FUNDAMENTAL AND APPLIED TOXICOLOGY 3 3 , 2 3 5 - 2 4 5 ( 1 9 % )
ARTICLE NO. 0161
Measurement of the Flux of Lead from Bone to Blood in a Nonhuman
Primate (Macaca fascicularis) by Sequential Administration
of Stable Lead Isotopes
M. J. INSKIP,*1 C. A. FRANKLIN.t C. L. B A C C A N A L E , * W. I. MANTON,t E. J. O ' F L A H E R T Y , §
C. M. H. E D W A R D S , * 1 J. B. BLENKINSOP, 1 AND E. B. E D W A R D S *
*Health Protection Branch, Health Canada, Ottawa, Ontario, Canada; ^Pest Management Regulatory Agency, Health Canada, Ottawa, Ontario,
Canada; %Mass Spectrometry Laboratory, University of Texas at Dallas, Richardson, Texas; ^Department of Environmental Health, University of
Cincinnati College of Medicine, Cincinnati, Ohio; and ^Department of Earth Sciences, Carleton University, Ottawa, Ontario, Canada
Received November 22, 1995; accepted April 26, 1996
in bone may be returned to blood using a physiologically
based pharmacokinetic model based on bone calcium kinetics. Measurements of other bone-seeking elements have
tration of Stable Lead Isotopes. INSKIP, M. J., FRANKLIN, C. A.,
shown that the predominant process is a slow "diffuse"
BACCANALE, C. L., MANTON, W. I., O'FLAHERTY, E . J., EDWARDS,
exchange
from bone, via the canaliculi, to blood, but also
C. M. H., BLENKINSOP, J. B., AND EDWARDS, E. B. (1996). Funimportant
is random structural remodeling, involving the
dam. Appl. Toxicol. 33, 235-245.
resorption of bone by osteoclast cells, followed by new minTo better understand the kinetics of the transfer of lead from eral apposition (O'Flaherty, 1993).
bone to blood, we have developed and tested a method in which
The association between bone lead remobilization and insequential doses of lead, each enriched with a different stable creased calcium metabolism in bone is supported by the
isotope, were administered in a nonhuman primate Macaca fascicobservation of higher blood lead levels in women after
ularis whose skeleton had been previously labeled with lead of
menopause (Silbergeld et al., 1988) when there is increased
known isotopic composition. Lead isotopic ratios of blood and
demineralization
due to decreasing estrogen levels. The posbone samples, analyzed by thermal ionization mass spectrometry
sibility
of
a
similar
remobilization of lead during pregnancy
(TIMS), were unmixed by isotope dilution techniques. The first
raises
concerns
because
of the susceptibility of the devellabel administered allows the contribution from historical bone
stores to be measured. Subsequent labels allow measurement of oping fetal brain to lead toxicity (Goyer, 1990), given the
both the historical bone stores and the previous labels that have poorly developed blood:brain barrier. Further exposure of
become recently incorporated into bone. The method may be ex- the infant to lead may occur during lactation, since there is
tended to studies of bone lead mobilization in pregnancy, lactation, mobilization of calcium from the maternal bone to breast
menopause, or in disease states such as postmenopausal osteoporomilk (Kalkwarf and Specker, 1995; Prentice, 1994), although
sis. O 1996 Soctety of Toiieotogy
the relative contribution of the skeletal calcium pool under
different dietary calcium intakes is not well understood
(King et al., 1992). Maternal health may also be affected
Lead and other bone volume-seeking elements, such as should skeletal lead be sufficiently elevated, resulting in high
radium and strontium, become incorporated into the crystal blood lead levels (Thompson et al., 1985).
matrix of bone (MacDonald et al., 1951; Aufderheide and
Given the concerns that even low levels of lead can be
Wittmers, 1992; Durbin, 1992) due to their similarity in fetotoxic and that prepregnancy blood lead levels give no
atomic structure and ionic radius to calcium. Although it has indication of the lead level to which the fetus could be exbeen known for some time that bone is not an irreversible posed, it is critical to be able to measure the amount of lead
"sink" for lead (Manton, 1977, 1985; Rabinowitz et al., that could be transferred from maternal bone to the fetus
1976), the kinetics governing the return of lead to the circula- during pregnancy (Mushak, 1993; Franklin et al., 1995). A
tion are not well known.
direct method must be developed that can distinguish beBy assuming that lead behaves similarly to calcium, tween lead from endogenous sources, such as bone, and lead
O'Flaherty (1991) has described the processes by which lead from dietary/environmental sources.
Such a method is possible in human lead studies because
naturally
occurring lead is a mixture of four stable isotopes
1
To whom correspondence should be addressed at Bioregional Health
whose
ratios
vary with the geological age of ore deposits
Effects Program Division, Rm 1106 Mains Stats Bldg., Tunney's Pasture
(Doe, 1970). As a result, the environmental lead isotopic
Ottawa, Ontario K1A 0K9, Postal Locator 0301 A l .
Measurement of the Flux of Lead from Bone to Blood in a
Nonhuman Primate (Macaca fascicularis) by Sequential Adminis-
235
0272-05SKV96 $18.00
Copyright O 19% by the Society of Toxicology.
All rights of reproduction in any form reserved
236
INSK1P ET AL.
Common
204Pb-enriched
0
206Pb-enriched
50
207Pb-enriched
100
Common
150
Number of days from switch to enriched isotope
Birth_/\/_
12 years
years
80.0
DO
§ 70.0
1
60.050.0-
CO
I
40.030.0
£> 60.0
©
f; 50.0.
204 Dosing period
206 Dosing period
* Common
204
1 40.0.
1 30.0-
s
I 20.0
1
207 Dosing period
Soft tissue
ion \
and bone contribution
n.-- - r
10.0
0.050
100
Number of days from switch to enriched isotope
150
237
BONE LEAD CONTRIBUTION TO BLOOD LEAD
70.0
60.0
o
o
50.0
40.0
JJ
30.0
Common
Oral dose
PQ
20.0
10.0
204 Oral
dose
0
207 Oral
dose
50
100
150
Number of days from switch to enriched isotope
FIG. 3. Total blood lead concentrations and lead apportioned to oral dose.
signature varies within and between countries, depending
upon the source of industrial lead. Manton (1985) used this
knowledge to study temporal changes in blood lead ratios
of a resident in the United States, who had grown up in South
Africa, and had lead in his skeleton different in isotopic ratio
from lead in the U.S. environment. Approximately 70% of
the subject's blood lead was estimated to be from bone lead,
and after 8 years residence in the United States, the lead
isotopic ratios in the subject's metabolically active, trabecular bone were greatly different from those in the cortical
bone. Gulson and colleagues (1995) studied women from
eastern Europe and the former U.S.S.R. who had emigrated
to Australia. In this study, the assumption was made that the
isotopic ratios of skeletal lead reflect historical exposure
exclusively, and that the lead isotopic ratios in their skeletons
were identical to those in their blood on arrival in Australia.
They calculated that 41 to 73% of lead in their blood comes
from bone. Such calculations may underestimate the total
bone contribution, since any return of recently deposited
lead is not included. Due to the apparent random nature of
osteoclastic bone resorption, it appears that bone recently
laid down and older, more established bone, is equally subject to resorption processes.
The ideal experiment is one in which the whole skeleton
contains lead of identical isotopic composition. This is unattainable in a human subject but feasible in an animal study.
We therefore circumvented the need for assumptions concerning the nature and constancy of historical exposure by
working with a 12-year-old female cynomolgus monkey
(Macaca fascicularis) which had been continuously exposed
to the same lead from an early age. To measure the flux of
the historic dose from bone, we replaced the original dose
(common lead) with an enriched stable isotope of lead, and
by isotope dilution techniques we were able to calculate the
FIG. 1. Sequence of lead dose administration to nonhuman primate. Measured concentrations of lead dose (IDMS) for study period were 1320, 1460,
1390, and 654 f/g/kg/day for common lead, ^Pb-enriched, 206Pt>-enriched, and ^"Pb-enriched doses, respectively. * indicates times of bone biopsy.
FIG. 2.(A) Total blood lead concentrations (IDMS). (B) Elimination curves of common lead, ^Pb-enriched, ^Ph-enriched, and '"Pb-enriched labels
from blood/soft tissues. Horizontal portions of curve represent steady-state mobilization of lead from bone.
238
INSKIP ET AL.
proportions of the historic dose and the new dose in blood.
Lead, however, has four stable isotopes. We therefore sequentially replaced the first enriched isotope with two others,
and by isotope dilution calculated the flux not only of the
historic dose in bone, but also the amount of recently deposited enriched doses from bone.
METHODOLOGY AND ANALYTICAL TECHNIQUES
Animal History and Care
The animal used was a 12-year-old female cynomolgus monkey (CF124)
from a colony of chronically lead-dosed animals that had been raised from
birth in the pnmate facility of the Health Protection Branch, Health Canada,
Ottawa, according to national recommendations for the care of laboratory
animals (Canadian Council on Animal Care, 1984). The animal had undergone a hysterectomy 2 months prior to the start of this investigation due
to pyometritis, but was in good health by the time this study began and
throughout its duration. Zinc protoporphyrin levels in blood, which may be
elevated as a result of undue lead exposure, were within the normal range
(37.5 /ig/dl for this animal, at a blood lead of 44.6 /xg/100 g) after a lifetime
of chronic lead exposure. The colony had previously been used within
Health Canada for neurobehavioral studies on the effects of lead (Rice,
1990) and details of its dosing history are provided below. The animal was
fed a certified diet of Primate Monkey Chow.2 Water was provided ad
libitum.
There is insufficient evidence in the literature to allow us to conclude
that the current blood lead and bone lead concentrations may be capable
of altering bone physiology and processes affecting lead/mineral release
from bone. However, the fact that this animal and the 17 other chronically
dosed animals in the colony have (i) been dosed from birth or from a young
age and developed a normal-sized skeleton and (ii) have no signs of skeletal
problems or unusual clinical chemistry leads us to assume that this is not
the case.
Dose Regimen and Experimental Design
(a) Historical dosing. The animal had received chronic doses of lead
acetate trihydrate3 (common lead isotopic signature) at a nominal dose of
1500 ng Pb/kg/day from the age of 10 months unul the time of the authors'
custody in 1990 (approximately 10 years). Actual dosing had been on
weekdays only, at a pro rata administration of 2100 /Jg Pb/kg/day. The
animal had been trained to voluntarily take the gelatin capsule in the moming prior to feeding.
(b) Dosing during experimental period. Since 1990, oral administration of lead continued, but in order to maintain an optimal steady-state
2
Type 5047 (PMI Feeds, Richmond, IN), which was slightly modified
with respect to an optimal VitD3 level (2 IU/g), calcium (1%) and low lead
content (<0.25 ^g/g).
3
Preparation of the common lead and isotopically enriched lead doses.
A stock solution of the common lead dose was prepared by dissolving
common lead acetate trihydrate (Aldrich Chemical Co., Milwaukee, Wl)
in a mixture of glycerol (BDH Chemicals Inc., Toronto, Ontario, Canada),
and distilled water (96:4%, respectively). Small volumes were dispensed
into gelatin capsules. ^ P b - , ""Pb-, and 2O7Pb-enriched lead acetate trihydrate which were converted from the carbonate form were purchased from
Martin Marietta Energy Systems Inc. (Oak Ridge, TN). Stock solutions of
the isotopically enriched doses were prepared by mixing determined quantities of each isotopically enriched salt with the common lead acetate trihydrate and then dissolving in a mixture of glycerol and deionized water to
give dosing solutions with the lead concentrations and isotopic abundances
shown in Fig 1 and Table 1.
TABLE 1
Abundance of Lead Isotopes in the Doses
Dose solution
Abundance
of^Pb
(%)
Abundance
of^Pb
(%)
Abundance
of^Pb
(%)
Abundance
of^Pb
(%)
Common lead
^Pb-enriched
^Pb-enriched
^Pb-enriched
1.4
17.9
0.98
1.1
24.8
22.5
45.4
19.5
21.7
18.1
17.1
38.3
52.1
41.5
36.5
41 1
relationship between blood and bone compartments, a 7-day-per-week dosing regimen was adopted. The isotopic composition of the common lead
dose solutions administered from 1988 to 1993 were analyzed. Since so
large a dose would overwhelm any environmental lead (with a different
isotopic signature) that might be ingested, and since the isotopic ratios of
two bone biopsies, taken 1 year apart during this period, reflect the common
lead signature, we concluded that all parts of the animal's skeleton had
identical (common) lead isotopic ratios prior to any isotope manipulation
that occurred as part of this study.
To investigate the flux of both the historic and short-term stores of lead
from bone to blood, the common lead dose was replaced (see Footnote 3)
(in February 1993) first by a ^Pb-enriched dose (50 days), then by a ^ P b enriched dose (50 days), and finally by a 2<r7Pb-enriched dose (50 days,
with a reduced concentration) (Fig. 1 and Table 1). This sequential dosing
maintained exposure to lead, while the use of different isotopes allowed
elimination of the previously administered isotope to be observed.
Sampling Procedures
Particular care was taken to avoid contamination during processing and
analysis of the samples of blood and bone. The techniques were similar to
those described in Everson and Patterson (1980) and Inskip el al. (1992).
Individual samples collected for lead analysis were small because of the
animal size and the need for duplicate samples. Blood volumes varied
between 0.5 and 1.5 ml. Bone samples weighed on average 15 mg, but
samples one-tenth this size were analyzed.
Blood. Samples were collected on a weekly basis (Table 2) and processed in a Class-100 air-clean room. All personnel handling the animal
and samples wore clean-room clothing (Tyvec suit, hair and shoe covers,
and powder-free Class-100 gloves). Prior to sampling, the animal's leg was
shaved and wiped clean with lint-free wipes using soap solution, deionized
water, acetone, 0.25 M HC1, deionized water, and 70% isopropyl alcohol.
Blood samples were collected from the saphenous vein in preweighed
Teflon containers4 and the whole sample was analyzed.
Bone. A bone biopsy (iliac crest or distal/proximal tibia) was performed' at each isotope change (approximately every 50 days, Table 3).
The samples were meticulously cleaned of all nonosseous material (Inskip
et al., 1992) and separated into cortical and trabecular bone prior to vacuum
drying. The samples ("dry marrow-free bone") were analyzed by TIMS
to confirm incorporation of the isotope(s) into bone mineral
Measurement of Lead Concentration and Isotopic Ratios
Ultrapure reagents (HC1, HBr, HNO3)6 were used for lead chemistry. The
samples were dissolved in concentrated or 8 M HN0 3 and spiked with
4
Savillex Corp., Minnetonka, MI.
Bone biopsy surgery included antibiotic prophylaxis, premedication
with glycopyrrolate (0.015 mg/kg, im), immobilization with ketamine hydrochlonde (10 mg/kg, im), then intubated and maintained on isoflurane
gas. The analgesic, buprenorphine, was administered postoperatively (0.01
mg/kg).
6
Seastar Chemicals, Sidney, British Columbia, Canada.
5
239
BONE LEAD CONTRIBUTION TO BLOOD LEAD
TABLE 2
Measured Total Lead Concentration and Isotope Ratios in Blood Samples from Nonhuman Primate and Apportioning
of the Contributions from the Different Uniquely Labeled Doses
Number of
days from
Total blood
Blood lead component attributable to each
dose 0*g/100
switch to
enriched
Dose
fingerprint
lead (jigj
loo gr
Common
-119
0
Common
Common
48 0
46.3
48.0
46.3
204
206
207
Measured isotope ratio of lead*
^Pb/^Pb
^Pb/^Pb
"'Pb/^Pb
17.65
17.68
15.47
15.50
36.97
37.02
2.17
1.97
1.63
173
1.50
1.44
1 50
1.82
1.65
1.34
1.43
1.22
1.18
1.22
4.25
3.85
3.11
3.32
2.83
2.73
2.83
5.46
6.03
17.66
23.50
23.74
25.75
24.24
21.69
2.96
3.37
7.83
10.00
10.22
10.93
10.58
9.92
6.65
7.62
17.18
21.80
22.35
23.83
23.18
21.94
3
6
13
20
27
34
45
204
204
204
204
204
204
204
Pb
Pb
Pb
Pb
Pb
Pb
Pb
62.3
44.6
44.1
42.3
51.4
56.5
48.1
26.7
16.4
10.0
11.6
8.2
7.1
7.6
35.6
28.2
34.1
30.7
43.2
49.4
40.5
52
55
64
69
76
83
90
97
206
206
206
206
206
206
206
206
Pb
Pb
Pb
Pb
Pb
Pb
Pb
Pb
31.2
33.3
50.9
59.6
47.8
46.2
54.4
51.6
6.8
9.6
8.2
7.8
7.1
6.2
92
11.6
9.5
8.7
37
2.6
2.0
16
2.1
2.3
14.9
14.9
39.0
49.2
38.8
38.4
43 2
37.7
101
104
111
118
125
132
146
207
207
207
207
207
207
207
Pb
Pb
Pb
Pb
Pb
Pb
Pb
58.1
40.8
35.9
55.7
39.2
36.3
39.7
9.2
91
6.5
10.4
8.5
7.8
8.3
1.8
1.8
1.1
.9
.2
.4
.2
17.2
11 8
5.0
4.7
2.3
2.2
1.6
29.8
18.0
23.4
38.8
27.1
25.0
28.6
17.37
15.18
14.62
13.45
13.35
12.65
13.33
17.76
14.74
19.22
19.31
19.62
18.48
20.42
25.63
22.61
25.71
25.24
25.89
24.43
26.47
150
153
160
167
175
182
189
196
203
225
252
371
553
588
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
Common
56.8
34.5
34.7
38.9
56.0
40.8
47.2
54.2
41.4
44.7
48.0
42.8
43.4
40.9
32.3
19.9
26.0
32.6
51.6
37.2
43.1
51.1
38.4
41.8
45.8
41.5
42.2
39.6
.3
.3
.3
.2
0.9
0.9
1.2
0.9
0.8
0.9
0.6
0.3
0.3
0.3
.7
.6
.6
.4
.1
.1
.3
.0
.0
.0
0.7
0.4
0.4
0.4
21.5
11.7
5.7
3.6
2.3
1.6
1.7
1.3
1.2
1.1
08
0.5
0.5
0.5
14.22
12.64
12.66
13.38
15.13
14.31
14.15
15.25
14.70
14.83
15.62
16.48
16.62
16.34
16.86
14.24
12.26
12.27
13.45
12.60
12.42
13.37
12.86
12.95
13.64
14.42
14.53
14.28
28.71
24.98
25.05
26.81
30.96
29.05
28.69
31.30
29.96
30.28
32.14
34.21
34.50
33.85
652
665
679
707
735
763
None
None
None
None
None
None
19.6
19.4
16.8
16.4
15.2
13.4
18.0
17 7
15.3
14.6
13.8
12.1
0.5
0.6
0.5
0.5
0.5
0.4
0.6
0.7
0.6
0.3
0.5
0.5
0.5
0.5
0.5
1.0
0.4
0.4
14.07
13.64
13.89
13.21
13.43
13.45
12.17
11.79
11.99
11.99
11.61
11.62
28.45
27.47
27.92
27.09
27.02
27.06
' For conversion to figjd] multiply by 1.038; for conversion to /zmol/liter multiply by 0.05.
* Calculated by unmixing of isotope ratios as described in Appendix I
c
Measured isotopic ratios are not corrected for mass fractionation.
240
INSKIP ET AL.
TABLE 3
Measured Total Lead Concentration and Isotope Ratios in Bone Samples from Nonhuman Primate and Apportioning
of the Contributions from the Different Uniquely Labeled Doses
Number of
days from
switch to
enriched
lead
-14
-14
45
45
97
97
148
148
870
870
Total
bone
Bone lead component attributable
to each dose (JJ.g/g)"
Bone site and type
Dose
fingerprint
lead
<//g/g)c
Common
Iliac crest/cort
Iliac crest/trab
Proximal tibia/con
Proximal tibia/trab
Left distal tibia/cort
Left distal tibia/trab
Right distal tibia/cort
Right distal tibia/trab
Left proximal femur/con
Left proximal femur/trab
Common
Common
204 Pb
204 Pb
206 Pb
206 Pb
207 Pb
207 Pb
None
None
271.6
295.3
131 6
312.7
125.0
192.9
67.8
73.6
167.6
175.9
271.6
295.3
129.5
285.8
114.9
170.7
66.3
65.5
156.0
153.0
204
2.2
26.9
4.3
107
0.6
2.9
4.7
7.9
206
6.4
11.5
0.6
30
4.0
10.8
Measured ratio of lead*
207
^Pb/ 204 !
03
2.2
2.9
4.3
17.74
17.71
1484
8.74
13.25
11.32
16.36
1261
13.65
12.29
•b
15.52
15.52
12.95
7.63
10.95
9.26
14.18
10.83
11.80
10.29
^Pb/^Pb
37.27
37.15
31.14
18.19
26.18
22 04
33.91
25.14
27 79
23.96
Noie. Cort, cortical bone; trab, trabecular bone.
" Calculated by unmixing of isotope ratios as described in Appendix 1.
* Measured isotopic ratios are not corrected for mass fractionation.
r
As "marrow-free dry bone."
2O8
Pb or 2O2Pb for concentration determination. Lead was separated using a
standard HBr—HC1 ion exchange column (Manhes et ah, 1978) (using 100to 200- or 200- to 400-mesh AG 1X-8 anionic exchange resin).7 Lead
concentrations and isotope compositions were measured with a 5-collector
Finnegan MAT 261 mass spectrometer. Total lead blanks were less than
200 pg for all samples. Reproducibiliry based on multiple analyses of a
standard reference material for common lead, SRM981 (National Institute
for Standards and Technology [NIST], Bethesda, MD), was <0.05%/amu
(atomic mass unit). Uncertainties (1 a) on total lead concentration and
isotopic composition were estimated at 1% or better and 0.2% or better,
respectively, for blood samples and 0.07 and 0 05% or better, respectively,
for bone samples (based on duplicate analyses of samples; SRM955a, a
NIST standard for lead concentration in blood; and an in-house bone standard) Isotopic ratios have not been corrected for mass fractionation because
the extent of mass fractionalion in the spectrometer for biological samples
is unknown. The extent of fractionation for SRM 981, a metal standard,
was 0.13%/amu. The matrix of SRM 981 is very different from that of the
biological samples and thus it is not a good comparison for assessing
fractionation effects on the samples. No isotopic standard for blood is
available.
The total lead concentration and isotope ratios of the blood and bone
samples have been unmixed to give the proportion of the total lead derived
from each dose (Appendix 1).
RESULTS AND DISCUSSION
The isotopic ratios and total lead concentrations in sequential blood and bone samples are shown in Tables 2 and 3.
Using end-member unmixing equations (Appendix 1), we
were able to apportion the contribution to blood lead from
historic bone stores (common lead) and short-term bone
stores (^Pb-enriched, ^Pb-enriched, and ^Pb-enriched
components) and the data are plotted in Fig. 2.
7
Bio-Rad Laboratories Ltd.. Mississauga. Ontario. Canada
Total blood lead. In contrast to the relatively smooth
blood lead curves obtained in experiments with humans
(Rabinowitz et ai, 1976), the total blood lead concentrations
fluctuate between wide limits (Fig. 2A). Explanation for this
centers on the animal's blood lead being artificially maintained at a high level; although dosed daily (self dosing),
the timing of blood collection relative to ingestion of the
lead capsule is not always constant. Also, with the high lead
dose, even a small change in fractional absorption could
result in a comparatively large change in blood lead concentration. (O'Flaherty et ai, 1996).
Blood lead from oral dose. That most of the variation
in the total blood lead concentration is attributable to the
current oral dose is illustrated in Fig. 3. The rise and fall of
total blood lead closely follows the blood lead apportioned
to the oral dose.
Blood lead from bone. Significantly, the concentration
of historic lead (common lead, which is contributed by bone)
does not exhibit the variable pattern of total blood lead (Fig.
2), which indicates that the contribution from bone was relatively constant and that there was no significant contamination of the samples during sampling or processing.
The rapid drop in concentration of each dose signature
(2O4pb 2O6pbi a n d 2o?pb) i m m e ( ji a tely after termination of
administration (Fig. 2B) graphically illustrates the lability
of lead in blood and soft tissue combined. This is consistent
with in vivo measurements (210Pb) carried out in the kidneys
of baboons (Cohen, 1970) where, beyond Day 30, the lead
remaining in the tissue at this point is determined by overall
body lead stores. From Day 30 onward, the four elimination
curves reflect the return of lead from bone to blood for each
241
BONE LEAD CONTRIBUTION TO BLOOD LEAD
of four doses (Fig. 2B). The only exogenous sources of
common lead during the period of administration of enriched-isotope doses were the negligible quantities present
in feed (<0.2 /xg/g, dry wt) and air (<0.001 fig/m3). Therefore, after the introduction of the 204Pb-enriched dose and
clearance of common lead from soft tissues, the sole source
of common lead in the blood is bone. Likewise, after cessation of administration of the 2O4Pb-enriched dose and its
clearance from soft tissues, the sole source for the 2O4Pbenriched label in blood is 204Pb-enriched labeled lead deposited in bone during the 50-day dosing period. Analogous
reasoning applies to the curves representing the other two
short-term doses. The 50% reduction in dose rate in the final
dosing period did not result in a detectable difference in
fractional elimination of the M7Pb-enriched label. By dosing
sequentially with the three lead doses we were able to repeat
the experiment three times in the same animal. In each case
we were able to distinguish between the oral dose and the
historic/recent lead which was derived from bone. The contribution of lead from bone to blood was relatively constant
from day to day for each of the four doses, consistent with
the perceived physiological processes involved (diffuse exchange and bone remodeling).
We confirmed through analysis of marrow-free dry bone
that each of the unique isotopic fingerprints was incorporated
into bone (Table 3). Elimination of recently deposited labels
was demonstrated by reduction of the 204Pb label in trabecular bone after each of the two subsequent biopsies, at 45
and 97 days, after cessation of the ^ P b dose (Table 3).
Nevertheless, its long residence time was confirmed by the
continuing appearance of very small amounts of the 204Pb
fingerprint in blood samples taken 23 months after cessation
of the dosing period (Table 2).
The method gave the magnitude of the lead flux from
bone to blood. Under the conditions of this study, an estimated 20% of the total blood lead originated from historical
bone stores, although this figure may vary according to interanimal differences and the magnitude of the exposure
regimen (Inskip et ai, 1996). The flux of lead released to
blood from bone was relatively constant and a baseline level
was established. This flux was clearly dominated by the
historic bone stores (common lead) which had accumulated
over the 12 years of the animal's exposure to common lead.
All three administered isotope labels were observed to
slowly decline over the period of study, whereas, in this
particular animal, no decline in common lead was observed.
If required, the "total" contribution of bone lead to blood
lead can be obtained by summing the recent lead and the
historic lead contributions.
For this type of longitudinal investigation with very expensive isotope tracers, the results obtained for a single animal are sufficient for demonstrating the usefulness of the
stable isotope technique. The use of three consecutive isotopes generated data which show how similar the flux from
bone to blood is for each 50-day period. The uniqueness of
this approach is that it effectively allows the repeating of
the experiment three times in the same animal, while the
steady-state kinetics of lead between bone and blood are not
perturbed. Data obtained from related experiments using one
and two isotope administrations for six other animals have
provided confirmatory evidence.
The elimination curves generated in the current study need
to be analyzed by a physiologically based pharmacokinetic
model for their applicability to humans. Use of such a model,
incorporating reasonable descriptions of the actual processes
governing uptake and release of lead from bone, would assist
in interpreting the observations on regional bone uptake, as
well as in differentiation of the contributions to blood lead
of recently deposited and older bone lead. Application of a
dynamic model would also eliminate the need to restrict
interpretations to the terminal portions of the elimination
curves because steady state would not be assumed. Application of a scaled-down physiologically based model of human
lead kinetics (O'Flaherty, 1993) to the current data set, and
to data sets from pregnant nonhuman primates, has been
carried out (Inskip et ai, 1994; Franklin et ai, 1995).
This approach is also seen as having potential for investigating the time course of blood lead changes in other physiological states (lactation, menopause) as well as in disease
states such as postmenopausal osteoporosis and other bone
metabolic disorders where endogenous lead might be remobilized from bone. Such knowledge can be used to refine the
lead biokinetic models in humans, for whom experimental
parameters on bone-to-blood transfer are difficult to acquire.
APPENDIX 1
Method of Unmixing Lead Isotopic Ratios to Yield Lead
Concentrations by Time-Dependent Dose
1.0. Introduction
Lead has four naturally occurring stable isotopes (204Pb,
^ P b , M7 Pb, and 208Pb). In this study, a nonhuman primate
was sequentially dosed four times with isotopically distinct
enriched doses (common-Pb, 2O4Pb-enriched, 206Pb-enriched,
2O7
Pb-enriched). Blood and bone samples taken throughout
the experiment thus contain a mixture of isotopes from two,
three, or four doses. If the doses were 100% pure in a single
isotope (e.g., 204Pb), then it would be trivial to unmix the
doses because the proportions of each dose would simply
be the ratios of the isotopes. However, this is not the case,
and in order to determine the contribution from each dose
it is necessary to set up a series of simultaneous equations
derived from a simple mixing equation, commonly used in
isotope dilution analysis.
Atomic proportions, i.e., numbers of atoms, are used in
the equations in all except the final stages. Weight ratios are
derived in the final stages of the calculations using the atomic
242
INSKIP ET AL.
weights of the individual isotopes of lead, and the specific
atomic weights of the doses.
To calculate the atomic abundances for each of the doses,
and the measured ratios of the sample, the formula is
1
,
206
207
208\
1 +
+
+
204
204
204/
(1)
subset of this one. With four isotopes for lead there are many
combinations of ratios possible for use in the equations;
however, in practice, the ratios are chosen to minimize instrumental effects and are dependent on the doses involved.
The unmixing equation is applied three times, to form a
set of simultaneous equations.
The first equation defines the contribution from each of
the four doses to the total 2O4Pb in the mix, and uses the
isotopes ^ P b and ^ P b :
where Aba* is the atomic abundance of isotope 204, and
from which, the other proportions may be obtained:
206\
204;
= Ab'204 "
206
Ab M 7 = A b 2
\2M)\
207
I7 2 O 6 \
= Ab 2
208
(si) -
"204
(6)
2.0. The Fundamental Equation
Consider a mixture of k doses of isotopes a, b, c, and d.
Let Z\ be the number of atoms of isotope i from source k.
Then, the measured ratio of isotopes a and b in the mixture,
m, is
(2)
where c = contribution from common dose, 4 = contribution
from 204Pb-enriched dose, 6 = contribution from ^Pb-enriched dose, 7 = contribution from 2O7Pb-enriched dose.
Dividing each side by 204c and rearranging gives:
2044
204c LV204A
where the X is for all doses. The ratio of isotopes a and b
in a single dose k is defined as:
/206\ ]
\204jJ
|
204 6 r/206\
204c |A204/
U04/J + 204 c *L\204 > / 7
(3)
=
\2O4)J
_r/206\ _ /206\ 1
IA204A \204ylJ
Substituting Eq. (3) into Eq. (2) gives:
(4)
The second equation defines the contribution from each of
the four doses to the total 2O6Pb in the mix, and uses the
M7
isotopes M7
Pb and
d ^Pb
and rearranging gives:
206/c
(5)
In all equations, 2 is for all doses. Equation (5) is expanded
to k = 2, 3, 4 depending on the number of doses involved.
U06/J
-©J
L206
©-© J
2.1. Unmixing the Doses
For the case of the nonhuman primate discussed in this
paper four doses were applied (common-Pb, ^Pb-enriched,
^Pb-enriched, and ^Pb-enriched) over the period of the
experiment. An example of the expanded equations for the
four-dose case is presented below, as all other cases are a
Using the relationship:
206
206t = 204t*f— I = 204t*\204jk
204,
BONE LEAD CONTRIBUTION TO BLOOD LEAD
and dividing each side by 204c and rearranging gives:
204 4 ^/206\ J 2 0 7
204c \204/ 4 L2064
* [206 6
206mJ
and dividing each side by 204c and rearranging gives:
2 0 7 ] , 2046 _ / 2 0 6 \
206mJ
204c \204/ 6
|"207_ _ _207_l
+
+
243
2044 /207\ f208 _ ^2081 204 6 /207\
204c A204/ 4 l207 4 207mJ + 204 c *\204j 6
2047^206^ + |" 207_ _
204 c * \ 2 0 4 / 7 [2067 206n
"208 _ J2081
2076
207
206
l
J
(9)
207 m J
204^ /207\
+
+
204 c *V204/ 7
- _(?&L\ J™.
I" 208_ _ 208 1
L2077
207mJ
_ 208 1 (ID
207
m
The third equation defines the contribution from the four
doses to the total 207Pb in the mix, and uses the isotopes
^ P b and ^ P b :
207 c *
„„_,
208
207c
208
207n
, 208
2074
204, 2046 2047
204c ' 204c ' 204c
207m
208 ,
= 0. (10)
•^ ' m '
which are the ratios of the number of atoms of 2O4Pb from
the ^Pb-enriched, ^Pb-enriched, and ^Pb-enriched doses
relative to the number of atoms of 2O4Pb from the commonPb dose.
In order to solve for the unknowns, Eqs. (7), (9), and (11)
are put in terms of a matrix notation:
Using the relationship:
207 t = 204 t * [-—)
V204A
= 204 t *
207
|~206 _ _206/l ^04
|_2044
204 J*204 4
I" 206 _ _206_l 204
|_2046 204mJ*2046
F207
207 _ 207 1 206
L206
206mJ 2044
2064
206,
[2O66
_ _207_l + 206
2 0 6 mJ*204
J 2 0 46
["208
208 _ 208 1 207
I" 208
208 "I t 207
L2074
Each of the Eqs. (7), (9), and (11) contain three unknowns:
I" 206
[204,
206 1 1 204
204mJ*2047
2044
206
206 1 204
204c
204 c
204mJ
T 207_ _ _207j 206
[2067 206
206mmJ 204,
2046
204c
207
206 c
207 1 206
206mJ 204c
[" 208
204,
208
204c
207c
208 1 207
207ro I *204c
207 m J *204 6
207mJ 2044
208 ^207
207m 1*2047
204 c
A*X = C
And X can be solved in terms of the inverse of A. In practice
a Gaussian elimination routine is used to maximize numerical precision. In the three-dose case, the matrix A reduces
to the first two rows and columns; in the two-dose case, the
matrix A reduces to the first row and column. Knowledge of
X(l), X(2), and X(3) allows the calculation of the fractional
contribution of 2O4Pb from each dose as
Total-Pb =
+
204c
204,
204.
204c
204c '
+
204c
TotalPbt =
where Total ^ P b = total ^ P b in the sample.
2.3. Conversion to Contribution by Dose
Continuing with the four-dose example and recalling that:
204c
204c
which are the ratios (atomic) of 2O4Pb from the doses, 204Pbenriched, ^Pb-enriched, and ^"Pb-enriched, relative to the
common dose.
Because 2O4Pb is a different proportion of the total lead in
each dose, these ratios are not equivalent to the ratios of
total lead from each dose. To calculate the ratios of total
lead (atomic) from the ^Pb-enriched, ^Pb-enriched, and
2O7
Pb-enriched doses, relative to the common dose, we use
the relationship
204c
204 t
Ab204 t '
where TotalPbt is the total lead from dose k in the sample,
204 t is the 2O4Pb (atomic) from dose k in the sample, and
Ab204 t is the abundance of ^ P b in the dose k.
244
INSK1P ET AL.
Therefore:
Xtot(2) =
Xtot(3) =
TotalPbc
Ab2044
TotalPbc
Ab2046
TotalPbc
Ab2047
PbConc4 = XWR(l)*PbConc c
Before the total lead (in /xg/g) contributed by each dose is
calculated, the ratios given above must be converted to
weight ratios. This is accomplished by multiplying the appropriate ratio Xtot(l, 2, or 3) by the appropriate ratio of
atomic weights of the two doses. Now the atomic weight of
each dose is:
AtomicWt* = X (Ab,*AtomicWt,)
where k = dose, the 2 is for all isotopes i, Ab, is the fractional
abundance of isotope i in dose k, and AtomicWt, is its atomic
weight.
The atomic weights of the individual isotopes are:
Atomic
Atomic
Atomic
Atomic
wt
wt
wt
wt
204
206
207
208
=
=
=
=
where PbConcc = concentration of lead from common-Pb
dose (in /ig/g) in the sample, and TotalPbConc = concentration of total lead of the sample (in /xg/g). From this, the
contributions from the other doses can be calculated:
203.973
205.974
206.976
207.977
The weight ratios are therefore:
XWR(l) = Xtot(l)*
AtomicWt^
Atomicwt,.
Atomicwt,,
XWR(2) = Xtot(2)*
Atomicwt,.
XWR(3) = Xtot(3)*
Atomicwt7
Atomicwt,- '
where XWR(l) = weight ratio of total lead from ^ P b enriched dose/common-Pb dose, XWR(2) - weight ratio of
total lead from 2O6Pb-enriched dose/common-Pb dose,
XWR(3) = weight ratio of total lead from ^"Pb-enriched
dose/common-Pb dose.
Finally, the contributions from each dose (in fig/g) to the
total lead concentration of the sample, are derived as follows:
To determine the total lead contribution from the commonPb dose:
PbConc c
1
*TotalPbConc,
1 + XWR(l) + XWR(2) + XWR(3)
PbConc6 = XWR(2)*PbConc c
PbConc, = XWR(3)*PbConc c ,
where PbConc4 = concentration of lead from 204Pb-enriched
dose (in fig/g) in the sample, PbConc6 = concentration of
lead from 206Pb-enriched dose (in fJ.g/g) in the sample, and
PbConc7 = concentration of lead from ^"Pb-enriched dose
(in /xg/g) in the sample.
Software for multicomponent unmixing is available in the
form of an EXCEL spreadsheet or Quick BASIC program.
Please send a diskette with your request, or contact E. B.
Edwards at [email protected]
for e-mail delivery.
3.0. Experimental Error
The greatest analytical uncertainty on the data is mass
fractionation in the spectrometer. As discussed in the text,
we have not corrected the measured isotopic ratios for fractionation, and had no way of assessing mass fractionation
for these samples. Mass fractionation results from the tendency of the lighter isotopes to evaporate preferentially from
the filament during thermal ionization. It is most simply
described by a linear law
/-,+„,, = rT+n,,*(\ +
nf),
where the ratio on the left is the corrected ratio, the m designates the measured ratio, a n d / i s the mass fractionation per
atomic mass difference.
Under standard conditions (i.e., for the NBS981 standard
on the Carleton University spectrometer) the fractionation is
0.13% per atomic mass unit (amu). Correcting the measured
ratios for this amount of fractionation produces a change in
the unmixed values for lead attributable to each dose of less
than 0.01 /xg/100 g. Furthermore, an additional 50% increase
in fractionation from the standard value (i.e., 0.2%/amu)
gives a change in the unmixed values for lead attributable
to each dose of less than 0.2 //g/100 g. In both cases the
errors are extremely small and are biologically insignificant.
The number of significant figures quoted in Table 2 reflect
these uncertainties.
ACKNOWLEDGMENTS
This work is part of a series of studies investigating lead biokineucs in
nonhuman primate pregnancy funded by the National Institute of Environmental Health Sciences under Contract N01-ES-O5285 to C.A.F. We thank
Drs. W Jameson and R. Goyer of NIEHS, and Dr. K. Mahaffey (now with
BONE LEAD CONTRIBUTION TO BLOOD LEAD
U S. EPA) for their advice and support, M. Tocchi for bone dissection,
M. J. Conboy and D. Schanzer for their assistance with data analysis and
interpretation, C. Ferrarotto, C. Leblanc. D. Johnston, and C. Harron for
their assistance with clean-room blood sampling procedures, Drs. C. Bihun
and J. Foumier for help with bone biopsy development, and Animal Resources Division staff, M. Norman. D. Parks. T. McCabe. C. Dawe, and
D. Demers. for animal care and welfare.
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