Contribution of Lead from Calcium Supplements to Blood Lead

Brian L. Gulson

We conducted a case-control study to determine the contribution of lead to blood from consumption of calcium supplements approximating the recommended daily intakes over a 6-month period. Subjects were males and females ages 21 to 47 years (geometric mean 32 years) with a geometric mean blood lead concentration of 2.5 [micro]g/dL. They were subdivided into three groups. One treatment group (n = 8) was administered a complex calcium supplement (carbonate/phosphate/citrate) and the other treatment group (n = 7) calcium carbonate. The control group (n = 6) received no supplement. The lead isotopic compositions of the supplements were completely different from those of the blood of the subjects, allowing us easily to estimate contribution from the supplements. The daily lead dose from the supplements at 100% compliance was about 3 [micro]g Pb. Three blood samples were taken at 2-month intervals before treatment to provide background values, and three were taken during treatment. Subjects in the treatment group were thus their own controls. Lead isotopic compositions for the complex supplement showed minimal change during treatment compared with pretreatment. Lead isotopic compositions in blood for the calcium carbonate supplement showed increases of up to 0.5% in the [sup.206]Pb/[sup.204]Pb ratio, and for all isotope ratios there was a statistically significant difference between baseline and treatment (p [is less than] 0.005). The change from baseline to treatment for the calcium carbonate supplement differed from that for both the control group and the group administered the complex supplement. Blood lead concentrations, however, showed minimal changes. Variations in blood lead levels over time did not differ significantly between groups. Our results are consistent with earlier investigations using radioactive and stable lead tracers, which showed minimal gastrointestinal absorption of lead in the presence of calcium ([+ or -] phosphorus) in adults. Even though there is no discernible increase in blood lead concentration during treatment, there are significant changes in the isotopic composition of lead in blood arising from the calcium carbonate supplement, indicating a limited input of lead from diet into the blood. Because calcium carbonate is overwhelmingly the most popular calcium supplement, the changes we have observed merit further investigation. In addition, this type of study, combined with a duplicate diet, needs to be repeated for children, whose fractional absorption of lead is considerably higher than that of adults. Key words: blood lead, calcium supplements, isotopes, lead. Environ Health Perspect 109:283-288 (2001). [Online 2 March 2001]

http://ehpnet1.niehs.nih.gov/docs/2001/ 109p283-288gulson/abstract.html

The increasing incidence of fractures related to osteoporosis in both females and males has led to efforts to increase bone mass, especially in young children and adolescents (1). It is recognized, even in developed countries such as the United States, that daily intakes of calcium among children are below recommended levels (2,3). One approach to increase bone mass has been with vitamin and mineral supplementation; another has been fortification of foodstuffs such as cereals.

In addition to the potential to increase bone mass, calcium supplementation offers other advantages at various stages of life. For example, calcium supplementation reduces the incidence of pregnancy-induced hypertension and preeclampsia (4). In addition, Koo et al. (5) showed that supplementation with 2 g calcium per day increased fetal bone density in subjects whose calcium intake was normally [is less than] 600 mg per day. Of relevance to our study is the recognition that lead absorption by the gastrointestinal tract is increased in calcium-deficient diets (6). Absorption of lead by the gastrointestinal tract is inversely related to the amount of calcium present (6,7). Furthermore, calcium supplements had a protective effect by significantly reducing blood lead levels in pregnant women whose diets were deficient in calcium (8,9). The increased mobilization of skeletal lead during pregnancy and lactation was attributed to low calcium intake (10).

As people have been encouraged to take calcium supplements, several investigators have drawn attention to their potential lead toxicity (11-15). Furthermore, the lead concentrations in calcium supplements were the subject of California legal action, Proposition 65 (16).

Any lead contained in calcium supplements will be introduced in conjunction with the calcium. Lead binds tightly to the same transport proteins used by calcium, but the binding affinity of lead is at least twice that of calcium (17). Because the same transport mechanism is operative for absorption of lead and calcium from the gastrointestinal tract, there is resulting competitive interaction between lead and calcium (18).

The agreement reached between the California Attorney General and supplement manufacturers for Proposition 65 was an acceptable daily intake of [is less than] 1.5 [micro]g Pb/g Ca for adults (15,16). Even at these levels, however, supplements potentially contribute a significant proportion of the daily intake of lead that is now [is less than] 5 [micro]g/day in the United States (19).

We were concerned about the scientific validity of the California judgment in light of earlier investigations from tracer studies in both animals and humans demonstrating limited uptake of lead from the gastrointestinal tract in the presence of calcium ([+ or -] phosphorus; Figure 1) (20-24). These experiments, however, were undertaken at times of considerably higher intakes of lead with small numbers of subjects and mostly over limited time periods. The publicity arising from the California legal action was of concern because we had just begun a clinical trial in which subjects were given calcium supplements to verify the hypothesis that calcium supplementation minimizes the mobilization of skeletal lead during pregnancy and lactation. To satisfy ourselves and participants in our trial of the potential safety of calcium supplements and the minimal contribution to blood lead, we undertook a case-control study in which adults consumed calcium supplements containing lead whose isotopic composition was different from that in their current blood.

[GRAPH OMITTED]

The question we asked was, does the amount of lead in calcium supplements increase blood lead concentration or change the isotopic composition of lead in blood at the recommended daily intake levels of calcium?

Methods

Rationale. We demonstrated previously that the lead isotopic composition of multigenerational Australians was different from that of subjects from most other countries (10). We used this concept, first suggested by Manton (25), in our longitudinal study of mobilization of lead from the maternal skeleton during pregnancy and lactation. Commercially available Australian calcium supplements, which are typically made from overseas components, were administered to the subjects.

Subjects and dosing. Subjects, 8 males and 13 females, ranged in age from 21 to 47 years with a geometric mean of 32 years. We attempted to assign subjects randomly to one of three groups, two treatment and one control group, but two subjects expressed a desire to be in the control group. This does not, however, confound our results, because the treatment subjects formed their own controls by having three baseline measurements. The others were randomly assigned to the treatment groups. Ethics clearance was obtained from Macquarie University.

Sampling. A trained phlebotomist obtained 2-3 mL of venous blood from each subject. Three samples were collected at quarterly intervals to establish a background value before treatment. During treatment, samples were collected bimonthly for 6 months of calcium supplementation. Thus, each subject in the treatment group acted as his or her own control. Because of changes observed by the group given the calcium carbonate supplement, an additional blood sample for this group was taken 2 months after the treatment ended to see if there was a trend toward the baseline values.

Treatment. We originally intended to use a popular calcium carbonate product, but it was not possible to obtain the required quantity of a single manufacturing batch from the supplier. However, for the two treatment supplements, sufficient product was obtained from the one batch of production to minimize variability in the source of calcium and hence lead. The possibility of supplying the control group with a placebo was investigated but discarded because of production difficulties for such a small batch. The product details are listed in Table 1. The complex supplement was to be taken 3 times daily, with meals and at bedtime, because the fasting state is a time when most parathyroid hormone-mediated bone resorption occurs (26). The calcium carbonate was taken twice daily after meals.

Table 1. Product information.

                                Complex product
                                                              Calcium
                      Calcium      Calcium       Calcium     carbonate
Composition           citrate     phosphate     amino acid   (chewable)

Weight Ca compound      300          325           200         1,250
  (mg)
Equivalent Ca (mg)       64          126            40           500
Daily total Ca (mg)                  920                       1,000
Daily dosage                    3 times after                 2 times
                                  meals; 1 on                  after
                                  retiring                     meals
[sup.206]Pb/                        20.14         19.70
  [sup.204]Pb
Pb ([micro]g/kg)                     293           700
Daily Pb intake                      2.8           3.2
  ([micro]g/day)

Two subjects experienced nauseous reactions which they possibly attributed to the calcium supplements. One subject, given the complex product, had just conceived and was therefore moved to the control group. The other experienced nausea taking the calcium carbonate product; the subject was switched to the alternative supplement and thereafter showed no adverse effects.

In this pilot investigation, we did not attempt to undertake a dietary study apart from a questionnaire. However, our previous and ongoing pregnancy research showed that the daily calcium intake of migrant and Australian subjects is low (about 500 mg Ca/day) compared with the recommended levels for subjects participating in U.S. studies on pregnancy and lactation (27-32). In these studies, based on diaries and recall, calcium intake was estimated at 1,200-1,800 mg Ca/day, compared with 300-800 mg Ca/day for our subjects (10).

Questionnaire. On one occasion we gave the participants a questionnaire that placed a special emphasis on the intake of potentially calcium-bearing foods.

Analyses. All sample preparation was performed in purpose-built low-contamination laboratories (clean rooms) incorporating features such as filtered air intake and laminar flow hoods. To minimize sample heterogeneity, the total blood sample was predigested in ultrapure concentrated nitric acid and an aliquot of [is less than] 1g removed to a clean Teflon vessel. A [sup.202]Pb spike solution of known isotopic composition and lead concentration (~10 ng/g) was added to the aliquot to obtain the concentration of lead and isotopic composition of the unknown sample in the one analysis (this is known as the isotope dilution method). ([sup.202]Pb is not naturally occurring and is produced in cyclotrons as a by-product of preparation of thallium used in treatment of thyroid abnormalities; it has a half-life of about 3 x [10.sup.5] years.) We analyzed the calcium supplements for their lead isotopic composition by first spiking with the [sup.202]Pb spike and then dissolving/digesting in ultra-pure nitric acid. An aliquot was removed to a clean Teflon vessel and its pH was adjusted using clean ammonia. We separated lead from the calcium by passing the solution through a chelex filter. We further separated lead from other interfering ions using anion exchange chromatography in a bromide medium.

For isotope ratio measurement, we loaded fractions of the purified lead samples onto a rhenium filament using the silica gel technique (a mix of dilute phosphoric acid and purified silica gel) and analyzed them for lead isotope composition (and lead concentrations by isotope dilution) on a thermal ionization mass spectrometer (VG-ISO-MASS 54E; VG, Winsford, Cheshire, UK) run in fully automatic mode. Isotopic ratios were measured as [sup.208]Pb/[sup.206]Pb, [sup.207]Pb/[sup.206]Pb, and [sup.206]Pb/[sup.204]Pb. Precision estimates on the isotopic ratios have been defined by a repetition of the digestion/lead separation/mass spectrometry stages of the same samples of blood, urine, and water. The precisions we allocate our data are [+ or -] 0.2% (2 [Sigma]) on the [sup.206]Pb/[sup.204]Pb ratio, and [+ or -] 0.1% on the [sup.208]Pb/[sup.206]Pb and [sup.207]Pb/[sup.206]Pb ratios. Data are normalized to the accepted values of the National Institute of Standards and Technology (NIST) SRM 981 by applying a correction factor of +0.08% atomic mass units to allow comparisons between laboratories. We obtained a measurement of the environmental lead acquired by the sample throughout the entire preparation analysis procedure in the form of a lead blank measurement. The amount of contamination detected in blanks was generally around 200 pg for blood and from 300 to 900 pg for the calcium supplements. Because the blanks contributed negligibly to the lead in the sample, no blank corrections to the data were performed.

Data treatment. We analyzed the three isotopic ratios ([sup.208]Pb/[sup.206]Pb, [sup.207]Pb/[sup.206]Pb, and [sup.206]Pb/[sup.204]Pb) and lead concentrations using a 2 (baseline and treatment) x 3 (days 1, 2, and 3 of baseline and treatment) x 3 (the control, calcium carbonate, and calcium citrate groups) factorial design. The first two factors were within-subject factors.

Results

Results are summarized in Table 2, and a comparison of means of the [sup.206]Pb/[sup.204]Pb ratio and blood lead concentrations for each group is shown in Figures 2 and 3. Compliance for the 3 months of the treatment period ranged from 0 to 100% for each month. It was expected that changes in pretreatment blood samples may have been larger because sampling covered the Christmas holidays, a time of increased dietary intake and consumption of unusual foods. We sampled approximately 1 month after Christmas to minimize potential effects of abnormal dietary intakes, because the mean life of lead in blood of adults is approximately 20 days (33, 34).

[GRAPHS OMITTED]

Table 2. Changes in lead isotopic composition and blood lead for
subjects.

                                                              Changes
                                                              during
                                         Pretreatment        treatment

                                    PbB                         PbB
                  Age            ([micro]g/   [sup.206]Pb/   ([micro]g/
Identifier       (years)   Sex      dL)       [sup.204]Pb       dL)

Calcium
    citrate/
    phosphate/
    chelate
  1303             31       M       3.5          17.19        3.2-2.9
  1304             38       M       2.1          17.22        2.1-1.4
  1309             34       M       1.4          17.21        1.3-1.4
  1311             47       F       5.8          16.66        5.2-5.7
  1313             21       F       2.5          17.32        2.3-2.6
  1314             30       F       3.1          17.49        3.1-3.8
  1315             44       F       2.2          17.22        1.5-1.4
  1321             27       F       1.8          17.45        1.5-1.5
Calcium
    carbonate
  1305(b)          39       M     4.5-7.8     16.76-16.39     6.9-4.5
  1306(b)          36       F     2.2-3.5     17.36-16.93     2.2-1.9
  1308             32       F     3.5-4.3        16.84        5.1-3.3
  1312             25       F       2.1          17.39        2.3-2.1
  1316             27       F       1.5          17.52        1.4-1.5
  1319             33       F       2.1          17.35        2.0-2.6
  1320             30       F       1.2          17.28        1.2-1.4
Controls
  1302(b)          38       M     3.2-6.7     17.39-17.10     3.8-6.0
  1307             32       M       2.3          17.26        2.9-2.1
  1317             23       F       1.8          17.37          1.5
  1318             39       M       1.7          17.20        1.3-1.5
  1322(b)          25       F       2.4          17.24        2.3-1.8
  1323(b)          31       M       2.4          17.36        2.6-2.2

                   Changes
                   during         Posttreatment sample
                  treatment
                                   PbB                        Comp-
                 [sup.206]Pb/   ([micro]g/   [sup.206]Pb/   liance(a)
Identifier       [sup.204]Pb       dL)       [sup.204]Pb       (%)

Calcium
    citrate/
    phosphate/
    chelate
  1303           17.21-17.25        --            --         70-100
  1304           17.22-17.32        --            --         87-94
  1309           17.19-17.34        --            --         73-78
  1311           16.68-16.70        --            --         76-90
  1313           17.24-17.33        --            --          0-50
  1314           17.42-17.52        --            --         21-59
  1315           17.24-17.28        --            --         86-92
  1321           17.36-17.54        --            --         29-40
Calcium
    carbonate
  1305(b)        16.48-16.84       4.1          16.88        94-100
  1306(b)        17.25-17.46       1.9          17.53        74-90
  1308           17.31-17.42       2.6          17.34        70-89
  1312           17.49-17.64       1.6          17.51        84-96
  1316           17.63-17.70       1.3          17.58        70-86
  1319           17.32-17.47       2.1          17.38        17-62
  1320           17.36-17.38       1.0          17.33        29-40
Controls
  1302(b)        17.58-17.21        --            --           --
  1307           17.23-17.24        --            --           --
  1317           17.37-17.38        --            --           --
  1318           17.21-17.26        --            --           --
  1322(b)        17.54-17.35        --            --           --
  1323(b)        17.43-17.35        --            --           --

(a) Compliance each month over the 3 months of treatment.

(b) Possible reasons for the larger variability in isotopic
composition and lead concentration are given in the text.

Blood lead concentrations. We observed a slight downward trend over time in blood lead concentrations (Table 2, Figure 3), which was reflected in a main effect of treatment ([F.sub.(1,14)] = 5.94, p = 0.029) and also in a day main effect ([F.sub.(2,28)] = 4.12, p = 0.027). However, the change over days did not differ for baseline and treatment (for the day-by-treatment interaction, [F.sub.(2,28)] = 0.34, p = 0.717), and, as will be seen below, there was no suggestion of a group-by, treatment interaction.

Blood lead isotopic ratios. Apart from the anomalies described below, there were minimal changes in isotopic ratios in the control subjects and those given the complex supplement (Figure 2). In contrast, there were detectable increases in [sup.206]Pb/[sup.204]Pb ratio during administration of the calcium carbonate toward the values in the supplement (Figure 2; p = 0.002). Significant changes in the calcium carbonate group from baseline to treatment also occurred for [.sup.208]Pb/[sup.206]Pb and [sup.207]Pb/[sup.206]Pb (p [is less than] 0.0005 and p = 0.001, respectively). The blood sample taken 2 months after treatment ended showed a decrease toward the baseline values for [sup.206]Pb/[sup.204]Pb in some cases, indicating that the increases toward the supplement noted above were a valid effect. There was also a small decrease in blood lead concentration after cessation of treatment (Figure 3).

Anomalies in subjects. As is becoming increasingly evident in human studies, individual susceptibility to toxins or treatments plays a major role in determining impacts on health, as do changes in the environment of subjects. In our study, such problems arose for several subjects. For example, male subject 1305 undertook renovations using unsafe methods to an external garage with lead paint just before the start of treatment. Both he and his wife (subject 1306) experienced a sharp decrease in [sup.206]Pb/[sup.204]Pb ratio in blood Pb and an increase in blood lead (PbB) concentration (Table 2). The low [sup.206]Pb/[sup.204]Pb ratio in their blood is consistent with that of much of the lead in paint used in the past on Australian houses (35). Such changes, especially in isotopic compositions, associated with renovation of leaded paint were documented earlier by Manton (36). Despite the changes associated with renovation, the increases in isotopic composition that occur while taking calcium carbonate are consistent with the increases noted above.

One control subject (1302) was a regular firearms shooter, and large changes in PbB and isotopic composition are consistent with lead exposure from this activity. His data were omitted from the discussion and will be documented separately.

A husband and wife control pair (1322, 1323) exhibited small changes in PbB and isotopic composition after an 11-day visit to the United States (Table 2). High [sup.206]Pb/[sup.204]Pb ratios are characteristic of U.S. subjects and many environmental and dietary media (39).

Statistical analyses. We omitted the data for the anomalous subjects 1302, 1305, and 1306 from the statistical analyses. Significant interactions between group and treatment were found for all blood lead isotopic ratios (p-values for F-ratios with 2 and 14 degrees of freedom ranged from 0.01 for [sup.208]Pb/[sup.206]Pb through 0.023 for [sup.207]Pb/[sup.206]Pb to 0.027 for [sup.206]Pb/[sup.204]Pb). The group-by-treatment interaction was not significant for lead concentrations (p = 0.402). We followed up the significant interactions by testing interaction contrasts to answer the question, does the change between baseline and treatment differ for different pairs of groups? Two contrasts compared the calcium carbonate product and the complex calcium citrate product to the controls. The third contrast compared the complex calcium citrate product to the calcium carbonate product. The results are listed in Table 3. Using an adjusted [Alpha] level of 0.0167 (0.05/3) to allow for the three contrasts, there was a significant difference in the change from baseline and treatment for the calcium carbonate product and the control group for the [sup.208]Pb/[sup.206]Pb ratio (p = 0.008) and close to significant differences for the [sup.207]Pb/[sup.206]Pb and [sup.206]Pb/[sup.204]Pb ratios (p = 0.028 and 0.030, respectively). In contrast, there was no suggestion of significant differences between the control and calcium citrate groups (p [is greater than] 0.5 for each measure). The third contrast showed that the change for calcium carbonate product differed significantly from that for the calcium citrate product for each of the three measures (p [is less than] 0.010 in each case). The overall result was that the change from baseline to treatment for the calcium carbonate group differed from that for both the control and calcium citrate groups.

Table 3. Interaction contrasts.

Analysis                  Coefficient     SE      t-Value    p-Value

[sup.208]Pb/[sup.206]Pb
  Calcium carbonate vs.      -0.01692   0.00548   -3.0898    0.00799
    control
  Calcium citrate vs.        -0.00011   0.00465   -0.02456   0.98075
    control
  Calcium citrate vs.        -0.01681   0.00500    3.3618    0.00465
    calcium carbonate
[sup.207]Pb/[sup.206]Pb
  Calcium carbonate vs.      -0.01015   0.00414   -2.4480    0.02815
    control
  Calcium citrate vs.        -0.00155   0.00352    0.4405    0.66631
    control
  Calcium citrate vs.         0.01169   0.00378    3.0918    0.00796
    calcium carbonate
[sup.206]Pb/[sup.204]Pb
  Calcium carbonate vs.       0.22127   0.09147    2.4190    0.02977
    control
  Calcium citrate vs.        -0.02776   0.07774   -0.35711   0.72633
    control
  Calcium citrate vs.        -0.24903   0.08350   -2.9823    0.00989
    calcium carbonate

Discussion

Contribution to blood of lead from supplements. Over the 6-month dosing period, the potential impact on blood lead from the two calcium supplements tested in this pilot study was not detectable in the case of the complex product and only a small contribution was detected in the case of the calcium carbonate product. For the calcium carbonate product, there was little or no change in blood lead concentration but detectable changes in isotopic composition toward the [sup.206]Pb/[sup.204]Pb value of 19.7 in the supplement. Changes in isotopic composition toward the contributing source but not in blood lead concentration were observed in some other studies on newborn infants (37) and percutaneous absorption of lead (38). This may indicate that the pharmacokinetics of lead in blood need to be reevaluated, as also indicated by the long half-lives of lead in blood of up to 38 months observed in several young infants unnaturally exposed to lead from home renovations (39). With the changes in isotopic composition in blood compared with those in the pretreatment phase of calcium carbonate relative to the values in the product, the maximum contribution to blood lead is [is less than] 0.5%.

Indirect evidence supporting the protective effect of calcium (calcium carbonate [+ or -] phosphorous) comes from our longitudinal investigation on mobilization of lead from the maternal skeleton during pregnancy and lactation. In four pregnant subjects who were given calcium supplements (calcium carbonate or calcium citrate from the present study), minimal changes in blood lead concentration and isotopic composition were observed during pregnancy and lactation (40).

Effect of type of supplement. Given the competition of calcium and lead, an additional factor contributing to the uptake of lead from the gastrointestinal tract is the relative absorption of calcium from different compounds. The absorption of calcium from calcium carbonate and calcium citrate alone is approximately 30% (41). The absorption may be higher for the complex product of calcium citrate/phosphate/chelate, but to our knowledge this has not been rigorously tested using isotopic tracing methods.

Effect of state of the gut. Another important factor affecting gastrointestinal absorption is the relative condition of the gut--that is, whether it is in a fasted or nonfasted state. Tracer studies have demonstrated that in the absence of additional calcium, gastrointestinal absorption of lead was greater when lead was ingested by fasting subjects given soluble lead compounds in distilled water than when lead was ingested in the presence of food. Lead absorption in nonfasted subjects on either a normal diet or a controlled diet, in which nutritional content was known, ranged from approximately 3% to 10%, whereas lead absorption in fasted subjects who received soluble lead in distilled water (with no additional calcium) ranged from approximately 30% to 76% (20-24,33,34). The absence of calcium and other minerals in the gastrointestinal tract at the time of lead ingestion is a major reason for increased lead absorption in fasting subjects compared with nonfasting subjects (20-24,33,34), although when calcium and other minerals are present, differences seen between fasting and nonfasting subjects essentially disappear (22,23,32).

Effect of minerals on absorption. The presence of other minerals than calcium is an important factor in lead absorption from the gastrointestinal tract. For example, lead absorption decreases as calcium ([+ or -] phosphorous) concentrations increase (Figure 1) (20,22). Lead absorption in fasting subjects given lead chloride in drinking water was reduced by 83% and 97% when the lead was given with calcium and phosphorous at intakes of 200 mg Ca + 140 mg P and 700 mg Ca + 500 mg P, respectively (22). Reductions in lead absorption and retention were noted with both calcium alone (as calcium carbonate) and phosphorous alone (as sodium phosphate), but calcium was much more effective than phosphorous (20,22). The larger decreases in absorption for calcium phosphate compared with calcium carbonate observed by Blake and Mann (20) (Figure 1) may explain the absence of any effect of the complex product used in our study.

Lead absorption and retention among infants and young children is also influenced by dietary calcium intake. For example, Ziegler et al. (42) observed an inverse relationship between dietary calcium and retention and lead absorption in young infants in metabolic balance studies. Dietary calcium and phosphorus were important predictors of blood lead concentrations in children 12-47 months of age from a low-income population in central Washington, D.C. (43). Likewise, Sorrell et al. (44) and Johnson and Tenuta (45) observed inverse correlations between lead and calcium intake, vitamin D, and milk-based foods. Reductions in lead absorption were also noted in subjects ingesting different foods, depending on the calcium and mineral content of the ingested meal (24).

Animal studies. Studies using fasted and nonfasted laboratory animals, including rats, mice, and monkeys, have produced results similar to those in humans (46-49). For example, Mahaffey-Six and Goyer (46) and Mahaffey et al. (47) reported that rats ingesting a low-calcium diet had blood lead concentrations approximately 4 times higher than rats consuming a normal calcium diet. Later studies showed that gastrointestinal absorption of lead decreases in the presence of a number of minerals (50-52). Increasing calcium concentrations were found to decrease lead retention exponentially as calcium concentrations, increased (49). As in the human studies, administration of phosphorous without calcium did not produce reductions in lead retention as great as that for calcium alone or for calcium with phosphorous (50).

Effect of dosing regime. An additional protective effect associated with the complex product may come from its dosing regime. This supplement was to be taken 4 times daily including at bedtime because the fasting state is a time when most parathyroid hormone-mediated bone resorption occurs (26). The calcium carbonate was taken twice daily after meals. Heaney (26) recommended a daily dosing regime of 4 times because, although the amount of a mineral absorbed continues to rise with the intake, the percentage absorbed falls.

Matrix effects. The minimal effects observed in our pilot study may also be caused partly by matrix effects of the products. In studies of fasted human subjects, lead was administered as dissolved salts or suspended in solution. Using absorption values for lead in a solution as representative of lead absorption from a complex matrix may produce an overestimate of lead absorption. Before absorption, lead must dissociate from the compound to which it is bound. This may explain the difference between the complex calcium citrate and the calcium carbonate; the latter was in a chewable form and more easily absorbed.

Effect of duration of investigations. The length of time over which a study is undertaken may also be an important factor in absorption of lead from the gastrointestinal tract. Unlike the investigations by Rabinowitz et al. (23,33), the other studies involving radioactive tracers were only of short duration--less than 7 days. Our treatment period was 6 months, but we were able to detect increases in isotopic composition for calcium carbonate supplement. Because calcium carbonate is the most popular supplement (53), the potential uptake of lead from this product should be verified in a more comprehensive investigation, especially of young children.

Daily intake of lead from supplements. The amounts ingested from the two supplements of approximately 3 [micro]g Pb/day are more than half the current daily lead intake of less than 5 [micro]g Pb/day for the United States (19)--despite the temporal decrease in the amount of lead in supplements illustrated by Scelfo and Flegal (15). Lanphear and colleagues recently demonstrated that lead shows detrimental effects in children at blood concentrations as low as 2.5 [micro]g/dL (54). Hence, any source contributing 50% or more to lead exposure--especially in young children, with lead absorption greater than that of adults--requires investigation and monitoring.

In conclusion, despite the fact that lead exposure from the calcium supplements tested in this study was approximately half the daily intake of lead, the contribution to blood lead concentration was minimal. However, the same may not apply to calcium-fortified foods. Furthermore, even though there is no discernible increase in blood lead concentration, there are significant changes in the isotopic composition of lead in blood arising from the calcium carbonate supplement, which indicate a limited input of lead from diet into the blood. Because calcium carbonate is the overwhelmingly most popular calcium supplement, the changes we have observed merit further investigation. The small contributions of lead from calcium supplements, however, should not discourage people from their usage given other significant benefits deriving from calcium.

REFERENCES AND NOTES

(1.) NIH. Osteoporosis and Fractures in Men. NIH Guide, Vol 25, No 39. PA-97-009. Bethesda, MD:National Institutes of Health, 1996.

(2.) NIH Consensus Development Panel on Optimal Calcium Uptake. JAMA 272:1942-1948 (1994).

(3.) Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. Food and Nutrition Board, Institute of Medicine. Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC:National Academic Press, 1997.

(4.) Bucher HC, Guyatt GH, Cook RJ, Hatala R, Cook DJ, Lang JD, Hunt D. Effect of calcium supplementation on pregnancy-induced hypertension and preeclampsia. JAMA 275:1113-1117 (1996).

(5.) Koo WW, Walters JC, Esterlitz J, Levine RJ, Bush AJ, Sibai B. Maternal calcium supplementation and fetal bone mineralization. Obstet Gynecol 94:577-582 (1999).

(6.) Mahaffey KR. Factors modifying susceptibility to lead toxicity. In: Dietary and Environmental Lead: Human Health Effects (Mahaffey KR, ed). Amsterdam:Elsevier, 1985;373-419.

(7.) Goyer RA. Nutrition and metal toxicity. Am J Clin Nutr 61(Suppl):646S-650S (1995).

(8.) Farias P, Borja-Aburte VH, Rios C, Hertz-Picciotto I, Rojas-Lopez M, Chavez-Ayala R. Blood lead levels in pregnant woman of high and low socioeconomic status in Mexico City. Environ Health Perspect 104:1070-1074 (1996).

(9.) Hernandez-Avila M, Gonzalez-Cossio T, Palazuelos E, Romieu I, Aro A, Fishbein E, Peterson KE, Hu H. Dietary and environmental determinants of blood and bone lead levels in lactating postpartum women living in Mexico City. Environ Health Perspect 104:1076-1082 (1996).

(10.) Gulson BL, Mahaffey KR, Jameson CW, Mizon KJ, Korsch MJ, Cameron MA, Eisman JA. Mobilization of lead from the skeleton during the post-natal Period is larger than during pregnancy. J Lab Clin Med 131:324-329 (1998).

(11.) Capar SG, Gould JH. Lead, fluorine and other elements in bonemeal supplements. J Assoc Off Anal Chem 62:1054-1061 (1979).

(12.) Boulos BM, Smolinski A. Alert to users of calcium supplements as hypertensive agents due to trace metal contaminants. Am J Hypertens 1:137S-142S (1988).

(13.) Bourgoin BP, Evans DR, Cornett JR, Lingard SM, Quattrone AJ. Lead content in 70 brands of dietary calcium supplements. Am J Public Health 83:1155-1160 (1993).

(14.) Rogan WJ, Ragan NB, Damokosh AL, Davolie C, Shaffer TR, Jones RL, Wilkens S, Heenehan MC, Ware JW, Henretig F. Recall of a lead-contaminated vitamin and mineral supplement in a clinical trial. Pharmacoepidemiol Drug Saf 8:343-350 (1999).

(15.) Scelfo GM, Flegal AR. Lead in calcium supplements. Environ Health Perspect 108:309-313 (2000).

(16.) California Attorney General's Office. Superior Court Settlement No. 984503. San Francisco, CA, 15 May 1997. Available: http://www.prestongates.com/newsroom/ dietary_supplement/consent_judgment.htm [cited 5 February 2001].

(17.) Heaney RP. Unpublished data.

(18.) Mushak P. Gastro-intestinal absorption of lead in children and adults: overview of biological and biophysico-chemical aspects. Chem Speciat Bioavaiabl 3:87-104 (1991).

(19.) Bolger M. Personal communication.

(20.) Blake KCH, Mann M. Effect of calcium and phosphorus on the gastrointestinal absorption of [sup.203]Pb in man. Environ Res 30:188-194 (1983).

(21.) Blake KCH, Barbezat GO, Mann M Effect of dietary constituents on the gastrointestinal absorption of [sup.203]Pb in man. Environ Res 30:182-187 (1983).

(22.) Heard MJ, Chamberlain AC. Effect of minerals and food on uptake of lead from the gastrointestinal tract of humans. Hum Toxicol 1:411-415 (1982).

(23.) Rabinowitz MB, Kopple JD, Wetherill GW. Effect of food intake and fasting on gastrointestinal lead absorption in humans. Am J Clin Nutr 33:1784-1788 (1980).

(24.) James H, Hilburn M, Blair J. Effects of meals and meal times on uptake of lead from the gastrointestinal tract in humans. Hum Toxicol 4:401-407 (1985).

(25.) Manton WI. Sources of lead in blood: Identification by stable isotopes. Arch Environ Health 32:149-159 (1977).

(26.) Heaney RP. Calcium supplements: practical considerations. Osteoporos Int 1:65-71 (1991).

(27.) Sowers MF, Corton G, Jannausch ML, Crutchfield M, Smith ML, Randolph JF, Hollis B. Changes in bone density with lactation. JAMA 269:3130-3135 (1993).

(28.) Sowers MF, Randolph J, Shapiro B, Jannausch M. A prospective study of bone density and pregnancy after an extended period of lactation with bone loss. Obstet Gynecol 85:285-289 (1995).

(29.) Cross NA, Hillman LS, Allen SH, Krause GF. Changes in bone mineral density and markers of bone remodelling during lactation and postweaning in women consuming high amounts of calcium. J Bone Miner Res 10:1312-1320 (1995).

(30.) Kalkwarf HJ, Specker BL. Bone mineral loss during lactation and recovery after weaning. Obstet Gynecol 86:26-32 (1995).

(31.) Kalwarf HJ, Specker BL, Bianchi DC, Ranz J, Ho M. The effect of calcium supplementation on bone density during lactation and after weaning. N Engl J Med 337:523-528 (1997).

(32.) Krebs NF, Reidinger CJ, Robertson AD, Brenner M. Bone mineral density changes during lactation, maternal, dietary, and biochemical correlates. Am J Clin Nutr 65:1738-1746 (1997).

(33.) Rabinowitz MB, Wetherill GW, Kopple JD. Kinetic analysis of lead metabolism in healthy humans. J Clin Invest 58:260-270 (1976).

(34.) Chamberlain AC, Heard MJ, Little P, Newton D, Wells AC, Wiffen RD. Investigations into Lead from Motor Vehicles. AERE-R9198. Harwell, UK:United Kingdom Atomic Energy Authority, 1978.

(35.) Gulson BL, Davis JJ, Bawden-Smith J. Paint as a source of recontamination of houses in urban environments and its role in maintaining elevated blood leads in children. Sci Total Environ 164:221-235 (1995).

(36.) Manton WI. Total contribution of airborne lead to blood lead. Br J Ind Med 42:168-172 (1985).

(37.) Gulson BL, Mizon KJ, Palmer JM, Patison N, Law AJ, Korsch MJ, Mahaffey KR, Donnelly JB. Longitudinal study of daily intake and excretion of lead in newly-born infants. Environ Res (in press).

(38.) Stauber JL, Florence TM, Gulson BL, Dale LS. Percutaneous absorption of inorganic lead compounds. Science Total Environ 145:55-70 (1994).

(39.) Manton WI, Angle CR, Stanek KL, Reese YR, Kuehnemann TJ. Acquisition and retention of lead by young children. Environ Res 82:60-80 (2000).

(40.) Gulson BL Mizon KJ, Palmer JM, Korsch MJ, Taylor AJ. Unpublished data.

(41.) Heaney RP. Availability of calcium from various sources. Bone 7:54-56 (1990).

(42.) Ziegler EE, Edwards BB, Jensen RL, Mahaffey KR, Fomon SI. Absorption and retention of lead by infants. Pediatr Res 12:29-34 (1978).

(43.) Mahaffey KR, Treloar S, Banks TA, Peacock BJ, Parekh LE. Difference in dietary intake of calcium phosphorus and iron of children having normal and elevated blood lead concentrations [Abstract]. J Nutr 106:7 (1976).

(44.) Sorrell M, Rosen JF, Roginsky MR. Interactions of lead, calcium, vitamin D and nutrition in lead-burdened children. Arch Environ Health 32:160-164 (1977).

(45.) Johnson NE, Tenuta K. Diets and blood leads of children who practice pica. Environ Res 18:369-376 (1979).

(46.) Mahaffey-Six KR, Goyer RA. Experimental enhancement of lead toxicity by low dietary calcium. J Lab Clin Med 76:933-941 (1970).

(47.) Mahaffey KR, Haseman JD, Goyer RA. Dose-response to lead ingested in rats fed low dietary calcium. J Lab Clin Med 83:92-100 (1973).

(48.) Quarterman J, Morrison NJ, Humphries WR. The influence of high dietary calcium and phosphate on lead uptake and release. Environ Res 17:60-67 (1978).

(49.) Meredith PA, Moore MR, Goldberg A. The effect of calcium on lead absorption in rats. Biochem J 16:531-537 (1977).

(50.) Barltrop D, Khoo HE. The influence of nutritional factors on lead absorption. Postgrad Med J 51:795-799 (1975)

(51.) Ragan HA. Effect of iron deficiency on the absorption and distribution of lead and cadmium in rats. J Lab Clin Med 90:700-706 (1977).

(52.) Fine B, Barth A, Sheffet A, Levenhar MA. Influence of magnesium on the intestinal absorption of lead. Environ Res 12:224-227 (1976).

(53.) Natural Resources Defense Council News Release. NRDC reaches legal settlement with largest calcium supplement manufacturer to provide "lead-free" calcium. New York:Natural Resources Defense Council, 27 January 1997.

(54.) Lanphear BP, Dietrich KN, Auinger P, Cox C. Subclinical lead toxicity in U.S. children and adolescents [Abstract]. Pediatr Res 47:894 (Part 2, suppl) (2000).

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

(1) Graduate School of the Environment, Macquarie University, Sydney, Australia; (2) CSIRO/Division of Exploration and Mining, North Ryde, Australia; (3) Department of Psychology, Macquarie University, Sydney, Australia

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, the participants in this study, and R. Setright of Blackmores for supplying one of the products.

Received 24 August 2000; accepted 13 October 2000.

COPYRIGHT 2001 National Institute of Environmental Health Sciences
COPYRIGHT 2004 Gale Group