The populations most sensitive to lead exposure from various sources are pregnant women

and young children (National Research

Council 1993). Ongoing lead exposure not

only directly affects health, but also allows the

accumulation of lead in tissues such as bone.

During pregnancy, the mobilization of bone

lead increases, apparently as the bone is

resorbed to produce the fetal skeleton (Franklin

et al. 1997; Gulson et al. 1997a, 1998b;

Manton 1985). Previous studies using only

lead concentrations for a limited number of

samples (usually three to five) generally showed

an increase in blood lead levels during the

third trimester, which was attributed to

increased bone resorption to meet the calcium

requirements of the developing fetus (HertzPicciotto et al. 2000; Lagerkvist et al. 1996;

Rothenberg et al. 1994; Schell et al. 2000,

2003; Schumacher et al. 1996; Sowers et al.

2002; West et al. 1994).

Recent studies, especially those employing

precision stable lead isotope methods, have

confirmed the early work of Manton (1985)

and demonstrated that extra lead is released

from the maternal skeleton during pregnancy

and lactation in cynomolgus monkeys (Franklin

et al. 1997; O’Flaherty et al. 1998) and in

humans (Gulson et al. 1997a, 1998b, 2003;

Manton et al. 2003). Furthermore, these

studies showed that the lead is transferred to

the infant.

Because recent evidence suggests that

there are detrimental neurologic effects on

young children whose blood lead levels are

< 10 μg/dL (Canfield et al. 2003; Lanphear

et al. 2000), it is critical to minimize the

exposure of the developing fetus and the newborn infant, especially lead from bone.

Results of several cross-sectional studies

have indicated that increased levels of calcium

during pregnancy have a protective effect,

reducing the amount of lead released from

bones (Farias et al. 1996; Hernandez-Avila

et al. 1996). Calcium carbonate supplementation of 1,200 mg elemental calcium/day gave

a modest reduction of 16% in blood lead levels among lactating women with relatively

high bone lead burden (Hernandez-Avila et

al. 2003). In our longitudinal study of lead

mobilization from bone during pregnancy

and for 6 months postpartum, we found that

both newly arrived migrants to Australia and

multigenerational Australian subjects consumed very low amounts of calcium in their

diets. Estimations were based on quarterly 6-

day duplicate diets and averaged about

400–600 mg calcium/day (Gulson et al.

2001), less than half the intakes recommended by the National Institutes of Health

(1994).

Two of the migrant subjects who consumed calcium supplements showed lower

amounts of lead mobilized from their skeleton

compared with the others on low calcium

intakes. In light of this finding and those of the

previous studies mentioned above, we undertook a longitudinal investigation in which subjects were provided with calcium supplements

during pregnancy and for 6 months postpartum. Our hypothesis was that if calcium

supplementation is effective in reducing the

mobilization of lead from the maternal skeleton, then there should be little or no change in

blood lead or isotopic ratios during pregnancy

and postpartum.

Materials and Methods

We employed the protocols refined during

previous investigations (Gulson et al. 1995,

1997a, 1998b).

Environmental Health Perspectives • VOLUME 112 | NUMBER 15 | November 2004 1499

Address correspondence to B.L. Gulson, Graduate

School of the Environment, Macquarie University,

Sydney, NSW 2109 Australia. Telephone: 61-2-

9850-7983. Fax: 61-2-9850-7972. E-mail: bgulson@

gse.mq.edu.au

We thank M. Salter for phlebotomy, L. Munoz for

technical assistance, the participants in this study,

English Language Schools in Sydney, B. Jameson and

B. Ragan of the National Institute of Environmental

Health Sciences (NIEHS) and P. Mushak for their

support and encouragement over many years, J. Fouts

for reviewing an earlier version of the manuscript, and

R. Setright of Blackmores for supply of one of the

products.

This research was largely funded by the NIEHS

through NO1-ES0252.

The authors declare they have no competing

financial interests.

Received 26 June 2003; accepted 27 July 2004.

Research | Article

Blood Lead Changes during Pregnancy and Postpartum with Calcium

Supplementation

Brian L. Gulson,1,2 Karen J. Mizon,1 Jacqueline M. Palmer,2 Michael J. Korsch,2 Alan J. Taylor,3

and Kathryn R. Mahaffey4

1Graduate School of the Environment, Macquarie University, Sydney, New South Wales, Australia; 2Commonwealth Scientific and

Industrial Research Organisation/Division of Exploration and Mining, North Ryde, New South Wales, Australia; 3Department of

Psychology, Macquarie University, Sydney, New South Wales, Australia; 4U.S. Environmental Protection Agency, Office of Prevention,

Pesticides and Toxic Substances, Washington, DC, USA

Pregnancy and lactation are times of physiologic stress during which bone turnover is accelerated.

Previous studies have demonstrated that there is increased mobilization of lead from the maternal

skeleton at this time and that calcium supplementation may have a protective effect. Ten immigrants

to Australia were provided with either calcium carbonate or a complex calcium supplement

(~ 1 g/day) during pregnancy and for 6 months postpartum. Two immigrant subjects who did not

conceive acted as controls. Sampling involved monthly venous blood samples throughout pregnancy

and every 2 months postpartum, and quarterly environmental samples and 6-day duplicate diets. The

geometric mean blood lead at the time of first sampling was 2.4 μg/dL (range, 1.4–6.5). Increases in

blood lead during the third trimester, corrected for hematocrit, compared with the minimum value

observed, varied from 10 to 50%, with a geometric mean of 25%. The increases generally occurred at

6–8 months gestation, in contrast with that found for a previous cohort, characterized by very low calcium intakes, where the increases occurred at 3–6 months. Large increases in blood lead concentration

were found during the postpartum period compared with those during pregnancy; blood lead concentrations increased by between 30 and 95% (geometric mean 65%; n = 8) from the minimum value

observed during late pregnancy. From late pregnancy through postpartum, there were significant

increases in the lead isotopic ratios from the minimum value observed during late pregnancy for 3 of

8 subjects (p < 0.01). The observed changes are considered to reflect increases in mobilization of lead

from the skeleton despite calcium supplementation. The identical isotopic ratios in maternal and cord

blood provide further confirmation of placental transfer of lead. The extra flux released from bone

during late pregnancy and postpartum varies from 50 to 380 μg lead (geometric mean, 145 μg lead)

compared with 330 μg lead in the previous cohort. For subjects replete in calcium, the delay in

increase in blood lead and halving of the extra flux released from bone during late pregnancy and postpartum may provide less lead exposure to the developing fetus and newly born infant. Nevertheless, as

shown in several other studies on calcium relationships with bone turnover, calcium supplementation

appears to provide limited benefit for lead toxicity during lactation. Key words: blood, bone remodeling, calcium supplementation, lead, lead isotopes, postpartum, pregnancy. Environ Health Perspect

112:1499–1507 (2004). doi:10.1289/ehp.6548 available via http://dx.doi.org/ [Online 27 July 2004]
Subjects. Participants were female immigrants to Australia who were of child-bearing

age (19–32 years) and whose skeletal lead

isotopic composition, based on our previous

investigations and an initial blood sample, was

different from that in their current environment. In essence, the lead isotopic composition or “fingerprint” in multigenerational

Australian residents is different from that in

subjects from most other countries, although

these differences are reducing over the past

decade because of globalization (Gulson 2000;

Gulson et al. 2001). Hence, by monitoring

migrant subjects who conceive after arrival in

Australia, it is possible to detect changes in

isotopic composition and lead during pregnancy and the postpartum period arising from

increased mobilization of skeletal lead. The

approach of using subjects whose skeletal lead

was different from that in their current environment to determine exchange phenomenon

was first explored by Manton (1977, 1985)

and later reiterated by Rabinowitz (1991).

Subjects were recruited by networking

through our previous cohort study (Gulston

et al. 1998b), from English language classes,

and via limited advertising in ethnic newspapers. Compared with the previous cohort,

there are some differences in demographics of

the subjects in our present study. For example,

in the previous cohort, most subjects were

from Eastern Bloc countries, such as the former Soviet Union and former Yugoslavia.

With changing migration patterns to Australia,

subjects in the present study were from South

Asia and the Middle East (Table 1). One subject (subject no. 1226) was approximately

4 months pregnant when recruited but was

retained because our previous investigations of

people from other countries showed that, in all

cases, their isotopic compositions were different from that of long-term Australians. Subject

1208 miscarried during the first trimester of

her first pregnancy. Subject 1231 returned to

China immediately after the birth of her child,

so no postpartum results are available. Two

nonpregnant control subjects were matched

mainly on country of origin as other parameters such as age, parity, and blood lead concentration were found not to be critical in

previous studies (Gulson et al. 1997a, 1998b).

Signed consent forms were obtained from

each volunteer. This consent form had been

reviewed and approved by the ethics committees of the Central Sydney Area Health Service,

Western Sydney Area Health Service, and

Macquarie University. As part of the entry

requirements into Australia, all subjects were

declared medically fit.

Sampling and analysis. Blood and urine

samples were obtained monthly during pregnancy and the postpregnancy period for

≥ 6 months. The urine samples were collected

to back up the blood samples in case isotopic

data could not be obtained from the blood

samples. Environmental samples of 6-day

duplicate diet, drinking water, house dust,

gasoline, and ambient air were collected quarterly. High-precision lead isotope ratios and

lead concentrations were obtained by thermal

ionization mass spectrometry. Details of analytical methods have been published previously (Gulson et al. 1995, 1997a, 1998b).

As in our previous reports (Gulson et al.

1995, 1997a, 1998b), our data are expressed as

206Pb:204Pb ratios for ease of identifying and

illustrating changes, especially for readers with

limited experience in isotopic methods and for

whom significant differences in the third decimal place of a ratio of, say 0.900, may seem

unrealistic. Although Manton and colleagues

(2003) express results as 206Pb:207Pb, direct

comparisons of variations can be made between

our data because 206Pb is the numerator in both

ratios. For the statistical analyses, both the

measured 207Pb:206Pb and 206Pb:204Pb ratios

were treated.

Hematocrit was measured in blood samples and creatinine in the urine samples using

standard methods.

Calcium supplements. Because it was not

viable to produce a custom supplement for

this study, commercially available Australian

calcium supplements, which are typically

made from overseas components, were administered to the subjects. Details of the supplements are given in Table 2. The supplements

were those with the lowest concentrations of

lead and were evaluated as suitable in the

following investigation.

Approximately 6 months into the present

study, we investigated the impact on blood lead

of the calcium supplements to be provided to

the subjects. This was necessary to satisfy ourselves that we were not “poisoning” the subjects and their developing fetuses because there

had been much recent publicity about the

potential toxicity of supplements (Boulos and

Smolinski 1988; Bourgoin et al. 1993; Capar

and Gould 1979; Rogan et al. 1999; Scelfo

and Flegal 2000). This publicity occurred

despite earlier work from the 1980s indicating

that calcium (plus phosphorus) inhibited the

uptake of lead from the gastrointestinal tract.

Furthermore, Rogan et al. (1999) discovered

that vitamin supplements used in the multicenter lead trial were contaminated with lead.

Article | Gulson et al.

1500 VOLUME 112 | NUMBER 15 | November 2004 • Environmental Health Perspectives

Table 1. Subject information.

Subject Country of Time to conception No. of Age Calcium Average compliance Initial Pb Sex of

identifier origin (months) after arrival children (years) supplement (%) during pregnancy (μg/dL) newborn Breast-feeding

1204 Bulgaria 13 1 31 Carbonate 68 4.0 M > 6 months

1207a Croatia NA 1 26 NA NA 1.0 NA NA

1208b Bosnia 34 1 23 Carbonate 73 1.9 M/F 0

1211c Bangladesh 8 1 25 Carbonate 95 1.6 M 3 months

1212c Turkey 20 1 31 Carbonate 86 (1st 2 months) 2.4 F > 6 months

1213 Lebanon 4 0 32 Complex 71 (1st 6 months) 2.9 M < 2 weeks

1214 Turkey 14 0 25 Complex 52 1.8 F 0

1224a Bulgaria NA 0 31 NA NA 2.4 NA NA

1225c Pakistan 3 0 28 Complex 72 6.5 F 0

1226c,d Iraq 1 0 20 Complex 56 1.4 M > 4

1229 Lebanon 18 0 19 Complex 52 2.3 M < 1 month

1231a,e China 2 0 32 Carbonate 100 (6 months) 1.7 F Unknown

NA, not applicable.

aNonpregnant controls. bGave birth to twins. cPregnant when recruited. dAbout 4 months pregnant on recruitment. eSubject 1231 returned to China immediately after giving birth.

Table 2. Product information.

Product 1 (complex product) Product 2

Calcium Calcium Calcium Calcium carbonate +

Composition citrate phosphate amino acid vitamin D3

Weight Ca compound (mg) 300 325 200 1,500

Equivalent Ca (mg) 64 126 40 600

Daily total Ca (mg) 920 1,200

Daily dosage 3 times after meals, 1 on retiring Twice daily

206Pb:204Pb 20.1 18.5

Pb [μg/kg (μg/tablet)] 293 (0.4) 940 (1.6)

Daily Pb intake (μg) 2.8 3.2
Using a cohort of generally younger adults,

we showed that an extra intake of 900–1,000

mg calcium/day for 6 months had minimal

impact on the blood lead concentration

(Gulson et al. 2001). However, we observed

changes in the isotopic composition of the

blood with a calcium carbonate supplement

indicating that lead from the supplement was

entering blood, but at this stage we have no

explanation as to why there were no changes

in blood lead concentration.

Oral and written instructions regarding

dosage were given to the subjects. Compliance

relied largely on the loyalty established between

the cohort coordinator and the subjects to

accurately report compliance, and this was

checked monthly by tablet count.

A control group taking a placebo or not

taking calcium supplements was decided

against because, first, although investigated,

producing a placebo for such a small group of

subjects was not considered viable; second, the

much larger cohort in our previous pregnancy

investigations had very low calcium intakes,

and the present study was one outcome of

this; and third, financial constraints given the

expense of running such a project.

Data treatment. After a visual inspection of

graphic presentations, we analyzed changes in

206Pb:204Pb (and 207Pb:206Pb) ratios and blood

lead concentration over time from the minimum value of blood lead concentration before

an increase during late pregnancy using a

regression analysis for time series that took into

account any autocorrelation of the residuals

from the sequences of observations (the autoregression procedure in SPSS 11.0; Chicago, IL,

USA). The autocorrelation was taken into

account when calculating the significance and

magnitude of time effects (Ostrom 1990).

To assess changes in 206Pb:204Pb (and

207Pb:206Pb) ratios and blood lead concentration between pregnancy and postpartum for

individual subjects, we used an interrupted

time-series analysis procedure (Crosbie 1995).

In this analysis, the intercept and slope of the

pregnancy (baseline) regression line are jointly

compared with those of the regression fitted to

the postpartum (treatment) phase, and if the

joint test is significant, differences between the

two intercepts and two slopes can be considered. This procedure takes account of autocorrelation when fitting the lines and testing the

differences, and provides better control of type

I error (i.e., an erroneous inference of a significant difference between phases/stages in experiments) than does visual inspection and has

acceptable power. It requires measurements

at regular intervals, as exemplified by the

monthly blood sampling in our investigation.

We calculated the extra amount of lead

added to blood during pregnancy and postpartum using a similar area under the curve

method that was used for our previous

cohort (Gulson et al. 1999). This comparative graphic technique is commonly used

for cumulative estimates of lead exposure

by integrating blood lead concentration

across exposure times in occupational exposure (Chia et al. 1996) or for comparative

lead dosings of test animals in the study of

such exposure parameters as the relative

bioavailability of lead (Casteel et al. 1997).

Estimates of extra lead flux mobilized were

possible only for migrant subjects whose

blood lead concentrations exhibited positive

variations during the monitoring period

(n = 10). We calculated the extra flux by

estimating the total area generated by serial

blood lead concentrations versus pregnancy

and postpregnancy time and subtracting the

estimated background area, the portion of

the area representing blood lead over time

that would have occurred in the absence of

added lead releases. Background area was

generated from the lowest blood lead level

during late pregnancy, in contrast to our

previous study (Gulson et al. 1998b), in

which the minimum blood lead level usually,

although not always, occurred in the first

trimester.

Results

Changes in blood lead during pregnancy and

6 months postpartum. The data for the present

case series have been subdivided into two

groups depending on the length of breastfeeding: those who breast-fed for longer periods (Figure 1), in this case ≥ 3 months, and

those who breast-fed for shorter periods, either

not at all or for < 1 month (Figure 2).

To account for the well-recognized

changes in blood volume during pregnancy

(Hytten 1985), increases of which may result

in decreases in blood lead concentration, the

blood leads can be adjusted for changes in

hematocrit. Data for subjects 1212 and 1214

are shown in Figure 3, which illustrate the

changes in hematocrit and the hematocritcorrected and uncorrected blood lead concentrations over time. Hematocrit values

throughout pregnancy in the present case series

show decreases, little change, or decreases and

then increases in late pregnancy (0–2 months

before parturition). As shown in Figure 3, the

uncorrected and corrected blood lead values

follow similar trends and, because of the

“return-to-normal” hematocrit values during

the postpartum period and the importance of

this period for our results, measured blood

lead values are shown in the accompanying

figures.

Changes in blood lead during pregnancy

followed similar U-shaped patterns for the

two groups. From early to mid-pregnancy

the hematocrit-corrected blood lead concentrations decreased for 8 of the 10 subjects

and for the second pregnancy of subject

1208. During late pregnancy, the blood lead

Article | Extra calcium and blood lead in pregnancy and postpartum

Environmental Health Perspectives • VOLUME 112 | NUMBER 15 | November 2004 1501

Figure 1. Changes in blood lead concentration (not

corrected for hematocrit) for subjects who breastfed for ≥ 3 months. The length of breast-feeding is

noted to the right of the individual lines. Increases

in blood lead concentration occurred in late pregnancy compared with our previous cohort (Gulson

et al. 1998b), whose calcium intakes were very low.

There are significant increases during the late

pregnancy–postpartum period.

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Figure 2. Changes in blood lead concentration (not

corrected for hematocrit) for subjects who breastfed for ≤ 1 month. The length of breast-feeding is

noted to the right of the individual lines. Increases

in blood lead concentration occurred in late pregnancy compared with our previous cohort (Gulson

et al. 1998b), whose calcium intakes were very low.

There are significant increases during the late

pregnancy–postpartum period.

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Figure 3. Plot illustrating the changes in hematocrit

values (HCt) and uncorrected (PbB) and hematocrit-corrected (HCt Corr) blood lead concentrations for subjects 1212 and 1214. The U-shaped

pattern for blood lead concentrations over time

persists in the hematocrit-corrected values. The “0

days” scale is offset for subject 1212 and is

denoted for all subjects by the breaks in the lines.
concentrations increased in all subjects

(Table 3); the increase in blood lead concentration above a minimum value, corrected for

hematocrit, ranged from 10 to 55% (geometric mean, 25%), although the values did not

exceed those determined in prepregnancy or

early pregnancy.

Changes in blood lead from late pregnancy through the 6 months postpartum

were variable and did not appear to be related

to length of breast-feeding and calcium

intakes. The maximum value during postpartum the concentration was almost double the

minimum blood lead value during late pregnancy in half the subjects; the percentage

increase varied from 30 to 95%. For half the

subjects (subjects 1204, 1211, 1213, and

1226), the blood lead increased until the second or third month postpartum and then

either leveled or decreased (Figures 1 and 2).

Only in subjects 1212 and 1208 was there a

continuous linear increase from late pregnancy

through postpartum. The increases postpartum in both groups commonly exceeded

the values measured in prepregnancy or early

pregnancy.

Autoregression analyses of blood lead concentration from the time of minimum value in

late pregnancy through postpartum for eight

subjects are presented in Table 4. There are

significant positive increases for half the subjects and none for the other four (subjects

1211, 1213, 1214, 1226). However, visual

inspection of the plots (Figures 1 and 2) shows

that for the latter four, there are increases from

late pregnancy to the first 1 or 2 months postpartum and, thereafter, either a leveling or

decrease in blood lead.

For the interrupted time-series analyses, the

overall test of change was significant for five of

eight subjects in blood lead concentration during the whole of the pregnancy phase compared with the postpartum phase (Table 5).

There were significant differences in the blood

lead slopes for half of the eight subjects during

pregnancy compared with postpartum, and for

six of eight subjects there were significant

differences in intercept.

Subject 1225 showed a major increase in

blood lead concentration soon after parturition, with the value increasing from 5.6 to

about 43 μg/dL. This increase was accompanied by a large decrease in the 206Pb:204Pb

ratio toward Australian environmental values

(Figure 4).

The blood lead concentrations for the

controls showed only small variations and an

overall decrease over time (Figure 5).

As is well documented in the literature,

the cord blood lead concentrations were lower

than those of the last maternal sample taken

before parturition. The geometric mean ratio

of cord:maternal blood lead was 0.72, with a

range from 0.55 to 0.93, and the correlation

of cord versus maternal blood lead had an

r2 of 0.93.

Changes in isotopic ratios during pregnancy

and 6 months postpartum. As was the case for

blood lead concentrations, changes in isotopic

ratios varied but not with consistent trends

(Figures 6 and 7). In some subjects (1212,

1214, 1225, 1229) it was not possible to estimate any changes in isotopic composition

during pregnancy, especially late pregnancy,

because of a change in the slope or scatter of

the data (Table 3). In the other subjects, any

changes during late pregnancy ranged from little difference from the experimental error of

± 0.2% up to 0.8% for subject 1226.

Autoregression analyses for the 206Pb:204Pb

(and 207Pb:206Pb) ratio from the time of minimum value in late pregnancy through postpartum for eight subjects are presented in

Table 4. The analyses for both ratios give

consistent results, except for subject 1229,

although those for the 207Pb:206Pb ratio tend

to show better correlation than those for the

206Pb:204Pb ratio, probably because of the

higher precision and ease of measurement of

the 207Pb:206Pb ratio (~ 0.9) compared with

the larger 206Pb:204Pb ratio. Four of the eight

subjects who were monitored during the

6 postpartum months (1204, 1208, 1213,

1229) showed significant increases in the

206Pb:204Pb ratio, whereas there were minimal

changes for the other subjects.

For the interrupted time-series analyses, in

no case was there a significant overall test of

change for 206Pb:204Pb (and 207Pb:206Pb ratio;

not shown in Table 5), nor was there any significant difference in the slopes or intercepts

for the isotopic ratios (Table 5).

There were small variations in the isotopic

compositions of the control subjects (Figure 5).

The ratios for the Bulgarian subject decreased

slowly, reflecting an ongoing exchange between

her long-term body stores and Australian

environmental lead, as we have observed in

migrant subjects who did not conceive (Gulson

et al. 1997b).

Isotopic ratios in the cord blood samples

were similar to those in the maternal blood

samples taken before parturition (r2 = 0.98),

as we found in our previous studies (Gulson

et al. 1998b) and also observed by Manton

(1977, 1985). The similarity in isotopic ratios

of the maternal and cord blood samples provides confirmation of the placental transfer

of lead.

Dietary samples. Lead concentrations in

duplicate diet samples vary considerably both

between and within subjects. For example, the

geometric mean concentration for all subjects

was 7.1 μg/kg with a range of 3.0–20.1 μg/kg

(n = 48). In contrast, the range in 206Pb:204Pb

ratio was surprisingly less than expected, from

17.79 to 18.81, with a geometric mean ratio

of 18.25.

Article | Gulson et al.

1502 VOLUME 112 | NUMBER 15 | November 2004 • Environmental Health Perspectives

Table 3. Changes during pregnancy and postpartum.

Percent increase in

Month PbB late pregnancy Percent increase Percent increase Extra

Country of increased PbB postpartum in PbB:min fluxd

Identifier origin > minimuma (HCt corr) 206Pb:204Pbb PbB 206Pb:204Pbb valuec (μg)

1204 Bulgaria 8 40 0.17 30 0.33 90 380

1208 Bosnia 8 55 0.11 40 1.22 95 150

1211 Bangladesh 6 10 0.51 50 ND 90 125

1212 Turkey 8 10 ND 55 ND 95 235

1213 Lebanon 8 25 0.28 30 1.65 70 140

1214 Turkey 8 40 ND 10 0.61 40 80

1225 Pakistan 6 50 ND NA NA NA NA

1226 Iraq 8 20 0.80 20 ND 50 200

1229 Lebanon 6 10 ND 40 0.2 30 50

1231 China 4 40 0.33 NS NS NA 165a

Abbreviations: (HCt corr), (hematocrit corrected); min, minimum; NA, not applicable; ND, not able to be estimated

because of change in slope (see figures); NS, no samples; PbB, blood lead concentration. 

aDuring pregnancy. bRelative to experimental error of ± 0.2%. cLate pregnancy to postpartum. dExtra lead released from

bone during late pregnancy and postpartum.

Table 4. Results for autoregression analyses.

R2-value p-Value

Subject 206Pb:204Pba 207Pb:206Pb PbB 206Pb:204Pb 207Pb:206Pb PbB

1204 0.87 (10) 0.91 0.63 (9) < 0.001 < 0.001 0.007

1208 0.94 (9) 0.94 0.85 (9) < 0.0001 < 0.00001 < 0.001

1211 0.12 (11) 0.25 0.22 (10) 0.32 0.15 0.38

1212 0.09 (9) 0.23 0.94 (8) 0.46 0.25 0.002

1213 0.91 (7) 0.85 0.40 (6) 0.006 0.01 0.30

1214 0.12 (10) 0.60 0.09 (9) 0.35 0.07 0.37

1226 0.04 (9) 0.16 0.14 (8) 0.56 0.31 0.45

1229 0.76 (8) 0.28 0.77 (7) 0.002 0.21 0.002

Numbers of data points in analysis are given in parentheses. 

aCord blood value incorporated in analysis, except for subject 1208 (no sample available).
There does not appear to be any relationship between calcium intake and changes in

isotopic composition of blood lead during

pregnancy and postpartum. For example, subjects 1208 and 1213 exhibited the largest

increases in 206Pb:204Pb ratio and had a high

compliance with calcium supplements.

Likewise, subjects 1204 and 1212 had a high

compliance and showed large increases in

blood lead concentration postpartum. There

was also no relationship between the time of

increase in blood lead during pregnancy and

calcium intake.

Environmental samples. Dustfall accumulation ranged from 6 to 94 μg/m2/month

with a geometric mean of 17 μg/m2/month

(n = 36). The 206Pb:204Pb ratios ranged from

16.50 to 17.85 and were similar to values

measured in Sydney air from high-volume air

filter samples (Chiaradia et al. 1997); the geometric mean was 17.16.

Lead concentration in fully flushed drinking water ranged from 0.03 to 1.1 μg/dL with

a geometric mean 206Pb:204Pb ratio of 16.80;

at these very low concentrations, water has no

impact on blood lead.

Discussion

Changes in blood lead concentration and isotopic ratios during pregnancy and 6 months

postpartum. Mean increases of approximately

25% in blood lead concentration during late

pregnancy for our present case series with

calcium supplementation are consistent with

increases observed in our previous cohort

(Gulson et al. 1998b) with low calcium intakes

and in several other studies (Hertz-Picciotto

et al. 2000; Lagerkvist et al. 1996; Rothenberg

et al. 1994; Schell et al. 2000, 2003;

Schumacher et al. 1996; Sowers et al. 2002;

West et al. 1994). However, the increases

found in the present study occurred later in

pregnancy than in our previous cohort

(Gulson et al. 1998b) and in many of the

other studies (Hertz-Picciotto et al. 2000;

Lagerkvist et al. 1996; Rothenberg et al. 1994;

Schell et al. 2000, 2003; Schumacher et al.

1996; Sowers et al. 2002; West et al. 1994).

The changes in blood lead during the

whole of pregnancy follow a U-shaped curve,

as previously observed by Rothenberg et al.

(1994), Hertz-Picciotto et al. (2000), Sowers

et al. (2002), and Schell et al. (2000, 2003).

Apart from the recent study of Manton et al.

(2003) and our investigations (Gulson et al.

1995, 1997a, 1998b), only limited numbers

of samples were collected in the other studies,

usually a maximum of one for each trimester,

one at delivery, and one postpartum. In our

present case series, the changes in blood lead

during pregnancy were obvious whether the

blood lead values were corrected or uncorrected for hematocrit. Hematocrit-corrected

values did not alter the findings of HertzPicciotto et al. (2000) and Schell et al.

(2000), but in the case of Schumacher et al.

(1996), the decrease in uncorrected blood

lead in the first trimester compared with

prepregnancy was not observed for the

hematocrit-corrected values. Furthermore,

blood lead values in the second trimester

(~ 20 weeks) showed average increases of

approximately 14%, and for the maximum

increase observed in the 32-week sampling,

the change from the minimum value at

8 weeks was 36%. Sowers et al. (2002)

observed increases in blood lead from the

20th week to delivery in African Americans

(1.20–1.49 μg/dL; an increase of 24%) and

Hispanic Americans (0.99–1.32 μg/dL; an

increase of 33%) but a barely detectable

decrease in blood lead of 0.03 μg/dL at

delivery for Caucasians. Schell et al. (2003)

observed smaller increases in blood lead from

the second trimester to delivery in whites

(1.6–1.8 μg/dL; an increase of 12%) compared with African Americans (1.8–2.5 μg/dL;

an increase of 39%).

Four of the eight subjects in the present

case series showed linear increases in blood

Article | Extra calcium and blood lead in pregnancy and postpartum

Environmental Health Perspectives • VOLUME 112 | NUMBER 15 | November 2004 1503

Table 5. Results for interrupted time-series analyses.

No. of measures Sum squares intercept Sum squares slope Overall test of change

Subject Pregnancy PP t-Value p-Value t-Value p-Value F-value p-Value

1204 7 7 1.40 0.194 0.45 0.664 0.14 (2,9) 0.87

8 6 4.12 0.003 –3.14 0.012 5.75 (2,9) 0.025

1208 7 6 0.52 0.617 1.41 0.197 1.16 (2,8) 0.36

7 6 4.79 0.001 –3.35 0.010 5.81 (2,8) 0.028

1211 8 7 –1.94 0.081 1.41 0.188 1.28 (2,10) 0.32

8 6 2.50 0.034 –2.15 0.06 4.91 (2,9) 0.036

1212 9 7 2.03 0.068 –1.45 0.175 2.01 (2,11) 0.181

9 6 2.70 0.022 –1.21 0.255 0.83 (2,10) 0.464

1213 9 5 –0.11 0.915 1.39 0.197 1.50 (2,9) 0.274

9 4 4.77 0.001 –4.0 0.004 8.01 (2,8) 0.012

1214 10 7 1.23 0.241 0.22 0.828 3.60 (2,12) 0.060

10 6 –1.21 0.253 1.04 0.320 0.64 (2,11) 0.547

1226 6 8 1.57 0.15 –1.02 0.34 1.05 (2,9) 0.39

6 7 3.94 0.004 –4.39 0.002 10.27(2,8) 0.006

1229 8 5 –0.05 0.964 1.11 0.301 1.09 (2,8) 0.380

8 4 1.32 0.229 –0.76 0.474 1.10 (2,7) 0.385

PP, postpartum. For all subjects, first row is 206Pb:204Pb and second row is PbB. Values in parentheses are degrees of

freedom. Cord blood value incorporated in postpartum analysis, except for subject 1208 (no sample available).

Figure 4. Plot of blood lead concentration (PbB)

and isotopic ratios for subject 1225, who experienced a major unknown lead exposure soon after

parturition (Part).

Figure 5. Plot of blood lead concentration (PbB) and

isotopic ratios for subjects 1207 and 1224, who acted

as nonpregnant controls.

Figure 6. Changes in isotopic ratio expressed as 206Pb:204Pb for subjects who breast-fed for ≥ 3 months.

The length of breast-feeding is noted to the right of

the individual lines.

Figure 7. Changes in isotopic ratio expressed as 206Pb:204Pb for subjects who breast-fed for ≤ 1 month.

The length of breast-feeding is noted to the right of

the individual lines.
lead concentration from the minimum in

late pregnancy through postpartum, but there

was no consistency with length of breastfeeding or compliance with calcium supplementation. Manton et al. (2003) also found

major increases in blood lead concentration by

up to a factor of 3 from a minimum value during pregnancy to postpartum. The magnitude

of the increases was attributed partly to breastor bottle-feeding by the mothers. In contrast

to the changes observed in the above investigations, Berglund et al. (2000) could not

detect any increase in blood lead during pregnancy, but they found a significant increase in

blood lead concentration during lactation.

Extra blood lead and calcium supplementation. For our present case series, with

subjects taking calcium supplements, the

increases in blood lead from the minimum

value during late pregnancy took place at

about 6–8 months in all subjects. In contrast, for the previous cohort (Gulson et al.

1998b), whose daily calcium intake was

generally very low, an increase in blood lead

from the minimum occurred at 3–4 months

for six of the nine subjects. Thus, even

though there is an increase of similar magnitude in blood lead from a minimum value

during pregnancy in both these cohorts, the

fetus in the group receiving calcium supplements would be exposed to considerably

lower lead flux than would those whose

mothers had a low calcium intake. This is

confirmed by the lower mean value of

145 μg lead for the extra flux to blood during late pregnancy through postpartum for

the present higher-calcium-intake subjects

compared with the mean of 330 μg lead for

the low-calcium-intake cohort. In breastfeeding mothers who were consuming close

to, or exceeding, what they recommended as

the daily calcium intake of 1,000 mg/day,

Manton et al. (2003) suggested there was no

tendency for increases in blood lead in late

pregnancy. However, inspection of their

Figure 2 shows that there are increases in

blood lead concentration in the last

2 months of pregnancy; increases in blood

lead in late pregnancy also occurred for at

least four bottle-feeding mothers with varying calcium intakes, consistent with our

observations.

We argued that the changes in blood lead

concentration and isotopic composition during pregnancy and postpartum in our previous cohort (Gulson et al. 1998b) reflected

increased mobilization of lead from the

maternal skeleton, probably associated with

the low calcium intakes of the subjects

(Gulson et al. 1998b). Any changes in blood

lead concentration during pregnancy and

postpartum are a function of plasma volume,

red cell mass, and variations in lead flux from

endogenous and exogenous sources. Low

blood lead concentrations during the first half

of pregnancy have been attributed to hemodilution (Rothenberg et al. 1994), which has

been associated mainly with an increased

plasma volume, calculated by Hytten (1985)

to be approximately 1,250 mL for a normal

pregnancy. Hytten (1985) showed that once

the “plateau” of increased plasma volume had

been attained at about week 30 of pregnancy,

there was minimal increase in plasma volume

and, as expected, little change during postpartum. That is, the dilution effects of increased

plasma volume on blood lead concentration

should be most marked during the second

and early third trimesters. Hytten (1985) also

drew attention to changes in red cell mass and

the impact of iron supplementation on this.

The changes in volume are mirrored in hematocrit, which decreases until third trimester

and then increases above normal values until

early in postpartum (Figure 3; also see Schell

et al. 2000, their Figure 2). During pregnancy, if the flux of lead into blood remains

constant, blood lead concentration will

decrease as blood volume increases. On the

other hand, if blood lead concentrations

remain constant or increase during late pregnancy, this suggests that there has been an

increase in lead flux into the blood.

The decreases in blood lead from early to

mid-pregnancy in our present and previous

cohort (Gulson et al. 1998b) are consistent

with a hypothesis of reduced flux from the

skeleton, as indicated by our estimations of

flux. Such a reduction in lead flux was noted by

Franklin et al. (1997) in cynomolgus monkeys

and in one subject in the recent study of

Manton et al. (2003). Although they did not

report data from early pregnancy, Manton et al.

(2003) hypothesized that in early (to mid) pregnancy, only trabecular bone of presumably low

lead content was resorbed, decreasing lead concentrations more than expected from hemodilution alone. In late pregnancy, more cortical

bone with a presumed higher lead content was

resorbed, increasing blood lead concentrations.

Such a hypothesis would appear to conflict with

measurements of bone lead over the past

decades showing that patella or calcaneus (trabecular) bone has higher concentrations than

does tibia (cortical) bone (e.g., Hernandez-Avila

et al. 2002; Oliveira et al. 2002; Rothenberg

et al. 2000) and that the trabecular/cortical bone

lead ratio ranges from 1 to 2. Nevertheless, the

total body burden of lead in cortical bone is

generally higher than in trabecular bone

because the proportion of cortical:trabecular

bone is about 4:1.

Bone research of relevance to the changes

in lead during pregnancy and lactation. The

linear increases in blood lead isotopic ratio,

blood lead concentration, and increased lead

flux to blood during late pregnancy and postpartum in half the subjects from the present

case series and most subjects in our previous

cohort (Gulson et al. 1998b) are attributed to

increased flux from the maternal skeleton,

despite increased calcium intake for the present case series.

Rather than cortical bone providing the

major contribution to increased blood lead, as

suggested by Manton et al. (2003), it may

well be trabecular bone. For example, based

on bone X-ray fluorescence measurements,

Schütz et al. (1987) and Hu et al. (1989) suggested that trabecular bone exerted the most

influence on blood lead and that this was consistent with the higher vascular and turnover

rates associated with trabecular compared

with cortical bone.

Bone mineral density and biochemical

markers of bone turnover and especially resorption during pregnancy and lactation appear

to affect trabecular more than cortical bone

(Black et al. 2000; Kolthoff et al. 1998; Naylor

et al. 2000; Sowers et al. 2000) as a result of

having more cancellous (trabecular) bone

surfaces available for turnover (Sowers et al.

2000). Even though Sowers et al. (2000) stated

that findings of previous studies were inconsistent, there was a convergence toward bone loss

during pregnancy and lactation. For example,

there are findings of increase at localized bone

sites (Drinkwater and Chestnut 1991), no

change (Sowers et al. 1991), and bone loss

(Aguado et al. 1998; Honda et al. 1998;

Kolthoff et al. 1998; Lamke et al. 1977; Laskey

et al. 1998; Naylor et al. 2000; Pires et al.

2002; Ritchie et al. 1998; Sowers et al. 1995).

Lactation would appear to be associated with a

larger decrease in bone density and net bone

loss of from 1 to 5% during 3–6 months postpartum compared with pregnancy (Laskey

et al. 1998; Pires et al. 2002; Ritchie et al.

1998; Sowers et al. 1995).

Bone research can provide further useful

information with respect to the observation in

lead pregnancy studies that the most significant increase in blood lead concentration (and

significant changes in isotopic ratios) occurs in

late pregnancy. For example, Sowers et al.

(2000) confirmed that there was increased

bone turnover in the third trimester of pregnancy compared with the first trimester, as

determined by measurement of the urinary

excretion of markers of type I collagen, but

suggested that the mechanisms of bone resorption during pregnancy are poorly understood.

Furthermore, the data of Black et al. (2000)

and Naylor et al. (2000) indicated that there

was a dissociation of bone formation and

resorption in the first two trimesters and well

into the third trimester of pregnancy. They

suggested that the gain in bone mineral density at cortical bone sites during pregnancy

may result from the redistribution of mineral

from trabecular to cortical bone sites and that

elevated bone turnover may explain trabecular

Article | Gulson et al.

1504 VOLUME 112 | NUMBER 15 | November 2004 • Environmental Health Perspectives
bone loss during pregnancy. On the other

hand, a study of primates using the stable lead

isotope method indicated that there was a

reduction in first-trimester bone mobilization

(Franklin et al. 1997). These conclusions are,

however, inconsistent with data from bone

mineral density and bone turnover indices

demonstrating that bone resorption increased

in the first trimester. For example, the bone

biopsy data of Purdie et al. (1988) in women

at the time of termination of pregnancy at

12–14 weeks of gestation showed that bone

resorption predominated in early pregnancy.

Likewise, Shahtaheri et al. (1999) reported

early bone loss in bone biopsy samples from

15 women in their first trimesters, whereas at

term in another 13 women they found new

and more numerous (but thinner) trabeculae.

Bone turnover and calcium and lead relationships during pregnancy and lactation.

Changes in blood lead during pregnancy and

lactation are inexorably linked with changes

in calcium. During pregnancy and lactation,

there is increased demand for calcium for

transport to the fetus. The maternal response

to the demand for calcium theoretically can

involve increased absorption of calcium from

the intestine, greater calcium conservation by

the kidneys, or greater bone turnover (Sowers

et al. 2000). There are, however, wide disparities in the literature on both bone/calcium

and lead about the efficacy of calcium intakes

during pregnancy and lactation.

Several authors consider that bone mineral changes especially during lactation are hormonally regulated and independent of

the amount of calcium in the woman’s diet;

provision of additional calcium has minimal

impact on preventing bone resorption at sites

such as the spine and femur (Cross et al.

1995; Kalkwarf 1999; Kolthoff et al. 1998;

Laskey et al. 1998; Prentice 2000). For example, Prentice (2000) states there are firm data

demonstrating that a low calcium intake

during lactation does not lead to impaired

lactational performance or to exaggerated

bone loss.

In contrast, previous evidence suggested

that bone loss during lactation in adolescents

may be prevented by adequate dietary calcium intakes (Chan et al. 1987). Later on,

Krebs et al. (1997) suggested that bone loss

during lactation may be attenuated by a

generous dietary ratio of calcium to protein.

Specker et al. (1994) concluded that even

when dietary intake of calcium exceeded

the recommended daily intake, the calcium

demands in lactation in humans were preferentially met by increased skeletal resorption of

calcium and probably increased renal conservation of calcium, but not by increased

intestinal absorption of calcium.

Like the complexity in the bone/calcium

literature, the relationships between bone lead,

calcium, bone turnover, and reproduction are

also controversial. For example, Rothenberg

et al. (2000) found calcaneus and tibia lead

were directly associated with prenatal third

trimester blood lead but only calcaneus lead

was associated with postnatal blood lead. They

also found that there was no effect of dietary

calcium on calcaneus lead despite the more

easily mobilized trabecular lead. In a crosssectional Mexico City study, Hernandez-Avila

et al. (1996) also found a significant association between trabecular bone lead (in the

patella) and postnatal blood lead and a significant effect of calcium on patella lead.

With respect to calcium intakes, several

studies have suggested that dietary calcium

may have a protective role against lead by

decreasing absorption of lead in the gastrointestinal tract and by decreasing the mobilization of lead from bone stores to blood,

especially during periods of high metabolic

activity of the bone such as pregnancy, lactation, and menopause (e.g., Farias et al. 1996;

Hernandez-Avila et al. 1996, 1997, 2003;

Hertz-Picciotto et al. 2000). However, most

of these studies were cross-sectional in nature,

and dietary calcium was estimated by questionnaire, diary, or recall. Furthermore, the

outcomes between studies of similar population groups were sometimes conflicting (e.g.,

Farias et al. 1996; Hernandez-Avila et al.

1996). In more recent studies in Mexico City,

calcium carbonate supplementation of

1,200 mg elemental calcium per day gave a

modest reduction of 16% in blood lead levels

among lactating women with relatively high

bone lead burden (Hernandez-Avila et al.

2003). In an earlier report apparently using

the same cohort but with lower numbers, there

did not seem to be any benefit from calcium

supplementation (Tellez-Rojo et al. 2002).

Using a sensitive biomarker of bone resorption, levels of cross-linked N-telopeptides of

type I collagen (NTx), Janakiraman et al.

(2003) observed that a bedtime 1,200 mg calcium supplement during the third trimester of

pregnancy reduced maternal bone resorption

by an average of 14%.

Contribution to blood lead from diet and

calcium supplements. In contrast to our previous cohort (Gulson et al. 1998b), where the

environmental and dietary contributions to

blood lead were considered minimal, conditions were somewhat different in the present

case series. For example, even though the

206Pb:204Pb ratios in the dust, air, and water

are still lower than observed in the blood of

our migrant subjects and would contribute

little to blood lead, the ratios in the 6-day

duplicate diet increased considerably, especially since our market basket surveys of 1990

when the 206Pb:204Pb ratio was about 17.0

(Gulson et al. 1996). This change has come

about with globalization, especially of the

food supply. Nevertheless, we do not attribute

the increase in blood lead during the last

2 months of pregnancy to diet; otherwise,

there should have been obvious effects earlier in the pregnancy. A potential source of

the increased lead may come from the calcium supplements. These contribute almost

50% of the daily lead intake, and even

though we observed minimal changes in the

6-month trial of a separate cohort taking

the same dose and same type of calcium

supplements (Gulson et al. 2001), physiologic processes during pregnancy may affect

the uptake of lead even in the presence of

sufficient calcium. For example, pregnant

swine absorb and retain more lead than do

nonpregnant swine (Casteel et al. 2001),

and there is convincing evidence especially

from stable calcium isotope studies, mentioned above, that there is increased intestinal absorption of calcium during pregnancy.

An increased absorption of lead from the

calcium supplement could partially explain

the increase in 206Pb:204Pb ratio observed

for three subjects during 6 months postpartum, but there was no such change in

the other three subjects for whom we have

data. Furthermore, there was no consistency

in changes relating to the two different

supplements.

The major decrease in 206Pb:204Pb ratio

and increase in blood lead for subject 1225

postpartum are remarkable (Figure 4).

Fortunately, this subject was not breastfeeding. A sample of the husband’s blood was

collected approximately 4 months after detection of the changes in his wife; it had a lead

concentration of 23 μg/dL and a 206Pb:204Pb

ratio of 16.62, consistent with an exposure

similar to that of the wife. Considerable effort

was devoted to determine the source of the

changes because they indicate a very high

acute dose of lead of Australian origin.

However, evaluation of the potential sources

(diet, traditional medicines, cosmetics, social

calendar) indicated that the only explanation

for the changes could be attributed to their

attendance at an ethnic fair where they

consumed foodstuffs different from their

regular diet.

In summary, despite the recent studies of

Hernandez-Avila et al. (2003) and Janakiraman

et al. (2003) pointing to benefits from calcium

supplementation during pregnancy and lactation, significant increases in blood lead during

late pregnancy and postpartum found in our

investigations and those of several other

authors appear to indicate that calcium supplementation is ineffective in minimizing the

mobilization of lead from the skeleton during

lactation—a position consistent with evidence

from the calcium literature using bone density

and bone turnover index measurements

(Prentice 2003). It does appear to offer some

Article | Extra calcium and blood lead in pregnancy and postpartum

Environmental Health Perspectives • VOLUME 112 | NUMBER 15 | November 2004 1505
Article | Gulson et al.

1506 VOLUME 112 | NUMBER 15 | November 2004 • Environmental Health Perspectives

protection, however, during pregnancy by

delaying the extra lead mobilized from bone

and reducing the extra flux. Despite the potential exposure of the infant to lead from breastfeeding, we have shown that the transfer of

lead to the infant from breast milk is low, especially at low blood lead concentrations (Gulson

et al. 1998a).

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Article | Extra calcium and blood lead in pregnancy and postpartum

Environmental Health Perspectives • VOLUME 112 | NUMBER 15 | November 2004 1507