Comp. Biochem. PhysioL, 1975, Vol. 5011,pp. 113 to 117. Pergamon Press. Printed in Great Britain
METABOLISM OF 6°Co IN CHICKENS: RETENTION AND DISTRIBUTION K. SALMINEN,* O.-P. OBERMEIER AND W. KREUZER tLehrstuhl for Hygiene und Technologie der Lebensmittel tierischen Ursprungs der Universitiit Mtinchen, Tieriirztliche Fakultiit, 8 Miinchen 22, Veteriniirstrasse 13, Germany (Received 3 January 1974)
Abstract--1. The retention and distribution of e°Co in chickens was studied for up to 46 days following the administration of a single peroral 800 or 400 nCi/kg e°CoCI2 dose. 2. The absorption of 6°Co was found to be low. After 5 days over 97 per cent of the administered radioactivity had been excreted. 3. The retention of 6°Co in organs and tissues can adequately be described by a power function of the form R(t)= a.t -b in which R(t) gives the retention after time (t) (days). The numerical values of a and b were determined for the organs and tissues of chickens. 4. The fractional distribution of °°Co in chickens was found to vary at various time intervals after the administration. High initial fractional distribution was recorded in the intestine, liver and kidneys. Low initial but steadily increasing fractional distribution was noted for musculature and skeleton. INTRODUCTION
MATERIALS AND METHODS
THE CHARACTERof cobalt as an essential nutrient is mainly due to its central role in vitamin BI~. The need for cobalt is especially pronounced in ruminants, which have a rapid propionate metabolism including an active methylmalonyl-CoA mutase and its necessary cobalamin coenzyme. If the animals ingest enough cobalt, the intestinal micro-organisms are able to synthesize the requisite cobalamin for them. A corresponding mechanism exists in the avian caeca, and the amount of vitamin BI~ present in the caeca, if absorbed, could contribute significantly to the bird's requirement (Coates et aL, 1968). In addition to its role in vitamin B12, cobalt is needed for glutamine synthesis by glutamine synthetase (E.C. 188.8.131.52) in chicken liver (Fazekas & Denes, 1966). Most of the animal data concerning the resorption, distribution and excretion of cobalt have been derived from investigations following administration of radioisotope cobalt to rats (Comar, 1948; Taylor, 1962; Strain etaL, 1965; Barnaby et al., 1968). Data on fowl are scanty, and studies have been confined to the early periods after administration (Monroe, et al., 1952; Lee & Wolterink, 1955; Wegorek et aL, 1963). The present studies were carried out to determine the distribution and retention of perorally administered e°Co in chickens.
Two groups of birds of both sexes, Leghorn Hyline breed, averaging 2.5 kg in weight at the onset of the experiments, were used. The number of males was twenty-six and that of females twelve. The radioactive isotope was obtained from Physi kalisch-Tectmische Bundesanstalt, Bratmschweig, and the specific activity of the e°CoCl~ in 0.1 n HCI was 25.59 /~Ci/mg. The dose was 800 nCi/kg (males) or 400 nCi/kg (females), corresponding to 34 (respectively 17)/zg stable Co/kg. Prior to administration the birds were kept fasting for 12 hr. The solution was administered perorally into the crop. The birds were fed a commercial full feed containing 0.419 mg Co/kg. After an appropriate period the chickens were sacrificed and dissected. The dates of slaughter and number of chickens are listed in Table 1.
Table 1. Number of test animals and time of slaughter
*Present address: Department of Biochemistry, College of Veterinary Medicine, Hiimeentie 57, 00550 Helsinki 55, Finland. t Vorstiinde: Prof. Dr. Dr. H. C. L. Kotter und Prof. Dr. G. Terplan 113
No. of chickens Time of slaughter (days)
2 4 8 14 20 29 46
3 3 3 3 3 2 3
2 2 2 2 I
K . SALMINEN, O . - P . OBERMEIER AND W . KREUZER
The crop, proventriculus and gizzard (all referred to as stomach), small and large intestine (consisting of caeca and rectum) were opened and rinsed thoroughly with warm water. These as well as the liver, kidneys, lungs, heart, spleen, ovaries, testes and blood were dried for 2-3 days at 105°C and pulverized. The skin, bones, musculature of trunk and extremities were separated and ashed at 550°C (bones at 650°C). The seven control animals were treated in a similar manner except for the administration of radioactive e0Co. The excreta of three individuallycaged chickens (males) were collected daily and ashed at 550°C. The 6°Co determinations were carried out with a Scipp Victoreen 400 multi-channel analyzer equipped with a 2 x 2 in. NaJ(T1) well crystal. The counting sensitivity and counting time were such that the measurement of an activity of 0.5 nCi involved a 3 per cent counting error. An activity of 0.05 nCi was taken to be the minimum level measurable with this counter.
ministered, and t the time in days after administration. The equation E ( t ) = 0.25exp(-0.54t) adequately describes the actual excretion data. Excretion takes place rapidly; after 5 days over 97 per cent of the radioactivity administered was excreted, and after 46 days 98"9 per cent of the original dose was recovered in the excreta. Tissue retention
Data for the retention of ~°Co in tissues are given in Table 2. The retention in all tissues investigated can be described by a power function of the form R ( t ) = exp(-)trt ) a . t -b between 1 and 46 days. Because the radioactive half-life of e°Co, 1.9 x 10a days, far exceeds the metabolic period t, the radioactive decay term has been
The daily excretion amounts following a single per oral administration of e°CoCI= at the 800 nCi/kg dose level, expressed in terms of per cent of the dose administered, are presented in Fig. 1. After the first
Table 2. Values of a and b which satisfy the retention equation R(t)= a . t -b for various organs following a single oral 800 nCi/kg or 400 nCi/kg administration of e0Co a
Small intestine Large intestine
Kidneys Stomach Liver Skin Lungs
0.086 0.12 0.41 0"12 0'038
0-47 0.66 0.76 0'56 0'57
Musculature of extremities Musculature of trunk Skeleton
0.11 0.072 0.11
0.40 0.29 0.32
4o c E 3O "o o
~ A ~ I ~ I ~ 5 I0 15 Dayspostadministration Fig. 1. Daily excretion of a°Co in chickens following peroral administration of a single 800 nCi/kg dose of 6°CoClv Individual points represent the mean values of results for three chickens. day and up to the end of the experiment, the excretion of e°Co can be adequately described by an exponential function of the form E(t)= a.exp(--bt), where E ( t ) represents the fraction excreted per day expressed as a function of the radioactivity ad-
omitted. The numerical values of a and b which satisfy the retention equation for various chicken organs are given in Table 2. Retention was shown to be similar for both the dose levels studied, and the results for both groups have been taken together to represent the retention of e°Co after peroral administration. Tissue distribution
Distribution figures for the e°Co retained in chickens at various time intervals after administration are presented in Table 3 and Fig. 2. The distribution patterns for both the dose levels studied were shown to be similar, and the results for both groups have been taken together to represent the distribution following peroral admininstration of t°Co. The share of 6°Co activity during the first few days is highest in the intestine, but decreases quickly. The fraction retained in the liver increases sharply during the first 4 days, but decreases thereafter. A somewhat similar fractional distribution was noted for the
Metabolism of ~°Co in chickens
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Fig. 2. The fractional distribution of retained 8°Co in intestine, liver, kidneys, skeleton and musculature of extremities in chickens following peroral administration of a single 800 or 400 nCi/kg dose of e°CoCl~. kidneys. N o 6°Co activity could be detected in the liver or kidneys after 29 days. A steady gradual increase in the share of the musculature was observed, remaining present the longest in the musculature of the extremities. The fraction of the activity retained in the skeleton was initially very low, but increased continuously, until finally most of the remaining activity was found in the skeleton. Low fractions of s°Co activity were found in the heart, spleen, lungs and testes. The skin, stomach and ovaries showed some activity, a noteworthy share being detectable in the skin as late as the forty-sixth day. The level of activity in the blood was very low, and has been omitted here due to the difficulty of obtaining a quantitative recovery of blood. DISCUSSION
Most of the e°Co administered was found to pass through the gut without retention. According to Lee & Wolterink (1955) 6°Co, as e°CoSO4, is both readily absorbed and returned to the intestinal tract through its wall in the chicken, e°Co is highly concentrated in the caecal contents, in the caecal wall and in the small intestine regardless of the route of administration. The present results are in accordance with these findings. After 2 4 h r the test chickens had already excreted over 50 per cent of the dose administered, which is in good accordance with the 54.2 per cent excretion reported by Monroe et aL (1952). After 72 hr about 94-5 per cent of the administered dose had been excreted, corresponding to the 95 per cent reported for pheasants (Wegorek et aL, 1963). After 8 days the excretion had been practically completed, the total fraction excreted being 98.7 per cent. When inorganic 6°Co was given orally to human subjects the fraction absorbed was dependent upon
the amount of stable cobalt which accompanied the 6°Co. When this was small ( < 1/~g) absorption was low ( < 5 per cent), but with larger quantities of stable cobalt (1.2 mg) absorption could be 20 per cent or more (Smith et al., 1972). One explanation is that with trace doses a considerable proportion of the e°Co becomes bound to gut contents and is not available for absorption. If there are a limited number of binding sites, however, a smaller amount of 6°Co would be bound if a larger quantity of stable cobalt were given with the n°Co dose. On the other hand, the feeding of 10-200 ppm levels of cobalt in the diet did not affect the level of absorption or the biological half-life of the isotope in chickens receiving 6°Co orally (Suso & Edwards, 1969). The relatively low fractional retention noted for the stomach indicates a minor role in e°Co absorption. There are differences between the slopes (b) of the retention lines for different tissues, indicating dissimilar rates of elimination of e°Co. In general, the lower the value of b, the slower is the elimination. Thus the tissues and organs studied can be roughly divided into three groups according to the numerical value of b. The highest observed values of b, in the range of 1.6-1.9, for the small and large intestine indicate a rapid elimination. The intermediate group, which includes the stomach, kidneys, liver, skin and lungs, shows values of b in the range of 0.47-0.76. The skeleton and musculature exhibit the longest retention, with the value of b varying in the range of 0.29-0.40. The liver initially retains the highest fraction of 6°Co taken up by the body, but after 5-6 days the rate of elimination of the radionuclide, when expressed as a proportion of the amount retained, is greater for liver than for any other organ except the gut. The 6°Co burden of this organ at its highest was almost 30 per cent of the total amount in the body. Since the liver averages only 2-3 per cent of the total
Metabolism of 60(20 in chickens body weight, a burden of the above proportion would indicate an approximate ten-fold activity concentration per g in the liver compared to the body as a whole. The main reason for the accumulation of cobalt in the liver is due to the portal transport, whereby the liver has a function of a collecting pool. The tendency for cobalt to concentrate in the liver has also been observed in other animals (Comar & Davis, 1947; Braude et al., 1949; Cuthbertson et al., 1950; Barnaby et aL, 1968; Smith et aL, 1971; H6bel et aL, 1972). The rapid decline of 6°Co activity in the liver shows the presence of relatively short-term retention components. The same is true for the kidneys, which, however, never reached the same fractional retention level as the liver. According to Vostal & Heller (1968), cobalt seems to resemble sodium and other essential ions in being able to pass into the urine not only by glomerular filtration but also by direct transport across the tubular wall in chickens. The remaining organs, lungs, heart, spleen and reproductive organs, contained measurable amounts of 6°Co in the beginning, but elimination was relatively rapid except in the ovaries, which showed an increasing fractional retention up to 14 days. The dissimilar distribution of 6°Co in testes and ovaries especially on days 2, 4 and 8 should cause some error in the share of other organs and tissues, but the observed values for these in females were within the range of variation observed in males. The skin showed a relatively low but long retention. The tissues showing long-term retention components, the skeleton and musculature, showed an increasing fractional retention. At the end of the experiment, i.e. on day 46, most of the e°Co activity still remaining was found in the skeleton, indicating the lowest elimination of the tissues and organs studied. The musculature of the extremities likewise contained considerable activity, whereas the musculature of the trunk contained only traces. Since the musculature of the extremities contains predominantly red fibres with a relatively large quantity of cytochrome c and myoglobin, it might be tempting to assume a correlation between cytochrome c and myoglobin on the one hand and cobalt on the other. The longevity of 6°Co in the skeleton may be due to the location of vitamin B ~ in the bone marrow, the site of hematopoiesis. Acknowledgements--This work was partly carried out during the time one of the authors (K. S.) worked as a research fellow of Deutscher Akademischer Austauschdienst (German Academic Exchange Service) in the University of Munich. The authors thank Mr. Hermann Lassal for his valuable technical assistance. REFERENCES
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VESTAL J. & HELLER J. (1968) Renal excretory mechanisms of heavy metals--I. Transtubular transport of heavy metal ions in the avian kidney. Environm. Res. 2, 1-10. WEGOREK W., GLOC,OWSKI K. & CZAPLICKIE. (1963) Possibility of contamination of insectivorous game birds during ecological investigations using insects labelled with e°Co. Prace Nauk. Inst. Ochrony RosL 5, 19-28. (In Polish.) Key Word Index--Cobalt; e°Co, radiocobalt; metabolism; heavy metals.