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BIOKINETICS OF ='Pu CITRATE AND NITRATE IN THE RAT IMPLICATIONS FOR Pu STUDIES IN MAN Environmental and Medical Sciences Division, Harwell Laboratory, Didcot, Oxfordshire OX 1IORA, United Kingdom Absrracr-Plutonium-237 decays mainly by electron capture with a half-life of 45 d. Alpha particles are emitted io only 5 X IO-'% of its disintegrations. Thiu nuclide can now be produced with relatively small amounts of aemitting contaminants so that, in principle, a7Pu can be used for studies of Pu biokinetics in man. However, because of its high specific activity, there was some doubt that its metabolism would be the same as that of the a- and 8emitting isotopes of Pu normally encountered in the nuclear industry. In this study, the biokinetics of nearly "pure," high specific activity ='Pu are compared with those of lower specific activity, "impure" ='Pu contdnhg significant amounts of a-emitting Pu,following administration to rats by inbnveaous injection as the citrate. Both the distribution lad excretion of the "pure" and "impure" "Pu used in the two studies were similar and also in good agreement with the results of previously reported studies using %d u'Pu citrate, thus validating the w of ='Pu for studies of Pu metabolism in man. Data on the biokinetics of ='Pu nitrate are also included. PLUTONIUM-237 decays predominantly by electron cap ture with a half-life of 45.2 d; a particles are emitted in only 0.005%of the disintegrations (ICRP 1983; Kocher 198 1 ). At the Harwell Laboratory, it is produced by the irradiation of highly enriched 235Uwith helium ions in the Variable Energy Cyclotron (Whittaker 1984). The irradiation conditions can be adjusted to give either a relatively large activity of "impure" 2 3 7 ~containing significant masses of other Pu isotopes (principally 236Pu, 237Puminimally contaminated with other Pu isotopes. Due to the low associated a activity, "pure" 237Pumay be used for studies of Pu biokinetics in volunteer subjects with a committed effective dose equivalent of less than the 0.5 mSv recommended by the World Health Organization for Category 1 studies (WHO 1977). For example, if 237Puwas administered as the citrate by intravenous injection, useful data could be obtained on the clearance of Pu from the bloodstream, on its excretion in urine and feces, and on its rate of uptake in liver and bone using in-vivo counting. Such infomation from healthy volunteers would improve and complement the data obtained by Langham et al. (1950) using "terminally ill" patients and also enable comparisons with data obtained from experimental animals (e.g., ICRP 1972). Plutonium-237 has already been used in a study with volunteer subjects ( Waite and Ramsden 197 1 ). In that (Manuscript received 21 September 1989; revised manuscript r e ceived 21 February 1990. accepted 16 March 1990) investigation, however, the objective was to use the x rays emitted by 237Pu(similar in energy to those emitted by 23%) to calibrate counters used for the determination of 23% in lung. As the 237Puwas incorporated into particles of iron oxide, the results are not relevant to Pu biokinetics Mass effects have been shown to occur in the distribution of Pu injected into beagle dogs (Lloyd et al. 1976) and in gastrointestinal absorption of Np (Metivier et al. 1986). Therefore, before studies of Pu metabolism using '"Pu can be considered in volunteers, it is necessary to establish that its biokinetics faithfully reproduce those of long-lived Pu isotopes. In this study, the metabolism of specific activity) and "pure" (high specific activity) forms is compared; the results are also compared with previously reported studies of 239Pu(Stather and Howden 1975) and 24'Pu(Priest 1977) distribution in rat following intravenous administration as the citrates. Although the rat is not an ideal model, if distribution and excretion patterns of injected 237Pushow independence of specific activity, this will support its use as a metabolic tracer in man. Additional information on the biokinetics of *"Pu nitrate MATERIALS AND METHODS Male Wistar rats (HMTstrain) about 20 wk old and weighing about 300 g were used for these experiments. Following injection, they were held, three per cage, in an airconditioned glove box and allowed pelleted diet and water ad libitum.The cages had wire grid floors that al-
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TI& 1. Relative radioactivity of long-lived acmitting Pu isotopes to *3’Pu at time of administration (WBq237Pu). Not detectable in DrcJena of much greater Pu a activity. lowed excreta to fall into collection trays lined with paper ”tray tidies.” Initially, these were changed daily and at least twice per week at later times. Hawell Variable Energy Cyclotron by the irradiation of highly enriched 235Uwith helium ions. The nuclear reactions are discussed in the Appendix. After separation from U target material, the Pu fraction was dissolved in respect to other Pu isotopesdetermined by a spectrometry. Details of the isotopic compositions of the “impure” and “pure” 131Pu are given in Table 1. Reparation and administration of 237 Pu citrate and nitrate ( w / v ) trisodium citrate was added to an equal volume of polymeric material that may have been present. The animals were anesthetized’ with 2-3% Halothane in 0 2 and aliquots (0.2 mL) of the filtrate injected via the left great saphenous vein. The wound site was closed with suture clips Similar 0.2-mL aliquots were dispensed to determine the activity of 237Puadministered and to prepare appropriate counting standards. The solution of “pure” 2 3 7 ~in 0.01 M HNO, was divided into two ‘1.5-mL aliquots. Trisodium citrate ( 1.5 mL, 2%) was added to one and 1.5 mL of 0.01 M HNO, was added to the other. Both solutions were passed through 0.025 pm membrane filters. Aliquots (0.2 mL) of the citrate or nitrate were each injected into eight rats as described above. I Analysis of !issues and excreta At predetermined times after injection, groups of rats were killed by exsanguination via the dorsal aorta while Millipore Corporation. Bedford, MA. August 1990, Volume 59, Number 2 under deep ether anesthesia. The following samples were dissected from each rat and placed in preweighed containers: kidneys, liver, testes, lungs (including the heart and external airways), gastrointestinal tract, spleen, and femora. About 50 g of muscle tissue was trimmed from the eviscerated carcass and weighed. To eliminate any bias introduced by preferential retention at the site of injection, no muscle was taken from the left leg. The remaining carcass was then skinned and finely chopped. The carcass and pelt were placed in separate containers. All excreta were collected from the time of administration to death. Feces were removed from the collection trays with forceps. Tbe 23’Pu remaining with the “tray tidies” was assumed to represent urinary excretion. Using this method, some crosscontamination between urine and feces was unavoidable so the results may not accurately represent relative urinary and fecal excretion rates, although estimates of total excretion are valid. In the studies with “impure” 237Pu,all samples were measured by y-ray spectrometry between coaxial NaI(Tl) all organ samples were counted in an automatic y counter* incorporating an 8 c m dam. NaI(Tl) well crystal. The 237Puactivities were measured by counting the emissions at about 60 and 100 Lev; calibration factors appropriate to the mass and volume ofeach sample were derived from measurements of standardized sources of 237Pu.Background counts, made using tissue samples from untreated rats, did not differ significantly from the intrinsic counter background. Tests of significance were made using the Eighteen rats were used in this study. Groups of three animals were killed at I , 2, 7, 14, 30, and 84 d after injection. Approximately 1.2 kBq of 237Puwas injected into each rat, accompanied by over 30 Bq of a-emitting Pu isotopes. The mass of Pu injected was about 37 ng kg-’. The mean fractions of administered 237Puexcreted in urine and feces by the groups of rats are shown in Table 2. It can be seen that, particularly after day 1, similar results were obtained from the different groups. By the creted in feces and about 5% in urine. The distributions of 237Puin various organs at death are shown in Table 3. Together with the excreted 237Pu, these accounted for 95 to 96% of the 237Puactivity administered (present in the noninjected aliquots). Initially, substantial fractions of the injected activity were found in the soft tissues, especially liver and pelt. However, by 84 d more than 90% of the residual Pu was found in the carcass and less than 4% in the liver. Additional measurements of 237Puin the right femur and in muscle tissue
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Table 2. Fecal (F) and urinary (U)excretion of '"Pu from diffettnt groups of three rats afier injection of "impure" *%I citrate. Period of cage 1 cage 2 Cage 3 cage 4 tag+ 5 cw 6 (d) FU FU FU FU FU FU indicated that, by that time, the 237Puin the carcass was predominantly (>98%) skeletal. Skeletal accumulation of 237Puwas essentially complete by 7 d after injection. Measurements of the pelt and muscle tissue of the injected left leg at 1 d after injection showed that less than 1% of the injected 237Puwas retained at the injection site at that time; at later times, no excess was detectable. Studies with "pure"237Pucitrate and nitrate In this study, eight rats were injected with 0.45 Bq of "pure" 237hcitrate and eight with an equal activity of "pure" 237Punitrate. Five animals from each group were killed at 7 d and three at 63 d. About 0.45 kBq of 2 3 ' ~ was injected associated with <O. I of aemining h isotopes. The mass of Pu injected was about 0.007 The distribution of 237Puin the organs at 7 and 63 d after injection is shown in Table 4, which includes the total activity excreted in urine and feces. The average of the summed radioactivities in the organs, carcass, and excreta was 101% of that in the noninstilled aliquots. The results of the study with "impure" 237Pugiven in Tables 2 and 3 showed that about 35% of the injected activity had been excreted by 84 d. Although 2% of the 237Puwas excreted in urine during the first 24 h, most of the subsequent excretion was in feces. There were large variations in the fecal:urinary excretion ratio; during the first day it was about I but increased to about 25 during the 7 to 14 d period, and then declined. The high fecal excretion rate in the early part of the study probably reflects the clearance of Pu from the liver and other soft tissue compartments that is apparent from Table 3. In the mouse, it has been shown that at early times the cumulative loss of Pu from the liver is very similar to the cumulative excretion of Pu in feces ( Schubert et al. 196I ). Initially, a significant proportion of the 237Pufound in the carcass was assoCiated with muscle, but by 84 d more than 98% of the carcass activity was present in bone. However, removal of 237Pufrom bone appeared during the second half of the study. Comparing the distribution of "pure" 237Puat 7 d Table 3. Tissue distribution and excretion of 237Puat various times after intravenous injection of "impure" 237Pucitrate. GI traa and contents. Includa spleen and esophrglu. 'Inclcludes hart, trachea, and thymus. e About 50 g of muscle WIS trimmed from the carcass. foul muscle weight was assumed lo be I80 &
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I86 Halth Physics August 1990, Volume 59, Number 2 Table 4. Tissue distribution and excretion of *"Pu at 7 and 63 d after intravenous injection of "pure" Tiaue/exmta Citrate Nitrate Citrate Nitrate GI tract and contents' 'Includes hean. trachea. and thymus. 'Rcprrscnts sum of femora, muscle sample, and residual carcass. 'About 50 g of m u c k was trimmed h m the eviscerated carcass. Total muscle weight assumed to be IS0 g at 7 and 63 to 84 d following injection (Table 4 ) with that of the "impure" at the same times (Table 3)shows that they were about the same. In addition, as shown in Table 5 , the distribution of 237Puat 7 d following administration as the citrate closely matches that observed for 239Pu ministered by intravenous injection to the same strain of Wistar rats in citrate form. Unfortunately, data for the distribution of '3% and '"Pu are only available at 7 d. The results are also similar to data obtained following injection of 2 3 9 ~citrate into Sprague-Dawley rats at 6 d (Seidel and Volf 1972) and at later times (Hollins and This study also provided a direct comparison of the biokinetics of '"Pu nitrate and citrate, given in ass0ciation with very small masses of Pu ( 7 pg per kg body weight). Because there was so little inter-animal variation in either the translocation or excretion of 2 3 7 ~within any of the treatment groups, even small inter-group differences could be demonstrated statistically. Following injection of the 237Punitrate solution, there was Little evidence of extensive polymerization /colloid formation that would have been expected had much larger masses of Pu been administered. Nevertheless, as indicated in Table 4, some differences in observed. The fractions of injected 237Puactivity deposited in the spleen were more than 50% higher for the 237Pu nitrate than for the citrate at both 7 and 63 d. Table 5. A comparison of 237Pu,239Pu,and '"Pu distribution in rat tissues 7 d after intravenous injection of citrate solutions. Percent of injected PU (mean f SEM)
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In addition, although the activities in the liver were similar at 7 d, they were greater for the 237Punitrate at the later time. Both liver and spleen are known to remove colloids from the bloodstream in preference to sotuble forms. The results support the findings of Lloyd et a]. (1976) and Bair et al. (1970) that 237F'unitrate is not a good indicator of the behavior of Pu at the mass loadings typical of experiments with 239Punitrate. However, they provide useful data on Pu metabolism at specific activities that would preclude the use of 2r)Pu.It appears that at sufficiently low mass concentrations,differences between the behavior of nitrate and citrate arc minimized from restricted potential for colloid formation. Despite its high specific activity, 237Pubehaves biokinetically in a similar manner to long-lived isotopes of Pu when administered as the citrate. Thus, subject to a p proval from the appropriate ethical organizations, there appear to be no contra-indicationsfor usage in Pu metabolism studies of healthy volunteers. Protection Core Program and by the Department of Energy.The ='Pu olism of 237Puand "'Pu in beagles. In: Pacific Northwest Laboratory Annual Report for 1968; Richland, WA: PNL; tonium by the tissues of the rat. Radiat. Res. 61:468477; tabolism of compounds of plutonium and other actinides. International Commission on Radiological Protection. Radionuclide transformations: Energy and intensity of emissions. Oxford: Pergamon Press; ICRP Publication 38; 1983. Kocher, D.C. Radioactive decay data tables. Springfield. VA: Technical Information Center; US.Department of Energy; tribution and excretion of plutonium administered intravenously to man. Los Alamos, NM: Los Alamos National Lab; Scientific Laboratory Report LA4 15 I ; 1950. '37pU(1V) citrate in beagles. Health Phys. 30:47-52: 1976. trointestinal absorption of neptunium in primates: Effm of I Oxford: Pergamon Press; ICRP Publication 19; 1972. ingested mass, diet and fasting. …