous decrease in blood lead seen in subject 107-1 (Fig. 5) is a manifestation of this process of remineralization of the skeleton.

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4.10. Concluding remarks We have seen increases in blood lead concentration in late pregnancy and in lactation. None has been large enough in absolute terms to cause concern, and with blood lead concentrations in the United States as low as they are today it is doubtful that lead stored in bone poses a significant threat to the health of the majority of pregnant women and their children. There will, however, always be cases both in the United States and in other countries in which a woman has been exposed to lead in childhood and has accumulated large stores in bone. The greatest probability of lead toxicity will be associated with her first pregnancy, but will occur at different times for her and her child. Her most dangerous time will be postpartum while her blood lead rises during nursing, but her child’s will be prenatal and especially in the last weeks of pregnancy. The child’s risk of toxicity is small during nursing because the intestinal absorption of lead from milk appears to be less than was previously thought, and blood leads of children born to mothers whose blood leads rise while nursing actually decline after delivery (Manton et al., 2000). We have made inferences regarding the amounts of lead derived from cortical and trabecular bone from seasonal changes in blood lead isotope ratios, which were discovered in this laboratory 25 years ago (Manton, 1977). Their correlation with cycles of 25hydroxyvitamin D appeared important at the time, but once it was realized that this particular metabolite was inactive their significance became questionable. Recently, however, Woitge et al. (2000) have taken up the question of seasonal changes in bone resorption and, besides confirming the strong seasonality of 25-hydroxyvitamin D, have found an equally strong but antiphasal seasonality in deoxypyridinoline excretion that unequivocally points to increased bone resorption in winter. This is the same conclusion as was reached by Manton (1977), making us confident that our inferences regarding the proportions of blood lead from cortical and trabecular bone rest on a physiologic process that is demonstrable and real, even though its cause may not be understood. Considerations of changes in lead isotope ratios have led us to postulate that resorption of trabecular and cortical bone are at times independent, which implies that each is regulated by a separate mechanism. This is not at variance with current thinking, as Gorski (1998) has argued that woven and lamellar bone are synthesized differently and Ninomiya et al. (1990) consider the two varieties of lamellar bone, trabecular and cortical, to be biochemically distinct. Reports of differing responses of bone to various treatment regimens appear to support such views (Rico, 1997). In the extreme case, our interpretations of lead isotope ratios taken together with those of Shahtaheri et al. (1999) of bone biopsies

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imply that in late pregnancy trabecular bone is in an anabolic state while cortical bone is catabolic. Again there is a clinical parallel: Riggs et al. (1990) conducted a 4-year clinical trial of sodium fluoride administered to 66 osteoporotic, postmenopausal women and found, compared to 69 women receiving a placebo, that a 35% increase in bone mineral density occurred in the lumbar spine while a 4% decrease occurred in the shaft of the radius. Research on lead has always been directed toward assessing its potential to do harm; that it might prove to be a unique index of bone resorption has never been the primary focus of any study. We hope that in this paper we have demonstrated that residues of lead in the environment may be turned to some advantage. It is doubtful, however, that in another prospective study we would find subjects like those we were fortunate enough to have for this study. The greatest chance of successfully using lead isotopes in bone research lies with primate studies, in which dosing can be controlled. The papers of Inskip et al. (1996) and Franklin et al. (1997) amply bear this out.

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