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Plasma and Blood Lead Concentrations, Lead Absorption, and Lead Excretion in Nonhuman Primates
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
138, 121–130 (1996)
0105
Plasma and Blood Lead Concentrations, Lead Absorption, and Lead Excretion in Nonhuman Primates1 E. J. O’FLAHERTY,*,2 M. J. INSKIP,† A. P. YAGMINAS,‡
AND
C. A. FRANKLIN†
*Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0056; and †Drugs Directorate and ‡Environmental Health Directorate, Health Protection Branch, Health Canada, Tunney’s Pasture, Ottawa, Ontario, Canada Received August 24, 1995; accepted January 30, 1996
relative efficiency of lead excretion in urine and feces. Plasma and Blood Lead Concentrations, Lead Absorption, and Lead Excretion in Nonhuman Primates. O’FLAHERTY, E. J., INSKIP, M. J., YAGMINAS, A. P., AND FRANKLIN, C. A. (1996). Toxicol. Appl. Pharmacol. 138, 121–130. In order to assess the comparability of lead disposition in the cynomolgus monkey to that in the human, we determined the relationships among blood lead concentration, plasma lead concentration, and lead excretion in monkeys. Six adult (3–5 kg) female cynomolgus monkeys (Macaca fascicularis) without previous experimental lead exposure were given single intravenous injections of from 750 to 3300 mg lead as lead nitrate, labeled with 210 Pb, per kilogram body weight. Four additional monkeys, fasted overnight, were administered single oral doses of either 750 or 1500 mg lead as lead nitrate, labeled with 210Pb, per kilogram for the assessment of fractional absorption. Blood and plasma lead concentrations (10 monkeys) and urinary and fecal excretion of lead (2 monkeys) were followed for up to 16 days after lead administration. Fractional absorption from an oral dose was 44% at the lower of the two doses and 22–28% at the higher dose. The relationship between plasma and blood lead concentrations was found to be similar to that in humans, with plasma lead concentration at most a few percent of total blood lead concentration at low concentrations. Partitioning of lead across the red cell membrane in the 2 monkeys given exceptionally high doses (3300 mg/kg) intravenously was distinctly lower than that in the 4 monkeys given lower intravenous doses. Urinary clearance of lead in these 2 monkeys was 19% of the estimated glomerular filtration rate, within the range of efficiencies reported for humans. Fecal clearance, however, was anomalous and appeared to be an artifact of the very high dose. Examination of published data for urinary and fecal lead excretion in three adult baboons showed that both functions in the baboons were quantitatively similar to those in humans. Urinary clearance in the baboons was 14–24% of the estimated glomerular filtration rate, and fecal clearance was 78– 85% of the urinary clearance. We conclude that nonhuman and human primates are comparable with respect to the relationship of plasma lead concentration to blood lead concentration and the
q 1996
Academic Press, Inc.
Partitioning of lead between blood plasma and the red cell is markedly dependent on the plasma concentration of lead in humans (DeSilva, 1981) and in rats (Klaassen and Shoeman, 1974). This partitioning can be described in terms of a capacity-limited binding of lead by the constituents of the red cell (Marcus, 1985; O’Flaherty, 1991, 1993). The implication of this concentration dependence is that while plasma lead concentration may increase linearly with whole-body lead uptake, the concentration of lead in the red cell will plateau at higher exposures. Although the relationship of plasma lead to lead exposure may be linear, the relationship of blood lead to exposure will be nonlinear. These relationships are particularly important for modeling lead kinetics, since whole blood lead concentration is conventionally measured experimentally while excretion and transfer into tissues are generally understood to take place from the plasma. When plasma lead concentration and blood lead concentration are not directly proportional to one another, the relationship of plasma lead to red cell lead must be characterized and quantified if the kinetic behavior of lead is to be fully understood. As part of a study whose broader purpose is to evaluate the contribution of bone lead to blood lead in nonhuman primates with well-documented lead exposure histories (Inskip et al., 1994), we undertook to examine the relationships between plasma lead and blood lead and between plasma lead and urinary and fecal lead excretion in cynomolgus monkeys with no known previous lead exposure. Fractional lead absorption from an oral dose was also estimated in this study. MATERIALS AND METHODS Animals
1
This work was supported by NIEHS Contract N01-ES-05285. To whom correspondence and reprint requests should be addressed at Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Cincinnati, OH 45267-0056. 2
Ten healthy adult female cynomolgus monkeys (Macaca fascicularis) without previous experimental lead exposure were assigned to the study from a research pool. Animals had either been bred in-house or had originally been caught in the wild and raised in the colony for at least 7 years.
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Body weights ranged from 3.1 to 5.1 kg. The animals were housed in the primate facility of the Health Protection Branch of Health Canada, Ottawa, Ontario. Each animal was housed individually in a controlled environment with a 12:12 hr light:dark cycle. Humidity was maintained at 40 { 10% and the room temperature at 22 { 27C. Access to food (Primate Monkey Chow, type 5047 (PMI Feeds, Richmond, IN) and water was on an ad libitum basis, with supplementary provision of vegetables and fruit. Lead content of food was certified by the manufacturer to be less than 0.25 mg/g. Drinking water lead content was less than 50 mg/liter. Dose Levels and Preparation Lead (210Pb) nitrate with a specific activity of 10 mCi/mg Pb and purity greater than 95% (Amersham International, Oakville, Canada) was received dissolved in 3 M HCl. Appropriate safety precautions were taken in dealing with radioactive materials and wastes in accordance with safety and handling guidelines issued by the Atomic Energy Control Board of Canada. Lead nitrate (Pb(NO3)2) (BDH Chemicals, Toronto, ON) was dissolved in 0.9% saline and mixed 9:1 with the tracer solution so that the 210Pb label was approximately 10% of the total lead. One measurement of total lead concentration in a serially diluted subsample of the dosing solution was performed by Dr. J. Blenkinsop at the Department of Earth Sciences, Carleton University. Analysis was by isotope dilution mass spectrometry using a stable 208Pb spike and a magnetic sector Finnegan-MAT 261 spectrometer. The dosing solution had a pH of 7.2 and an osmolality of approximately 300 mOsm. Doses of 750 or 1500 mg Pb/kg body weight were administered either intravenously (0.5 ml/kg body weight) or orally to the eight animals in Study 1. Selection of dose levels was based partly on the design of the parent study with a separate cohort of monkeys given a lifetime oral daily dose of 1500 mg Pb/kg/day, and partly on consideration of the probable relative oral/intravenous bioavailability. The two animals in Study 2 received intravenously approximately 3300 mg Pb/kg body weight. The same dosing solution was used in both studies. Due to the time difference between the two studies, adjustments were made to the total dose in Study 2 to allow for decay of the 210Pb label. The half-life of 210Pb is approximately 22 years. Major decomposition products are 210Bi and 210Po, noted by the supplier to be present initially at less than 1 mCi. Baseline blood lead concentration is low in these monkeys, of the order of 2 mg/dl (0.04 mg/dl of plasma if plasma lead concentration is 2% of blood lead concentration), and is swamped by the administered carrier lead. Experimental Design Two separate studies were conducted. In the first, only the relationship between plasma and blood lead was monitored. In the second, which was designed primarily to determine urinary and fecal lead clearance, the plasma/blood lead measurements were repeated. Study 1. The subjects were eight female cynomolgus monkeys without previous experimental lead exposure. The animals were placed in a neck restraint apparatus for the period covering intravenous dose administration and collection of the first four blood samples. Subsequently, the restraining device was used only at the times blood samples were required. Four monkeys were given the lead solution intravenously via the saphenous vein over a period of 1 min. Four were administered lead orally by capsule. Blood samples were obtained from the saphenous vein using heparinized Vacutainer tubes (Becton–Dickinson, Franklin Lakes, NJ) at time points of 0.05 (intravenous only), 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 12, 24, 36, and 48 hr and subsequently every 24 hr for a total of 13 days. The blood samples were divided into two equal portions, one of which was counted as whole blood and also used for determination of the hematocrit. The other portion was separated into plasma and cell fractions by centrifugation for 5 min at 2400 rpm. The cell fraction was washed twice with physiologic saline and centrifuged. Each of the samples (blood, blood plasma, cell fraction, and saline washes) was placed in a 5-ml lead-free polypropylene tube (Sarstedt Canada, St. Laurent, PQ) for counting.
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Study 2. The subjects were two female cynomolgus monkeys without previous experimental lead exposure. The intravenous dose was administered over an approximately 15-sec interval via the cephalic vein using an intravenous catheter placement unit, size 24 (Deseret Medical, UT). Blood samples were obtained from the femoral vein at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hr and then every 24 hr for a total period of 16 days. Particular procedures were incorporated to minimize the risk of red cell hemolysis. Extreme care was taken during the sampling by slowly withdrawing the blood using a heparinized disposable syringe. After removal of the needle and inversion of the syringe to prevent clotting, the blood was slowly ejected into a 2-ml heparinized Becton–Dickinson centrifuge tube with vacuum released. Exactly 1.0 ml of blood was transferred by pipet to a 5-ml polypropylene tube (Sarstedt Canada, St. Laurent, PQ) and centrifuged in a fixed-angle rotor at 2000 rpm for 30 min. A 200-ml aliquot of plasma was carefully pipetted from the top of the tube, avoiding contact with the tube walls, transferred to another 5-ml polypropylene tube, and retained for counting along with the tube containing the remaining blood plasma, red cells, and white cells/platelets. Stool samples were collected daily using screened collection trays. The entire stool sample was weighed and mixed well for 2 to 3 min by physical manipulation through a double, heavy-walled polyethylene bag. Using a wooden spatula, a representative subsample was prepared for counting by placing 1 to 2 g of material in a 5-ml polypropylene tube. Complete 24-hr stool collections were also made on 6 days for each monkey. Urine was collected in underlying polyethylene-lined collection trays screened from contamination by stool. The urine was poured into a polyethylene container and mixed well, and a subsample placed into a 5-ml polypropylene tube. In general, urine collections of recorded duration were made. In addition, spot urine samples were collected as close as possible to the times of blood sampling. Twenty-four-hour collections were made daily in order to establish the volume of urine excreted daily by each animal. Measurement of Radioactivity Blood, blood plasma, urine, and stool samples were all counted in 5-ml lead-free polypropylene containers (Sarstedt Canada, St. Laurent, PQ) in order to achieve consistent geometry. Activity was determined by counting the 47 kev gamma radiation in a gamma counter (LKB-1282 CompuGamma, Fisher Scientific, Ottawa, ON). In Study 1, the counts for plasma, saline, and cells from the divided aliquot were summed and the mean value of this total and the count obtained for the whole blood aliquot was considered the blood lead count. In Study 2, whole blood lead counts were calculated as the weighted average of counts in the plasma aliquot and in the combined remaining plasma, red cell, and white cell/platelet fraction. To obtain the concentration of lead in whole blood, counts were transformed using a conversion factor calculated from the original 210Pb/carrier lead dosing solution. The activity of the dosing solution was determined at the time of assay of the biological samples. Calibrations were obtained from serial aqueous dilutions covering the anticipated concentration range. In Study 2, dilutions were carried out both in saline and in heparinized whole blood and urine from pooled samples collected from non-lead-exposed monkeys. No differences due to diluent were detectable. Data Analysis Plasma and blood lead concentrations for the six monkeys given lead intravenously were fit by the expression PbB Å (1 0 HCT) 1 PbP / HCT 1 PbP 1 (A / BIND/(KBIND / PbP)),
(1)
where PbB is blood lead concentration, PbP is plasma lead concentration, HCT is the hematocrit, BIND is the maximum red cell lead binding capacity, KBIND is the dissociation constant for this binding, and A is a constant
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FIG. 1. The relationship of plasma lead to blood lead in Monkey 86163 (Study 1) at blood lead concentrations less than 1600 mg/dl.
of proportionality between plasma lead and unbound red cell lead. The assumptions on which this expression is based are that plasma lead and unbound red cell lead are directly proportional to one another and that red cell lead exchanges between the unbound fraction and a group of binding sites that can reasonably be characterized by a single maximum capacity and dissociation constant (Marcus, 1985; O’Flaherty, 1993). The fit of this expression to plasma and blood lead concentrations was optimized in two steps. Concentration pairs above PbP Å 100 mg/dl (PbB É 200 mg/dl) were fit by linear regression to obtain the value of A. A was then held constant while the entire data set was fit by nonlinear regression to estimate the values of BIND and KBIND . Data were weighted by the reciprocal of the dependent variable, PbB in this case. For the estimation of excretion clearances in the two monkeys in Study 2 for whom urine and feces were also monitored, only plasma lead concentrations below 3 mg/dl (blood lead concentrations below 100 mg/dl) were considered. Above this concentration range, both urinary and fecal excretion were erratic and tended to fall off relative to plasma lead. The relationship between urinary or fecal lead excretion rate and plasma lead concentration was determined by linear regression constrained to pass through the origin. Because plasma lead concentrations fell rapidly after administration to levels near the detection limit, measured plasma concentrations were not used directly. Instead, plasma concentrations were calculated at the measured blood lead concentrations using the line of best fit of the relationship of plasma lead to blood lead for each monkey (Expression 1). At some time points of urine or fecal collection, a measured blood lead concentration was not available. Plasma lead concentration at these time points was estimated by interpolation. Urinary lead excretion rate was estimated by multiplying the concentration of lead in a timed urine sample by the total volume of the sample. Some of the urine samples were timed 24-hr collections, while some were collections of measured volume taken over shorter time periods and some were spot samples taken simultaneously with a blood sample. Within the rather large variability in this data set, lead excretion rates estimated directly from the timed urine samples were not different from those calculated using daily urine volumes. Fecal lead excretion rate was estimated by multiplying the concentration of lead in a fecal sample by the mean daily total weight of feces excreted, determined based on the measurements from 6 sampling days for each monkey.
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An approximate average total (urinary and fecal) clearance was also estimated for the six monkeys given lead intravenously, by dividing the administered dose by the trapezoidal area under the plasma lead concentration curve from time zero to the end of the study 13 or 16 days later. A physiologically based model of lead kinetics in the monkey (Inskip et al., 1994) was visually fit to blood and plasma lead concentrations in the four monkeys given lead orally in order to estimate the fraction of the lead dose that had been absorbed. The model, to be presented in greater detail in a separate paper, is structurally identical to a physiologically based model of human lead kinetics (O’Flaherty, 1993, 1995). For this study, the human model was scaled to the body weight of the adult cynomolgus monkey. Tissue weights and bone mass bear the same relationship to body weight in the scaled model as they do in the human model. Cardiac output was scaled from the human on the basis of adult body weight to the 34 power. Glomerular filtration rate was set at 2 liters plasma/day/kg, scaled from the rhesus monkey (Bourne, 1975). Partitioning of lead into tissues including bone surface and the values of all bone uptake parameters were set identical to those in humans. In particular, bone turnover rate was set to the value 15%/year used for adult humans (O’Flaherty, 1995). The increased rates of bone growth and turnover that characterize the growth spurt of human adolescence were not included in the scaled model, since the monkeys were adults at the time of lead administration. The simulations were carried out by modeling parallel systemic absorption and fecal excretion (gut transit) processes acting on the total oral dose in the gastrointestinal tract. The absolute and relative magnitudes of the two processes were varied to obtain a good visual fit to the concentration data for each monkey and to estimate the corresponding total amount absorbed.
RESULTS
The relationship between plasma and red cell lead concentration in the six monkeys (four from Study 1, two from Study 2) given lead intravenously is illustrated for two of the monkeys in Figs. 1 and 2, and the parameter values and other pertinent information for all six monkeys
FIG. 2. The relationship of plasma lead to blood lead in Monkey 84117 (Study 1) at blood lead concentrations less than 1600 mg/dl.
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TABLE 1 Kinetic Parameters for Lead Partitioning and Excretion Study 1 Monkey:
86163
Weight, kg Intravenous lead dose, mg Initial plasma lead concentration, mg/dl Initial blood lead concentration, mg/dl A BIND, mg/dl of red cell volume KBIND, mg/dl of red cell volume AUC,c mg-hr/ml plasma Total clearance, dose/ AUC, liters plasma/ day Urinary clearance, liters plasma/day Estimated glomerular filtration rate, liters plasma/dayg Urinary clearance, fraction of glomerular filtration rate Fecal clearance, liters plasma/day
Study 2
86058
84117
84020
84042
83647
3.10
3.48
5.12
3.43
4.73
4.17
4495
2714
6912
2710
15470
13890
1362
544
1542
770
8023
7601
1664 1.12 (0.035)a
745 1.29 (0.072)
1841 1.15 (0.023)
977 1.19 (0.018)
5154 0.602 (0.0102)
4310 0.554 (0.066)
154 (14)
240b
211 (18)
221 (3)
240 (77)
349 (74)
4.9 (1.1) 33.5
5b 25.2
0.99 (1.9) 44.4
11.4 (3.6) 24.0
4.5 (2.9) 322.3
3.0 (1.6) 253.2
3.2
2.6
3.7
2.7
1.2
1.3
1.3d
1.4d
1.8d
1.4d
1.75 (0.24)e, f
1.60 (0.23)e
6.7
7.3
9.8
7.2
9.2
8.4
—
—
—
—
0.19
0.19
1.9 h
1.2h
1.9 h
1.3h
43.8 (5.5)e
18.7 (1.8)e
a
Standard error in parentheses. Mean of the other five values of this parameter. Data from Monkey B were not fit well by nonlinear regression using Expression 1, although visually the fit with these parameter values was good. c Trapezoidal area under the plasma concentration curve, calculated as described in the text. d Estimated by scaling mean urinary clearance of 0.62 liters/kg/day from Study 2 on the basis of the 2/3 power of body weight (intraspecies scaling). e Experimental value. f One excretion value, of 198 mg/day at a plasma lead concentration of 1.4 mg/dl, was excluded from this regression. g Estimated by scaling measurements made in the rhesus monkey (Bourne, 1975) on the basis of the 3/4 power of body weight (interspecies scaling). h Estimated as total clearance minus urinary clearance. b
are given in Table 1. The values of BIND and KBIND are determined by the shape and positioning of the curvature of this relationship at blood lead concentrations below about 200 mg/dl. At higher concentrations, the relationship between blood lead and plasma lead is linear, with slope A (Expression 1). In the four monkeys from Study 1, whose maximum (initial) blood lead concentrations did not exceed 1840 mg/dl, the slope A varied from 1.1 to 1.3. In the two monkeys from Study 2, whose initial blood lead concentrations were 4310 and 5150 mg/dl, the optimized values of A were 0.55 and 0.60. Table 2 gives the estimated fractional lead absorption by the four monkeys given lead orally. Fractional absorption was dose-dependent, being lower at the higher of the two doses.
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TABLE 2 Lead Absorption Monkey: Weight, kg Oral lead dose, mg Amount of lead absorbed, mga Fraction oral dose absorbed
83681
83145
83189
83472
3.40 2700
3.14 5400
3.35 2700
3.56 6300
1060
2290
1080
2360
0.44
0.22
0.44
0.28
a Calculated, as described in the text, by visually fitting a physiologically based model to blood and plasma lead concentrations, optimizing the magnitude of systemic absorption in order to achieve a good fit.
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FIG. 3. The relationship of urinary lead excretion rate to plasma lead concentration in Monkey 84042 (Study 2). As described in the text, plasma lead concentration was estimated by interpolation at urine collection times at which a measured blood lead concentration was not available.
FIG. 5. The relationship of fecal lead excretion rate to plasma lead concentration in Monkey 84042 (Study 2). As described in the text, plasma lead concentration was estimated by interpolation at feces collection times at which a measured blood lead concentration was not available.
Figures 3 and 4 illustrate the relationship between urinary lead excretion and plasma lead concentration in the two Study 2 monkeys. Figures 5 and 6 illustrate the comparable relationships for fecal lead excretion in the two Study 2 monkeys. The slopes, or urinary and fecal lead clearances, are given in Table 1.
DISCUSSION
The human plasma lead/blood lead concentration relationship reported by DeSilva (1981) is satisfactorily described by Expression 1 with values of A Å 1, BIND Å 270 mg/dl red cell volume, and KBIND Å 0.75 mg/dl red cell volume
FIG. 4. The relationship of urinary lead excretion rate to plasma lead concentration in Monkey 83647 (Study 2). As described in the text, plasma lead concentration was estimated by interpolation at urine collection times at which a measured blood lead concentration was not available.
FIG. 6. The relationship of fecal lead excretion rate to plasma lead concentration in Monkey 83647 (Study 2). As described in the text, plasma lead concentration was estimated by interpolation at feces collection times at which a measured blood lead concentration was not available.
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(O’Flaherty, 1993). The corresponding parameter values estimated (O’Flaherty, 1991) for the rat plasma lead/blood lead concentration relationship reported by Klaassen and Shoeman (1974) are A Å 1, BIND Å 1000 mg/dl red cell volume, and KBIND Å 6.7 mg/dl red cell volume. Comparison with the values of A, BIND, and KBIND in the cynomolgus monkeys (Table 1) suggests that there are not marked interspecies differences in the characteristics of lead binding by constituents of the red cell. There is considerable interanimal variability in the estimated values of KBIND in the present study (Table 1), and large uncertainties are associated with them. However, their range lies among the values that have been assigned to this parameter for rats and humans. The interanimal differences appear to be real, as illustrated by a comparison of the data from monkeys 86163 and 84117 in Figs. 1 and 2. These monkeys were given the same intravenous dose in Study 1. For simplicity, the red cell constituents that bind lead have been treated as if they presented a single set of identical binding sites. This is known not to be the case. Lead is bound to a variety of components of the human red cell including hemoglobins and low-molecular-weight proteins, some of which are inducible, as well as to sites on the red cell membrane. Binding to many of these sites is capacitylimited, causing shifts in the pattern of binding with changing blood lead concentration (Bruenger et al., 1973; Raghavan and Gonick, 1977; Ong and Lee, 1980a,b; Lolin and O’Gorman, 1988). There is no reason to suppose that binding in the red cell of the nonhuman primate is less complex. However, no quantitative data exist to suggest the magnitudes of parameters of a red cell model with more than one kind of binding site. Addition of a second class of red cell binding sites to the plasma/red cell partitioning model results in a cubic equation (not shown) that is not more satisfactory than Expression 1 as an expression of the partitioning of lead between plasma and red cell. The criterion of parsimony dictates use of the simpler model. For hematocrit HCT Å 0.45, A Å 1.2, maximum red cell binding capacity BIND Å 240 mg/dl red cell volume, and dissociation constant KBIND Å 1–10 mg/dl red cell volume, plasma lead concentration (Expression 1) in these cynomolgus monkeys would range from about 0.8% to about 7% of total blood lead concentration at low blood lead concentrations. In humans, plasma lead concentrations up to and exceeding 5% of the blood lead concentration have been reported at blood lead concentrations below about 50 mg/dl (see discussion in Everson and Patterson, 1980), but the most reliable estimates are in the range of 1% or less (Everson and Patterson, 1980; Manton and Cook, 1984). Some of the variability in reported plasma lead/blood lead concentration ratios is probably due to slight hemolysis of blood samples, which, at these concentration ratios, could significantly contaminate the plasma fraction.
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It is of interest whether there is a demonstrable time lag for achievement of a steady state between plasma and red cell lead when lead is administered intravenously. The plasma/red cell lead concentration ratio is responsive to changes in lead uptake over short time frames. DeSilva (1981) observed a rapid rise in the ratio of plasma to red cell lead during the first few hours after ingestion of 100 mg of lead by an adult human subject. Some of this increase can be attributed to the expected shift in partitioning that accompanied the increase in total blood lead to a maximum of 78 mg/dl. Some of it may have been due to a time lag in achievement of plasma/red cell steady state because the highest plasma/red cell concentration ratio recorded, greater than 3%, was larger than the steadystate ratio expected at the peak blood lead concentration. Nonetheless, the time lag for equilibration of lead between plasma and red cell does not appear to be large. In vitro, equilibrium between human red cell and plasma lead is achieved within 15 min, with an apparent half-time of less than 5 min (Hursh and Suomela, 1968; Barltrop and Smith, 1975). Chamberlain et al. (1978) administered 203Pb intravenously to adult humans and monitored its disappearance from the plasma. They also carried out an in vitro experiment in which human blood was incubated with 203Pb. The in vivo and in vitro loss rates from the plasma were strikingly similar, with half-times of about 3 min. There is no indication in the current study of a significant time lag to steady state. The first blood samples in Study 1 were taken 3 min after intravenous administration of the lead salt. The mean plasma lead/blood lead concentration ratio at this time in the two monkeys given 750 mg lead/kg was 0.76. This value is actually slightly lower than the mean ratio, 0.85, observed 1 hr later in the two Study 1 monkeys given 1500 mg lead/kg, at a time when concentrations had fallen to the initial values observed in the monkeys given the smaller dose. Fractional absorption of lead by the four Study 1 monkeys given lead orally (Table 2) is similar to that seen in fasted humans given soluble salts of lead. Rabinowitz et al. (1980), administering stable lead isotope tracers to five healthy men, found that a mean of 8% (SD 3%) was absorbed when the tracers were ingested with food, and a mean of 35% (SD 13%) when the tracers were taken after a 9-hr fast. Absorption in the two monkeys given the lower dose in the present study was 44%, while absorption in the two monkeys given the higher dose was 22 and 28% (Table 2). This observation suggests dose-dependent fractional absorption, which has been documented in rats (Aungst et al., 1981; Pola´k et al., 1996). The two Study 2 monkeys displayed lead partitioning very different from that of the Study 1 monkeys. While the values of BIND and KBIND are not greatly dissimilar in the two studies, the values of A in Study 2 are significantly smaller
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FIG. 7. The relationship of urinary lead excretion rate to blood lead concentration in Baboon B-100 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
than those in Study 1, implying that while binding of lead within the red cell is essentially the same in the two studies, partitioning of lead across the red cell membrane is not. Simons (1986b, 1993) has shown that lead, in association with bicarbonate, is passively transferred by the anion-exchange carrier in both directions across the red cell membrane and that the distribution of unbound lead across the membrane is close to equilibrium. The mean value of A, 1.2, in the four Study 1 monkeys is lower than the equilibrium ratio proposed by Simons (1986a), who noted that the red cell membrane potential of 012 mV would be consistent with a red cell-to-plasma unbound lead equilibrium ratio of 2.5:1. The impact of plasma binding of lead on the equilibrium ratio expressed in terms of total plasma lead concentration rather than unbound lead concentration, as it is in the present study, has not been quantified. In Study 2, plasma lead concentration was much higher than the red cell unbound lead concentration. Lead affects the permeability of the red cell membrane to sodium and potassium (Simons, 1986a). It is plausible that high plasma concentrations of lead might interfere with normal lead transfer into the red cell. The measured initial plasma lead concentrations in these two monkeys were extraordinarily high, around 8000 mg/dl, more than twice those predicted by extrapolation of the initial concentration/dose relationship from Study 1. It appears probable that movement of lead into the red cell was inhibited by factors related to the magnitude of the plasma lead concentration. In view of the disproportionately high initial concentrations of plasma lead in Study 2, it may be that lead distribution into other tissues at early times after lead
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administration was also impaired. Estimated total plasma clearance in these two monkeys, calculated as the dose divided by the trapezoidal area under the plasma concentration curve, was lower than that in the four monkeys in Study 1 (Table 1), although the measured urinary clearance, compared to that observed by Cohen (1970) in baboons, scaled allometrically as the 34 power of body weight (see discussion below). Urinary and fecal lead clearances were calculated from data collected after Day 10 (Monkey 84042) or Day 6 (Monkey 83647) following administration, when plasma lead was less than 3 mg/dl. In both monkeys, urinary lead clearance was 19% of the glomerular filtration rate estimated for a monkey of the same body weight (Table 1). This is within the range of efficiency of urinary lead excretion in adult humans, with values from 4 to 28% reported (Chamberlain et al., 1978; Manton and Malloy, 1983). Observations on the urinary excretion of lead by baboons have been published. Cohen (1970) administered 210Pb to three adult (12–14 kg) female baboons by single intravenous injection of carrier-free tracer and monitored the amounts of radiolabel in blood and urine for 48 to 62 days. The excretion data were reported. The blood concentrations were not reported, but a two-exponential expression that generated the observed concentrations was given for each baboon. Figures 7–9 illustrate the rate of excretion of radiolabel in the urine as a function of the concentration of radiolabel in the blood, based on a reconstructed blood activity curve, for each of the three baboons. Linear regressions are shown. Their slopes, the urinary clearances of lead from blood, are 59 ml
FIG. 8. The relationship of urinary lead excretion rate to blood lead concentration in Baboon B-106 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
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FIG. 9. The relationship of urinary lead excretion rate to blood lead concentration in Baboon B-158 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
FIG. 10. The relationship of fecal lead excretion rate to blood lead concentration in Baboon B-100 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
blood/day for baboon B-100, 45 ml blood/day for baboon B-106, and 177 ml blood/day for baboon B-158 (Figs. 7– 9). (The relationship between lead excretion rate and blood lead concentration for baboon B-158 (Fig. 9) is clearly curvilinear, and not fit well by a single straight line.) If plasma lead concentration is 2% of the blood lead concentration, urinary lead clearance would be a mean of 2.6 liters plasma/ day in the two similar baboons, and 8.8 liters/day in the third baboon. Glomerular filtration rate in a 12-kg baboon, estimated by scaling on the basis of the 34 power of body weight from measurements made in the rhesus monkey (Bourne, 1975), should be around 19 liters/day. Thus, urinary clearance of lead would be about 2.6/19, or 14%, of the glomerular filtration rate in baboons B-100 and B-106, and 46% of the glomerular filtration rate in baboon B-158. The two lower excretion efficiencies lie within the range of values noted above for humans. Cohen (1970) also monitored fecal excretion of lead in the three baboons administered 210Pb by intravenous injection. Figures 10–12 illustrate these relationships. None of the three data sets exhibits any obvious nonlinearity, and all three were analyzed by linear regression. Their slopes, the clearance of lead from blood into the feces, are 46 ml blood/ day for baboon B-100 (2.3 liters plasma/day, or 78% of the urinary clearance), 42 ml blood/day for baboon B-106 (2.1 liters plasma/day, or 93% of the urinary clearance), and 80 ml blood/day for baboon B-158 (4.0 liters plasma/day, or 45% of the urinary clearance). The ratios of fecal to urinary clearance are comparable to values reported for humans,
which have ranged from 0.33 to 1.0 (Chamberlain et al., 1978). The fecal lead clearances measured in the two very highdose monkeys in Study 2 (Table 1) are not consistent with the values reported for humans and baboons. They are very
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FIG. 11. The relationship of fecal lead excretion rate to blood lead concentration in Baboon B-106 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
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given the substantial interanimal differences in the values of the binding parameters for red cell lead in the monkeys in the present study, there appear to be only minimal differences between human and nonhuman primates in absorption, red cell/plasma partitioning, and urinary and fecal excretion of lead. ACKNOWLEDGMENTS The authors thank Catherine Ferrarotto and Ross Layberry (University of Ottawa) for carrying out the 210Pb determinations conducted during Study 2, and Dena Schanzer for assistance with data management. Ann Dorward (McMaster University) carried out the analysis of samples from Study 1. Assistance from animal health technologists C. LeBlanc, A. St.-Pierre, and S. Stals and the animal care staff at the Animal Resources Division of the Health Protection Branch, Health Canada, is greatly appreciated.
REFERENCES FIG. 12. The relationship of fecal lead excretion rate to blood lead concentration in Baboon B-158 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
large, greatly exceeding the estimated rate of blood flow to the liver in a 4-kg animal. Very little lead was excreted in the feces of either of these monkeys during at least the first 3 days after administration. Shortly after, the rate of fecal lead excretion was as high as 900 mg/day in Monkey 84042 and 400 mg/day in Monkey 83647. Essentially 100% of the dose had been excreted by Monkey 84042 by Day 17 after administration, 29% in the urine and 71% in the feces. Less than 60% of the dose, of which 24% had appeared in the urine and 76% in the feces, had been excreted at this time by Monkey 83647. Total plasma clearance by these two monkeys was lower than plasma clearances by the other four monkeys given lead intravenously (Table 1). It may be that a substantial fraction of the lead lost from the plasma of these two monkeys during the first day or two following administration was sequestered in a tissue pool from which delayed excretion into the feces subsequently occurred or was otherwise temporarily excluded from access to elimination processes. In spite of the anomalous fecal excretion behavior of very large amounts of lead injected intravenously, the results of this study combined with the results of Cohen’s (1970) studies in baboons support the interpretation that nonhuman and human primates are comparable with respect to the efficiency of both urinary and fecal lead excretion. In addition, fractional absorption of ingested lead by cynomolgus monkeys is comparable to its fractional absorption by humans, and partitioning of lead between blood plasma and red cell is very similar to that in humans and not greatly different from blood plasma/red cell partitioning in the rat. Particularly
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Aungst, B. J., Dolce, J. A., and Fung, H.-L. (1981). The effect of dose on the disposition of lead in rats after intravenous and oral administration. Toxicol. Appl. Pharmacol. 61, 48–57. Barltrop, D., and Smith, A. (1975). Kinetics of lead interaction with human erythrocytes. Postgrad. Med. J. 51, 770–773. Bourne, G. H. (1975). Collected Anatomical and Physiological Data from the Rhesus Monkey. In The Rhesus Monkey, Vol. I, Anatomy and Physiology, (G. H. Bourne, Ed.), Ch. 1. Academic Press, New York. Bruenger, F. W., Stevens, W., and Stover, B. J. (1973). The association of 210 Pb with constituents of erythrocytes. Health Phys. 25, 37–42. Chamberlain, A. C., Heard, M. J., Little, P., Newton, D., Wells, A. C., and Wiffen, R. D. (1978). Investigations into Lead from Motor Vehicles. AERE R9198, HMSO, London. Cohen, N. (1970). The Retention and Distribution of Lead-210 in the Adult Baboon. Dissertation submitted to the Graduate Division of the School of Engineering and Science, New York University. University Microfilms, Ann Arbor, MI. DeSilva, P. E. (1981). Determination of lead in plasma and studies on its relationship to lead in erythrocytes. Br. J. Ind. Med. 38, 209–217. Everson, J., and Patterson, C. C. (1980). ‘‘Ultra-clean’’ isotope dilution/ mass spectrometric analyses for lead in human blood plasma indicate that most reported values are artificially high. Clin. Chem. 26, 1603– 1607. Hursh, J. B., and Suomela, J. (1968). Absorption of 212Pb from the gastrointestinal tract of man. Acta Radiol. 7, 108–120. Inskip, M. J., Franklin, C. A., Baccanale, C. L., O’Flaherty, E. J., Schanzer, D. L., Blenkinsop, J., and Manton, W. I. (1994). The contribution of bone-derived and oral sources of lead to blood lead in a non-human primate Macaca fascicularis: An investigation utilizing stable isotope tracers. Toxicologist 14, 85. [Abstract]. Klaassen, C. D., and Shoeman, D. W. (1974). Biliary excretion of lead in rats, rabbits and dogs. Toxicol. Appl. Pharmacol. 29, 434–446. Lolin, Y., and O’Gorman, P. (1988). An intra-erythrocytic low molecular weight lead-binding protein in acute and chronic lead exposure and its possible protective role in lead toxicity. Ann. Clin. Biochem. 25, 688– 697. Manton, W. I., and Malloy, C. R. (1983). Distribution of lead in body fluids after ingestion of soft solder. Br. J. Ind. Med. 40, 51–57. Manton, W. I., and Cook, J. D. (1984). High accuracy (stable isotope
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dilution) measurements of lead in serum and cerebrospinal fluid. Br. J. Ind. Med. 41, 313–319. Marcus, A. H. (1985). Multicompartment kinetic model for lead. III. Lead in blood plasma and erythrocytes. Environ. Res. 36, 473–489. O’Flaherty, E. J. (1991). Physiologically based models for bone-seeking elements. II. Kinetics of lead disposition in rats. Toxicol. Appl. Pharmacol. 111, 313–331. O’Flaherty, E. J. (1993). Physiologically based models for bone-seeking elements. IV. Kinetics of lead disposition in humans. Toxicol. Appl. Pharmacol. 118, 16–29. O’Flaherty, E. J. (1995). Physiologically based models for bone-seeking elements. V. Lead absorption and disposition in childhood. Toxicol. Appl. Pharmacol. 131, 297–308. Ong, C. N., and Lee, W. R. (1980a). Interaction of calcium and lead in human erythrocytes. Br. J. Ind. Med. 37, 70–77. Ong, C. N., and Lee, W. R. (1980b). Distribution of lead-203 in human peripheral blood in vitro. Br. J. Ind. Med. 37, 78–84.
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Pola´k, J., O’Flaherty, E. J., Freeman, G. B., Johnson, J. D., Liao, S. C., and Bergstrom, P. D. (1996). Evaluating lead bioavailability data by means of a physiologically based lead kinetic model. Fundam. Appl. Toxicol. 29, 63–70. Rabinowitz, M. B., Kopple, J. D., and Wetherill, G. W. (1980). Effect of food intake and fasting on gastrointestinal lead absorption in humans. Am. J. Clin. Nutr. 33, 1784–1788. Raghavan, S. R. V., and Gonick, H. C. (1977). Isolation of low-molecularweight lead-binding protein from human erythrocytes. Proc. Soc. Exp. Biol. Med. 155, 164–167. Simons, T. J. B. (1986a). Passive transport and binding of lead by human red blood cells. J. Physiol. 378, 267–286. Simons, T. J. B. (1986b). The role of anion transport in the passive movement of lead across the human red cell membrane. J. Physiol. 378, 287– 312. Simons, T. J. B. (1993). Lead transport and binding by human erythrocytes in vitro. Pflu¨gers Arch. 423, 307–313.
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0105
Plasma and Blood Lead Concentrations, Lead Absorption, and Lead Excretion in Nonhuman Primates1 E. J. O’FLAHERTY,*,2 M. J. INSKIP,† A. P. YAGMINAS,‡
AND
C. A. FRANKLIN†
*Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0056; and †Drugs Directorate and ‡Environmental Health Directorate, Health Protection Branch, Health Canada, Tunney’s Pasture, Ottawa, Ontario, Canada Received August 24, 1995; accepted January 30, 1996
relative efficiency of lead excretion in urine and feces. Plasma and Blood Lead Concentrations, Lead Absorption, and Lead Excretion in Nonhuman Primates. O’FLAHERTY, E. J., INSKIP, M. J., YAGMINAS, A. P., AND FRANKLIN, C. A. (1996). Toxicol. Appl. Pharmacol. 138, 121–130. In order to assess the comparability of lead disposition in the cynomolgus monkey to that in the human, we determined the relationships among blood lead concentration, plasma lead concentration, and lead excretion in monkeys. Six adult (3–5 kg) female cynomolgus monkeys (Macaca fascicularis) without previous experimental lead exposure were given single intravenous injections of from 750 to 3300 mg lead as lead nitrate, labeled with 210 Pb, per kilogram body weight. Four additional monkeys, fasted overnight, were administered single oral doses of either 750 or 1500 mg lead as lead nitrate, labeled with 210Pb, per kilogram for the assessment of fractional absorption. Blood and plasma lead concentrations (10 monkeys) and urinary and fecal excretion of lead (2 monkeys) were followed for up to 16 days after lead administration. Fractional absorption from an oral dose was 44% at the lower of the two doses and 22–28% at the higher dose. The relationship between plasma and blood lead concentrations was found to be similar to that in humans, with plasma lead concentration at most a few percent of total blood lead concentration at low concentrations. Partitioning of lead across the red cell membrane in the 2 monkeys given exceptionally high doses (3300 mg/kg) intravenously was distinctly lower than that in the 4 monkeys given lower intravenous doses. Urinary clearance of lead in these 2 monkeys was 19% of the estimated glomerular filtration rate, within the range of efficiencies reported for humans. Fecal clearance, however, was anomalous and appeared to be an artifact of the very high dose. Examination of published data for urinary and fecal lead excretion in three adult baboons showed that both functions in the baboons were quantitatively similar to those in humans. Urinary clearance in the baboons was 14–24% of the estimated glomerular filtration rate, and fecal clearance was 78– 85% of the urinary clearance. We conclude that nonhuman and human primates are comparable with respect to the relationship of plasma lead concentration to blood lead concentration and the
q 1996
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Partitioning of lead between blood plasma and the red cell is markedly dependent on the plasma concentration of lead in humans (DeSilva, 1981) and in rats (Klaassen and Shoeman, 1974). This partitioning can be described in terms of a capacity-limited binding of lead by the constituents of the red cell (Marcus, 1985; O’Flaherty, 1991, 1993). The implication of this concentration dependence is that while plasma lead concentration may increase linearly with whole-body lead uptake, the concentration of lead in the red cell will plateau at higher exposures. Although the relationship of plasma lead to lead exposure may be linear, the relationship of blood lead to exposure will be nonlinear. These relationships are particularly important for modeling lead kinetics, since whole blood lead concentration is conventionally measured experimentally while excretion and transfer into tissues are generally understood to take place from the plasma. When plasma lead concentration and blood lead concentration are not directly proportional to one another, the relationship of plasma lead to red cell lead must be characterized and quantified if the kinetic behavior of lead is to be fully understood. As part of a study whose broader purpose is to evaluate the contribution of bone lead to blood lead in nonhuman primates with well-documented lead exposure histories (Inskip et al., 1994), we undertook to examine the relationships between plasma lead and blood lead and between plasma lead and urinary and fecal lead excretion in cynomolgus monkeys with no known previous lead exposure. Fractional lead absorption from an oral dose was also estimated in this study. MATERIALS AND METHODS Animals
1
This work was supported by NIEHS Contract N01-ES-05285. To whom correspondence and reprint requests should be addressed at Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Cincinnati, OH 45267-0056. 2
Ten healthy adult female cynomolgus monkeys (Macaca fascicularis) without previous experimental lead exposure were assigned to the study from a research pool. Animals had either been bred in-house or had originally been caught in the wild and raised in the colony for at least 7 years.
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Body weights ranged from 3.1 to 5.1 kg. The animals were housed in the primate facility of the Health Protection Branch of Health Canada, Ottawa, Ontario. Each animal was housed individually in a controlled environment with a 12:12 hr light:dark cycle. Humidity was maintained at 40 { 10% and the room temperature at 22 { 27C. Access to food (Primate Monkey Chow, type 5047 (PMI Feeds, Richmond, IN) and water was on an ad libitum basis, with supplementary provision of vegetables and fruit. Lead content of food was certified by the manufacturer to be less than 0.25 mg/g. Drinking water lead content was less than 50 mg/liter. Dose Levels and Preparation Lead (210Pb) nitrate with a specific activity of 10 mCi/mg Pb and purity greater than 95% (Amersham International, Oakville, Canada) was received dissolved in 3 M HCl. Appropriate safety precautions were taken in dealing with radioactive materials and wastes in accordance with safety and handling guidelines issued by the Atomic Energy Control Board of Canada. Lead nitrate (Pb(NO3)2) (BDH Chemicals, Toronto, ON) was dissolved in 0.9% saline and mixed 9:1 with the tracer solution so that the 210Pb label was approximately 10% of the total lead. One measurement of total lead concentration in a serially diluted subsample of the dosing solution was performed by Dr. J. Blenkinsop at the Department of Earth Sciences, Carleton University. Analysis was by isotope dilution mass spectrometry using a stable 208Pb spike and a magnetic sector Finnegan-MAT 261 spectrometer. The dosing solution had a pH of 7.2 and an osmolality of approximately 300 mOsm. Doses of 750 or 1500 mg Pb/kg body weight were administered either intravenously (0.5 ml/kg body weight) or orally to the eight animals in Study 1. Selection of dose levels was based partly on the design of the parent study with a separate cohort of monkeys given a lifetime oral daily dose of 1500 mg Pb/kg/day, and partly on consideration of the probable relative oral/intravenous bioavailability. The two animals in Study 2 received intravenously approximately 3300 mg Pb/kg body weight. The same dosing solution was used in both studies. Due to the time difference between the two studies, adjustments were made to the total dose in Study 2 to allow for decay of the 210Pb label. The half-life of 210Pb is approximately 22 years. Major decomposition products are 210Bi and 210Po, noted by the supplier to be present initially at less than 1 mCi. Baseline blood lead concentration is low in these monkeys, of the order of 2 mg/dl (0.04 mg/dl of plasma if plasma lead concentration is 2% of blood lead concentration), and is swamped by the administered carrier lead. Experimental Design Two separate studies were conducted. In the first, only the relationship between plasma and blood lead was monitored. In the second, which was designed primarily to determine urinary and fecal lead clearance, the plasma/blood lead measurements were repeated. Study 1. The subjects were eight female cynomolgus monkeys without previous experimental lead exposure. The animals were placed in a neck restraint apparatus for the period covering intravenous dose administration and collection of the first four blood samples. Subsequently, the restraining device was used only at the times blood samples were required. Four monkeys were given the lead solution intravenously via the saphenous vein over a period of 1 min. Four were administered lead orally by capsule. Blood samples were obtained from the saphenous vein using heparinized Vacutainer tubes (Becton–Dickinson, Franklin Lakes, NJ) at time points of 0.05 (intravenous only), 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 12, 24, 36, and 48 hr and subsequently every 24 hr for a total of 13 days. The blood samples were divided into two equal portions, one of which was counted as whole blood and also used for determination of the hematocrit. The other portion was separated into plasma and cell fractions by centrifugation for 5 min at 2400 rpm. The cell fraction was washed twice with physiologic saline and centrifuged. Each of the samples (blood, blood plasma, cell fraction, and saline washes) was placed in a 5-ml lead-free polypropylene tube (Sarstedt Canada, St. Laurent, PQ) for counting.
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Study 2. The subjects were two female cynomolgus monkeys without previous experimental lead exposure. The intravenous dose was administered over an approximately 15-sec interval via the cephalic vein using an intravenous catheter placement unit, size 24 (Deseret Medical, UT). Blood samples were obtained from the femoral vein at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hr and then every 24 hr for a total period of 16 days. Particular procedures were incorporated to minimize the risk of red cell hemolysis. Extreme care was taken during the sampling by slowly withdrawing the blood using a heparinized disposable syringe. After removal of the needle and inversion of the syringe to prevent clotting, the blood was slowly ejected into a 2-ml heparinized Becton–Dickinson centrifuge tube with vacuum released. Exactly 1.0 ml of blood was transferred by pipet to a 5-ml polypropylene tube (Sarstedt Canada, St. Laurent, PQ) and centrifuged in a fixed-angle rotor at 2000 rpm for 30 min. A 200-ml aliquot of plasma was carefully pipetted from the top of the tube, avoiding contact with the tube walls, transferred to another 5-ml polypropylene tube, and retained for counting along with the tube containing the remaining blood plasma, red cells, and white cells/platelets. Stool samples were collected daily using screened collection trays. The entire stool sample was weighed and mixed well for 2 to 3 min by physical manipulation through a double, heavy-walled polyethylene bag. Using a wooden spatula, a representative subsample was prepared for counting by placing 1 to 2 g of material in a 5-ml polypropylene tube. Complete 24-hr stool collections were also made on 6 days for each monkey. Urine was collected in underlying polyethylene-lined collection trays screened from contamination by stool. The urine was poured into a polyethylene container and mixed well, and a subsample placed into a 5-ml polypropylene tube. In general, urine collections of recorded duration were made. In addition, spot urine samples were collected as close as possible to the times of blood sampling. Twenty-four-hour collections were made daily in order to establish the volume of urine excreted daily by each animal. Measurement of Radioactivity Blood, blood plasma, urine, and stool samples were all counted in 5-ml lead-free polypropylene containers (Sarstedt Canada, St. Laurent, PQ) in order to achieve consistent geometry. Activity was determined by counting the 47 kev gamma radiation in a gamma counter (LKB-1282 CompuGamma, Fisher Scientific, Ottawa, ON). In Study 1, the counts for plasma, saline, and cells from the divided aliquot were summed and the mean value of this total and the count obtained for the whole blood aliquot was considered the blood lead count. In Study 2, whole blood lead counts were calculated as the weighted average of counts in the plasma aliquot and in the combined remaining plasma, red cell, and white cell/platelet fraction. To obtain the concentration of lead in whole blood, counts were transformed using a conversion factor calculated from the original 210Pb/carrier lead dosing solution. The activity of the dosing solution was determined at the time of assay of the biological samples. Calibrations were obtained from serial aqueous dilutions covering the anticipated concentration range. In Study 2, dilutions were carried out both in saline and in heparinized whole blood and urine from pooled samples collected from non-lead-exposed monkeys. No differences due to diluent were detectable. Data Analysis Plasma and blood lead concentrations for the six monkeys given lead intravenously were fit by the expression PbB Å (1 0 HCT) 1 PbP / HCT 1 PbP 1 (A / BIND/(KBIND / PbP)),
(1)
where PbB is blood lead concentration, PbP is plasma lead concentration, HCT is the hematocrit, BIND is the maximum red cell lead binding capacity, KBIND is the dissociation constant for this binding, and A is a constant
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FIG. 1. The relationship of plasma lead to blood lead in Monkey 86163 (Study 1) at blood lead concentrations less than 1600 mg/dl.
of proportionality between plasma lead and unbound red cell lead. The assumptions on which this expression is based are that plasma lead and unbound red cell lead are directly proportional to one another and that red cell lead exchanges between the unbound fraction and a group of binding sites that can reasonably be characterized by a single maximum capacity and dissociation constant (Marcus, 1985; O’Flaherty, 1993). The fit of this expression to plasma and blood lead concentrations was optimized in two steps. Concentration pairs above PbP Å 100 mg/dl (PbB É 200 mg/dl) were fit by linear regression to obtain the value of A. A was then held constant while the entire data set was fit by nonlinear regression to estimate the values of BIND and KBIND . Data were weighted by the reciprocal of the dependent variable, PbB in this case. For the estimation of excretion clearances in the two monkeys in Study 2 for whom urine and feces were also monitored, only plasma lead concentrations below 3 mg/dl (blood lead concentrations below 100 mg/dl) were considered. Above this concentration range, both urinary and fecal excretion were erratic and tended to fall off relative to plasma lead. The relationship between urinary or fecal lead excretion rate and plasma lead concentration was determined by linear regression constrained to pass through the origin. Because plasma lead concentrations fell rapidly after administration to levels near the detection limit, measured plasma concentrations were not used directly. Instead, plasma concentrations were calculated at the measured blood lead concentrations using the line of best fit of the relationship of plasma lead to blood lead for each monkey (Expression 1). At some time points of urine or fecal collection, a measured blood lead concentration was not available. Plasma lead concentration at these time points was estimated by interpolation. Urinary lead excretion rate was estimated by multiplying the concentration of lead in a timed urine sample by the total volume of the sample. Some of the urine samples were timed 24-hr collections, while some were collections of measured volume taken over shorter time periods and some were spot samples taken simultaneously with a blood sample. Within the rather large variability in this data set, lead excretion rates estimated directly from the timed urine samples were not different from those calculated using daily urine volumes. Fecal lead excretion rate was estimated by multiplying the concentration of lead in a fecal sample by the mean daily total weight of feces excreted, determined based on the measurements from 6 sampling days for each monkey.
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An approximate average total (urinary and fecal) clearance was also estimated for the six monkeys given lead intravenously, by dividing the administered dose by the trapezoidal area under the plasma lead concentration curve from time zero to the end of the study 13 or 16 days later. A physiologically based model of lead kinetics in the monkey (Inskip et al., 1994) was visually fit to blood and plasma lead concentrations in the four monkeys given lead orally in order to estimate the fraction of the lead dose that had been absorbed. The model, to be presented in greater detail in a separate paper, is structurally identical to a physiologically based model of human lead kinetics (O’Flaherty, 1993, 1995). For this study, the human model was scaled to the body weight of the adult cynomolgus monkey. Tissue weights and bone mass bear the same relationship to body weight in the scaled model as they do in the human model. Cardiac output was scaled from the human on the basis of adult body weight to the 34 power. Glomerular filtration rate was set at 2 liters plasma/day/kg, scaled from the rhesus monkey (Bourne, 1975). Partitioning of lead into tissues including bone surface and the values of all bone uptake parameters were set identical to those in humans. In particular, bone turnover rate was set to the value 15%/year used for adult humans (O’Flaherty, 1995). The increased rates of bone growth and turnover that characterize the growth spurt of human adolescence were not included in the scaled model, since the monkeys were adults at the time of lead administration. The simulations were carried out by modeling parallel systemic absorption and fecal excretion (gut transit) processes acting on the total oral dose in the gastrointestinal tract. The absolute and relative magnitudes of the two processes were varied to obtain a good visual fit to the concentration data for each monkey and to estimate the corresponding total amount absorbed.
RESULTS
The relationship between plasma and red cell lead concentration in the six monkeys (four from Study 1, two from Study 2) given lead intravenously is illustrated for two of the monkeys in Figs. 1 and 2, and the parameter values and other pertinent information for all six monkeys
FIG. 2. The relationship of plasma lead to blood lead in Monkey 84117 (Study 1) at blood lead concentrations less than 1600 mg/dl.
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TABLE 1 Kinetic Parameters for Lead Partitioning and Excretion Study 1 Monkey:
86163
Weight, kg Intravenous lead dose, mg Initial plasma lead concentration, mg/dl Initial blood lead concentration, mg/dl A BIND, mg/dl of red cell volume KBIND, mg/dl of red cell volume AUC,c mg-hr/ml plasma Total clearance, dose/ AUC, liters plasma/ day Urinary clearance, liters plasma/day Estimated glomerular filtration rate, liters plasma/dayg Urinary clearance, fraction of glomerular filtration rate Fecal clearance, liters plasma/day
Study 2
86058
84117
84020
84042
83647
3.10
3.48
5.12
3.43
4.73
4.17
4495
2714
6912
2710
15470
13890
1362
544
1542
770
8023
7601
1664 1.12 (0.035)a
745 1.29 (0.072)
1841 1.15 (0.023)
977 1.19 (0.018)
5154 0.602 (0.0102)
4310 0.554 (0.066)
154 (14)
240b
211 (18)
221 (3)
240 (77)
349 (74)
4.9 (1.1) 33.5
5b 25.2
0.99 (1.9) 44.4
11.4 (3.6) 24.0
4.5 (2.9) 322.3
3.0 (1.6) 253.2
3.2
2.6
3.7
2.7
1.2
1.3
1.3d
1.4d
1.8d
1.4d
1.75 (0.24)e, f
1.60 (0.23)e
6.7
7.3
9.8
7.2
9.2
8.4
—
—
—
—
0.19
0.19
1.9 h
1.2h
1.9 h
1.3h
43.8 (5.5)e
18.7 (1.8)e
a
Standard error in parentheses. Mean of the other five values of this parameter. Data from Monkey B were not fit well by nonlinear regression using Expression 1, although visually the fit with these parameter values was good. c Trapezoidal area under the plasma concentration curve, calculated as described in the text. d Estimated by scaling mean urinary clearance of 0.62 liters/kg/day from Study 2 on the basis of the 2/3 power of body weight (intraspecies scaling). e Experimental value. f One excretion value, of 198 mg/day at a plasma lead concentration of 1.4 mg/dl, was excluded from this regression. g Estimated by scaling measurements made in the rhesus monkey (Bourne, 1975) on the basis of the 3/4 power of body weight (interspecies scaling). h Estimated as total clearance minus urinary clearance. b
are given in Table 1. The values of BIND and KBIND are determined by the shape and positioning of the curvature of this relationship at blood lead concentrations below about 200 mg/dl. At higher concentrations, the relationship between blood lead and plasma lead is linear, with slope A (Expression 1). In the four monkeys from Study 1, whose maximum (initial) blood lead concentrations did not exceed 1840 mg/dl, the slope A varied from 1.1 to 1.3. In the two monkeys from Study 2, whose initial blood lead concentrations were 4310 and 5150 mg/dl, the optimized values of A were 0.55 and 0.60. Table 2 gives the estimated fractional lead absorption by the four monkeys given lead orally. Fractional absorption was dose-dependent, being lower at the higher of the two doses.
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TABLE 2 Lead Absorption Monkey: Weight, kg Oral lead dose, mg Amount of lead absorbed, mga Fraction oral dose absorbed
83681
83145
83189
83472
3.40 2700
3.14 5400
3.35 2700
3.56 6300
1060
2290
1080
2360
0.44
0.22
0.44
0.28
a Calculated, as described in the text, by visually fitting a physiologically based model to blood and plasma lead concentrations, optimizing the magnitude of systemic absorption in order to achieve a good fit.
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FIG. 3. The relationship of urinary lead excretion rate to plasma lead concentration in Monkey 84042 (Study 2). As described in the text, plasma lead concentration was estimated by interpolation at urine collection times at which a measured blood lead concentration was not available.
FIG. 5. The relationship of fecal lead excretion rate to plasma lead concentration in Monkey 84042 (Study 2). As described in the text, plasma lead concentration was estimated by interpolation at feces collection times at which a measured blood lead concentration was not available.
Figures 3 and 4 illustrate the relationship between urinary lead excretion and plasma lead concentration in the two Study 2 monkeys. Figures 5 and 6 illustrate the comparable relationships for fecal lead excretion in the two Study 2 monkeys. The slopes, or urinary and fecal lead clearances, are given in Table 1.
DISCUSSION
The human plasma lead/blood lead concentration relationship reported by DeSilva (1981) is satisfactorily described by Expression 1 with values of A Å 1, BIND Å 270 mg/dl red cell volume, and KBIND Å 0.75 mg/dl red cell volume
FIG. 4. The relationship of urinary lead excretion rate to plasma lead concentration in Monkey 83647 (Study 2). As described in the text, plasma lead concentration was estimated by interpolation at urine collection times at which a measured blood lead concentration was not available.
FIG. 6. The relationship of fecal lead excretion rate to plasma lead concentration in Monkey 83647 (Study 2). As described in the text, plasma lead concentration was estimated by interpolation at feces collection times at which a measured blood lead concentration was not available.
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(O’Flaherty, 1993). The corresponding parameter values estimated (O’Flaherty, 1991) for the rat plasma lead/blood lead concentration relationship reported by Klaassen and Shoeman (1974) are A Å 1, BIND Å 1000 mg/dl red cell volume, and KBIND Å 6.7 mg/dl red cell volume. Comparison with the values of A, BIND, and KBIND in the cynomolgus monkeys (Table 1) suggests that there are not marked interspecies differences in the characteristics of lead binding by constituents of the red cell. There is considerable interanimal variability in the estimated values of KBIND in the present study (Table 1), and large uncertainties are associated with them. However, their range lies among the values that have been assigned to this parameter for rats and humans. The interanimal differences appear to be real, as illustrated by a comparison of the data from monkeys 86163 and 84117 in Figs. 1 and 2. These monkeys were given the same intravenous dose in Study 1. For simplicity, the red cell constituents that bind lead have been treated as if they presented a single set of identical binding sites. This is known not to be the case. Lead is bound to a variety of components of the human red cell including hemoglobins and low-molecular-weight proteins, some of which are inducible, as well as to sites on the red cell membrane. Binding to many of these sites is capacitylimited, causing shifts in the pattern of binding with changing blood lead concentration (Bruenger et al., 1973; Raghavan and Gonick, 1977; Ong and Lee, 1980a,b; Lolin and O’Gorman, 1988). There is no reason to suppose that binding in the red cell of the nonhuman primate is less complex. However, no quantitative data exist to suggest the magnitudes of parameters of a red cell model with more than one kind of binding site. Addition of a second class of red cell binding sites to the plasma/red cell partitioning model results in a cubic equation (not shown) that is not more satisfactory than Expression 1 as an expression of the partitioning of lead between plasma and red cell. The criterion of parsimony dictates use of the simpler model. For hematocrit HCT Å 0.45, A Å 1.2, maximum red cell binding capacity BIND Å 240 mg/dl red cell volume, and dissociation constant KBIND Å 1–10 mg/dl red cell volume, plasma lead concentration (Expression 1) in these cynomolgus monkeys would range from about 0.8% to about 7% of total blood lead concentration at low blood lead concentrations. In humans, plasma lead concentrations up to and exceeding 5% of the blood lead concentration have been reported at blood lead concentrations below about 50 mg/dl (see discussion in Everson and Patterson, 1980), but the most reliable estimates are in the range of 1% or less (Everson and Patterson, 1980; Manton and Cook, 1984). Some of the variability in reported plasma lead/blood lead concentration ratios is probably due to slight hemolysis of blood samples, which, at these concentration ratios, could significantly contaminate the plasma fraction.
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It is of interest whether there is a demonstrable time lag for achievement of a steady state between plasma and red cell lead when lead is administered intravenously. The plasma/red cell lead concentration ratio is responsive to changes in lead uptake over short time frames. DeSilva (1981) observed a rapid rise in the ratio of plasma to red cell lead during the first few hours after ingestion of 100 mg of lead by an adult human subject. Some of this increase can be attributed to the expected shift in partitioning that accompanied the increase in total blood lead to a maximum of 78 mg/dl. Some of it may have been due to a time lag in achievement of plasma/red cell steady state because the highest plasma/red cell concentration ratio recorded, greater than 3%, was larger than the steadystate ratio expected at the peak blood lead concentration. Nonetheless, the time lag for equilibration of lead between plasma and red cell does not appear to be large. In vitro, equilibrium between human red cell and plasma lead is achieved within 15 min, with an apparent half-time of less than 5 min (Hursh and Suomela, 1968; Barltrop and Smith, 1975). Chamberlain et al. (1978) administered 203Pb intravenously to adult humans and monitored its disappearance from the plasma. They also carried out an in vitro experiment in which human blood was incubated with 203Pb. The in vivo and in vitro loss rates from the plasma were strikingly similar, with half-times of about 3 min. There is no indication in the current study of a significant time lag to steady state. The first blood samples in Study 1 were taken 3 min after intravenous administration of the lead salt. The mean plasma lead/blood lead concentration ratio at this time in the two monkeys given 750 mg lead/kg was 0.76. This value is actually slightly lower than the mean ratio, 0.85, observed 1 hr later in the two Study 1 monkeys given 1500 mg lead/kg, at a time when concentrations had fallen to the initial values observed in the monkeys given the smaller dose. Fractional absorption of lead by the four Study 1 monkeys given lead orally (Table 2) is similar to that seen in fasted humans given soluble salts of lead. Rabinowitz et al. (1980), administering stable lead isotope tracers to five healthy men, found that a mean of 8% (SD 3%) was absorbed when the tracers were ingested with food, and a mean of 35% (SD 13%) when the tracers were taken after a 9-hr fast. Absorption in the two monkeys given the lower dose in the present study was 44%, while absorption in the two monkeys given the higher dose was 22 and 28% (Table 2). This observation suggests dose-dependent fractional absorption, which has been documented in rats (Aungst et al., 1981; Pola´k et al., 1996). The two Study 2 monkeys displayed lead partitioning very different from that of the Study 1 monkeys. While the values of BIND and KBIND are not greatly dissimilar in the two studies, the values of A in Study 2 are significantly smaller
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FIG. 7. The relationship of urinary lead excretion rate to blood lead concentration in Baboon B-100 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
than those in Study 1, implying that while binding of lead within the red cell is essentially the same in the two studies, partitioning of lead across the red cell membrane is not. Simons (1986b, 1993) has shown that lead, in association with bicarbonate, is passively transferred by the anion-exchange carrier in both directions across the red cell membrane and that the distribution of unbound lead across the membrane is close to equilibrium. The mean value of A, 1.2, in the four Study 1 monkeys is lower than the equilibrium ratio proposed by Simons (1986a), who noted that the red cell membrane potential of 012 mV would be consistent with a red cell-to-plasma unbound lead equilibrium ratio of 2.5:1. The impact of plasma binding of lead on the equilibrium ratio expressed in terms of total plasma lead concentration rather than unbound lead concentration, as it is in the present study, has not been quantified. In Study 2, plasma lead concentration was much higher than the red cell unbound lead concentration. Lead affects the permeability of the red cell membrane to sodium and potassium (Simons, 1986a). It is plausible that high plasma concentrations of lead might interfere with normal lead transfer into the red cell. The measured initial plasma lead concentrations in these two monkeys were extraordinarily high, around 8000 mg/dl, more than twice those predicted by extrapolation of the initial concentration/dose relationship from Study 1. It appears probable that movement of lead into the red cell was inhibited by factors related to the magnitude of the plasma lead concentration. In view of the disproportionately high initial concentrations of plasma lead in Study 2, it may be that lead distribution into other tissues at early times after lead
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administration was also impaired. Estimated total plasma clearance in these two monkeys, calculated as the dose divided by the trapezoidal area under the plasma concentration curve, was lower than that in the four monkeys in Study 1 (Table 1), although the measured urinary clearance, compared to that observed by Cohen (1970) in baboons, scaled allometrically as the 34 power of body weight (see discussion below). Urinary and fecal lead clearances were calculated from data collected after Day 10 (Monkey 84042) or Day 6 (Monkey 83647) following administration, when plasma lead was less than 3 mg/dl. In both monkeys, urinary lead clearance was 19% of the glomerular filtration rate estimated for a monkey of the same body weight (Table 1). This is within the range of efficiency of urinary lead excretion in adult humans, with values from 4 to 28% reported (Chamberlain et al., 1978; Manton and Malloy, 1983). Observations on the urinary excretion of lead by baboons have been published. Cohen (1970) administered 210Pb to three adult (12–14 kg) female baboons by single intravenous injection of carrier-free tracer and monitored the amounts of radiolabel in blood and urine for 48 to 62 days. The excretion data were reported. The blood concentrations were not reported, but a two-exponential expression that generated the observed concentrations was given for each baboon. Figures 7–9 illustrate the rate of excretion of radiolabel in the urine as a function of the concentration of radiolabel in the blood, based on a reconstructed blood activity curve, for each of the three baboons. Linear regressions are shown. Their slopes, the urinary clearances of lead from blood, are 59 ml
FIG. 8. The relationship of urinary lead excretion rate to blood lead concentration in Baboon B-106 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
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FIG. 9. The relationship of urinary lead excretion rate to blood lead concentration in Baboon B-158 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
FIG. 10. The relationship of fecal lead excretion rate to blood lead concentration in Baboon B-100 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
blood/day for baboon B-100, 45 ml blood/day for baboon B-106, and 177 ml blood/day for baboon B-158 (Figs. 7– 9). (The relationship between lead excretion rate and blood lead concentration for baboon B-158 (Fig. 9) is clearly curvilinear, and not fit well by a single straight line.) If plasma lead concentration is 2% of the blood lead concentration, urinary lead clearance would be a mean of 2.6 liters plasma/ day in the two similar baboons, and 8.8 liters/day in the third baboon. Glomerular filtration rate in a 12-kg baboon, estimated by scaling on the basis of the 34 power of body weight from measurements made in the rhesus monkey (Bourne, 1975), should be around 19 liters/day. Thus, urinary clearance of lead would be about 2.6/19, or 14%, of the glomerular filtration rate in baboons B-100 and B-106, and 46% of the glomerular filtration rate in baboon B-158. The two lower excretion efficiencies lie within the range of values noted above for humans. Cohen (1970) also monitored fecal excretion of lead in the three baboons administered 210Pb by intravenous injection. Figures 10–12 illustrate these relationships. None of the three data sets exhibits any obvious nonlinearity, and all three were analyzed by linear regression. Their slopes, the clearance of lead from blood into the feces, are 46 ml blood/ day for baboon B-100 (2.3 liters plasma/day, or 78% of the urinary clearance), 42 ml blood/day for baboon B-106 (2.1 liters plasma/day, or 93% of the urinary clearance), and 80 ml blood/day for baboon B-158 (4.0 liters plasma/day, or 45% of the urinary clearance). The ratios of fecal to urinary clearance are comparable to values reported for humans,
which have ranged from 0.33 to 1.0 (Chamberlain et al., 1978). The fecal lead clearances measured in the two very highdose monkeys in Study 2 (Table 1) are not consistent with the values reported for humans and baboons. They are very
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FIG. 11. The relationship of fecal lead excretion rate to blood lead concentration in Baboon B-106 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
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given the substantial interanimal differences in the values of the binding parameters for red cell lead in the monkeys in the present study, there appear to be only minimal differences between human and nonhuman primates in absorption, red cell/plasma partitioning, and urinary and fecal excretion of lead. ACKNOWLEDGMENTS The authors thank Catherine Ferrarotto and Ross Layberry (University of Ottawa) for carrying out the 210Pb determinations conducted during Study 2, and Dena Schanzer for assistance with data management. Ann Dorward (McMaster University) carried out the analysis of samples from Study 1. Assistance from animal health technologists C. LeBlanc, A. St.-Pierre, and S. Stals and the animal care staff at the Animal Resources Division of the Health Protection Branch, Health Canada, is greatly appreciated.
REFERENCES FIG. 12. The relationship of fecal lead excretion rate to blood lead concentration in Baboon B-158 (Cohen, 1970). As described in the text, blood lead concentrations were reconstructed from the equation given by Cohen.
large, greatly exceeding the estimated rate of blood flow to the liver in a 4-kg animal. Very little lead was excreted in the feces of either of these monkeys during at least the first 3 days after administration. Shortly after, the rate of fecal lead excretion was as high as 900 mg/day in Monkey 84042 and 400 mg/day in Monkey 83647. Essentially 100% of the dose had been excreted by Monkey 84042 by Day 17 after administration, 29% in the urine and 71% in the feces. Less than 60% of the dose, of which 24% had appeared in the urine and 76% in the feces, had been excreted at this time by Monkey 83647. Total plasma clearance by these two monkeys was lower than plasma clearances by the other four monkeys given lead intravenously (Table 1). It may be that a substantial fraction of the lead lost from the plasma of these two monkeys during the first day or two following administration was sequestered in a tissue pool from which delayed excretion into the feces subsequently occurred or was otherwise temporarily excluded from access to elimination processes. In spite of the anomalous fecal excretion behavior of very large amounts of lead injected intravenously, the results of this study combined with the results of Cohen’s (1970) studies in baboons support the interpretation that nonhuman and human primates are comparable with respect to the efficiency of both urinary and fecal lead excretion. In addition, fractional absorption of ingested lead by cynomolgus monkeys is comparable to its fractional absorption by humans, and partitioning of lead between blood plasma and red cell is very similar to that in humans and not greatly different from blood plasma/red cell partitioning in the rat. Particularly
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Pola´k, J., O’Flaherty, E. J., Freeman, G. B., Johnson, J. D., Liao, S. C., and Bergstrom, P. D. (1996). Evaluating lead bioavailability data by means of a physiologically based lead kinetic model. Fundam. Appl. Toxicol. 29, 63–70. Rabinowitz, M. B., Kopple, J. D., and Wetherill, G. W. (1980). Effect of food intake and fasting on gastrointestinal lead absorption in humans. Am. J. Clin. Nutr. 33, 1784–1788. Raghavan, S. R. V., and Gonick, H. C. (1977). Isolation of low-molecularweight lead-binding protein from human erythrocytes. Proc. Soc. Exp. Biol. Med. 155, 164–167. Simons, T. J. B. (1986a). Passive transport and binding of lead by human red blood cells. J. Physiol. 378, 267–286. Simons, T. J. B. (1986b). The role of anion transport in the passive movement of lead across the human red cell membrane. J. Physiol. 378, 287– 312. Simons, T. J. B. (1993). Lead transport and binding by human erythrocytes in vitro. Pflu¨gers Arch. 423, 307–313.
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