Absorption and Tissue Distribution of Radiozinc in Steers Fed High-Zinc Rations J. P. Feaster, Sam L. Hansard, J. T. McCall, F. H.

Skipper and G. K. Davis J ANIM SCI 1954, 13:781-788.

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ABSORPTION AND TISSUE D I S T R I B U T I O N OF RADIOZINC I N STEERS FED HIGH-ZINC RATIONS 1 J. P. FEASTER, SAM L. HANSARD, J. T. McCALL, F. H. SKIPPER 3 AND 2 G. K. DAVIS
Florida Agricultural Experiment Station 4

has been shown by Bertrand by I TElvehjem and associates (1934, and Bhattacherjee a(1934) andcon1935) that zinc is necessary stituent of the diet for the normal growth of mice and rats. Excess dietary zinc was reported by Sutton and Nelson (1937) to produce anemia, subnormal growth and reproductive failure in rats. Gray and Ellis (1950) noted that the inhibitory effect of molybdenum on growth of rats was aggravated by the addition of zinc to the diet, but observed no retardation of growth upon addition of zinc alone. Scott and Fisher (1938) observed weight losses in mature cats fed on a high zinc diet. McCance and Widdowson (1942) observed that in humans, excess dietary zinc was excreted in the feces, with no corresponding increase in urinary zinc excretion. The excretion and tissue distribution of injected radioactive zinc 65 has been studied in dogs and mice by Sheline et al. (1943). The radioisotope has also been employed in studies of the relationship of zinc to the pancreas (Montgomery and co-workers, 1943; Heath and Liquier-Milward, 1950; Lowry, Baldwin and Harrington, 1954) and blood plasma and cells (Ebaugh and Ross, 1952; Tupper and Watts, 1952). The present investigation was undertaken to study the movement of zinc through the animal body and to observe possible effects of excess dietary zinc on the condition of cattle and on the absorption and distribution of radioactive zinc 65 administered orally and intravenously to young growing steers. Experimental Four purebred Jersey steers were maintained from the age of one month on a ration of grain (from ~/2 pound at one month to 6 pounds
z Florida Agricultural Experiment Station Journal Series, No. 281. Published with the approval of the Director of the Florida Agricultural Experiment Station. This work was supported in part by funds granted by the Nutrition Foundation, Inc. Present address UT-AEC Agricultural Research Program, Oak Ridge, Tennessee. a Present address University of New Mexico, Albuquerque, New Mexico. 4 Gainesville.

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DAVIS

at one year), 1 pound cottonseed oil meal and prairie hay according to the calculated requirements of the animal. Zinc carbonate was added at levels of 1000 ppm. zinc to the total ration of two of the steers and 50 ppm. to the total ration of the other two (control) steers. At 13 and 18 months of age, respectively, the high zinc and control animals were placed in metabolism units for the quantitative separation and collection of urine and feces. After the animals had become adjusted to the unit and were on a constant feed intake they were given orally or intravenously (Hansard et al., 1951) a single dose of zinc 65, and concurrent six-day chemical and radioisotope balance studies were started3 Blood, feces and urine samples were taken at regular intervals for assay. At the end of 6 days, the animals were slaughtered. Tissue samples were carefully selected, weighed and ashed. The ash was dissolved in acid and the solution made to volume. Radiozinc measurements were made with dipping type Geiger-Muller Counters and conventional scalers. Total zinc content of selected tissues was determined by means of the Leeds and Northrup polarograph after extraction of zinc by the dithizone procedure of Menzel and Jackson (1940). Results and Discussion

Absorption and Excretion o] Zinc

The results of the 6-day balance studies indicated that only 3-10 percent of the dietary zinc was absorbed from the gastrointestinal tract. There were no gross symptoms of toxic effects observed in the two animals receiving the high levels of dietary zinc, and although their weight gains were slightly lower than those of the controls, the difference was not statistically significant. The high-zinc animals at slaughter weighed 427 and 480 pounds at 378 and 409 days of age, while the control steers weighed 540 and 560 pounds at 505 and 539 days of age. Likewise, there were no significant effects noted on zinc ~ excretion that could be attributed to the difference in dietary zinc intake. Following oral administration, however, the high-zinc steer excreted slightly more of the isotope in the feces (73.2%) than the control animal (65.8%). Urinary excretion accounted for 0.24 and 0.39 percent of the administered dose. Following intravenous injection of zinc, fecal values were 20.6 and 20.7 percent and urine values were 0.33 and 0.13 percent of the injected dose for the high-zinc and control animals, respectively.
5The labeled zinc~ was obtained from the Oak Ridge National Laboratory, Oak Ridge, Tennessee, after allocation by the Isotope Division of the Atomic Energy Commission.

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These differences between animals are within the expected range of individual variation and would indicate little effect of dietary zinc intake upon zinc 6~ behavior in this species. Sheline et al. (1943) have reported that in mice 50 percent of intravenously injected zinc 65 appeared in the feces in 7 days, and in dogs 25 percent appeared in the feces in 15 days, less than 5 percent being excreted in the urine during these periods. Following oral administration of radiozinc, peak excretion of the isotope in the feces occurred during the 24- to 48-hour interval. After intravenous administration there was no such clearly defined peak, although radiozinc was excreted at a slightly higher rate from 24 to 120 hours after dosing than during the remainder of the dosage-tosacrifice interval.
Zinc 65 in the Blood

In order to study the movement and exchange of radiozinc between blood and tissues, blood samples were drawn at frequent intervals during the 6-day period. Analysis of 18 blood samples from the steers dosed orally indicated that zinc ~ entering the blood from the gastrointestinal tract never reached concentrations sufficiently high to measure. Following intravenous administration of the isotope, however, the level of zinc 65 decreased rapidly from 100 percent of the dose in the total blood at 3 minutes to 10 percent after 4 hours. Total blood was calculated as 7 percent of body weight (Hansard et al., !953). Equilibrium between blood and tissue appeared to have been reached after about 10 hours, with approximately 3 percent of the administered dose present in the blood. Ebaugh and Ross (1952) reported that 11 percent of the zinc 65 dose remained in the plasma of man at 30 minutes after intravenous administration. Since zinc is known to be a constituent of the enzyme carbonic anhydrase, found in red blood cells, an attempt was made to follow the in vivo uptake of radioactive zinc by the red cells. Packed cell volume was determined by centrifuging the blood samples, and blood cell zinc G~ activity calculated by subtracting from the counts per minute in a given volume of whole blood the counts found in the volume of plasma present in this volume of blood according to the cell-plasma ratio for the sample. Figure 1 shows the rate of disappearance and the partition of zinc 6~ between bovine plasma and cells as a function of time folIowing intravenous isotope administration. Zinc 65 values are expressed as counts per minute in the plasma and cell fractions making

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FEASTER,HANSARD, ~[cCALL, SKIPPER~ AND DAVIS

up 100 ml. of whole blood. The values at 3 and 10 minutes represent averages of those obtained with blood from both steers; the remaining points are based on single values for individual samples drawn alternately from the two animals.
9896-

~,

o_ 52

54[
28 ]

I q9 ~, 9 PLASMA 0 CELLS

~ 20 o 0 12-

"------1

0
i

212 HOURS AFTER DOSING

2'6

5C)

Figure 1. The disappearance of zinc65 from plasma and red cells of

bovine blood following intravenous administration. (Counts per minute per 100 ml. whole blood equivalent.)

In the samples drawn 3 minutes after isotope administration, 22 percent of the radioactive zinc in the blood was found in the cell fraction and 78 percent in the plasma. A steady increase in the cell-to-plasma zinc ~5 ratio was noted during the next several hours, equilibrium between the two fractions being reached about 6 hours after dosage. Thereafter, with plasma zinc ~ activity showing a slightly more rapid decrease than that of the cells, cellular activity predominated. These findings indicate that the red cell wall is permeable to the zinc cation, a concept in keeping with the essential nature of this element with respect to the red blood cell enzyme carbonic anhydrase. Keilin and Mann (1939) found the zinc content of carbonic anhydrase prepared from beef corpuscles to be directly proportional to the enzyme activity, and were of the opinion that all the zinc present in the erythrocytes was combined with carbonic anhydrase; they concluded that this enzyme is a zinc-protein complex. Tupper, Watts and

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Wormal (1952) corroborated the fact that zinc in carbonic anhydrase is firmly bound to the protein, noting that, in vitro at least, little if any of the zinc G5 used in labeling red ceils became firmly attached to the carbonic anhydrase. Berfenstam (1952) observed that intravenous injections of zinc raised the zinc content of the erythrocytes without a concomitant increase in carbonic anhydrase activity. Whether or not in the present study the zinc 6~, which apparently crossed the red cell membrane, actually exchanged with the zinc ions already attached to the carbonic anhydrase is problematical.
Tissue Distribution o] Zinc 65

Excretion data previously presented indicate that retention of zinc 6~ averaged 30 and 79 percent for oral and intravenous administration, respectively. This was reflected in the zinc 65 content of the tissue in that the intravenous values were from two to three times the corresponding oral values. As discussed earlier, the dietary zinc level did not appear to influence the behavior of radiozinc. Therefore, in order to eliminate effects due to differences in methods of administration, absorption from the tract or body weight, all tissue distribution values for zinc 6~ have been calculated on the basis of percentage of retained zinc 65 dose, and corrected to a standard body weight of 500 pounds. Thus the values tabulated in the second column of table 1 for individual tissues reflect the handling by the body of that radiozinc made available to it. Since there was no apparent influence of dietary zinc levels either upon total tissue zinc or upon the behavior of the administered radiozinc, the summary of the distribution of zinc 65 in selected tissues 6 days after oral and intravenous administration to all steers is presented in table 1. Concentration values for total zinc are expressed as rag. of zinc per kg. fresh weight, and tissues are arranged in decreasing order of concentration. I t is of particular interest that the pituitary contained the highest level of zinc and values for the incisors ranked along with the soft tissue values. The blood value of 8.8 rag. per kg. is in close agreement with the findings of Vallee and Gibson (1948). Scott and Fisher (1938) noted that in cats fed excess zinc at a slightly lower level per kg. of body weight than that received by these steers, zinc concentrations in the pancreas and liver were 480 and 900 micrograms per gram, respectively, as compared with 72 and 58 micrograms in the controls. This dietary influence was not noted for ruminants in this study where excess zinc was fed for more than a year.

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T h e accumulation of radiozinc, in general, was highest in the soft tissues. T h e pancreas, liver, pituitary, k i d n e y and adrenals showed the highest concentration of retained dose. However, the turnover rate as shown b y specific activity ( r a p i d i t y of u p t a k e and loss) was highest in the liver, adrenals and pancreas. I n decreasing order of concentration, the gastrointestinal tract and contents, muscle, spleen, hide and hair, brain, thymus, lungs, heart and spinal cord contained from .03 to 0.2 percent of the absorbed dose per kg. of fresh tissue. T h e bile and bone marrow contained no radiozinc after 6 days. TABLE 1. SUMMARY OF ZINC 65 DISTRIBUTION IN YEARLING CATTLE AT 6 DAYS AFTER ADMINISTRATION Mg. Zinc per Kg. Fresh Weight 247 130 125 121 55 45 33 28 25 8.8 Percentage of Zn 65 DoseRetained per Kg. Fresh Tissue o. 98 .17 1.58 .86 .75 .80 1.20 .15 .35 .04

Tissue Pituitary Incisors Pancreas Kidney Adrenals Rib Sternal End Liver Rib Shaft Mandible Angle Whole Blood

Specific Activity1 4.0 1.3 12.6 7.1 13.6 17.8 36.3 5.4 14.0 4.5

1 Specificactivity expressedas percent of retained zinc dose per milligramzinc X 10a. ~ Radiozinc accumulation in bones more or less paralleled their metabolic activity, p r o b a b l y reflecting the blood supply to the particular areas. T r a b e c u l a r bone retained more zinc 65 than cortical bone and the turnover rate, as shown b y the specific activity values, was higher for the sternal end of the rib than for the rib shaft; turnover rate was higher for the mandible than for the metatarsus shaft and was lowest for the incisor teeth. T h e pancreas and liver showed the highest concentrations of radiozinc in all 4 animals. This finding with respect to the pancreas bears out the close chemical relationship between insulin and zinc. Sheline and co-workers (1943) found t h a t the tissues showing the greatest turnover following intravenous administration of zinc 65 to dogs were the liver, pancreas and kidney. Slowest turnover occurred in red blood cells, brain, skeletal muscle and skin. H e a t h and Liquier-Milward (1950) observed that zinc 65 was present in relatively large amounts in the pancreas as well as the liver, spleen and k i d n e y of humans

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following intramuscular injection of the isotope, and zinc65 was found by Montgomery et al. (1943) to be eliminated in the pancreatic juice of the dog. Lowry, Baldwin and Itarrington (1954) noted that in alloxan-diabetic rats the uptake of zinc 65 by the pancreas was considerably lower than in normal rats. Summary Radiochemical procedures have been employed to study the movement of zinc through the body of four yearling steers. Absorption and tissue distribution in two steers maintained for more than a year on i000 ppm. zinc were compared with control animals following oral and intravenous administration of the radioisotope. Concurrent chemical and radiozinc balance studies indicated that most of the zinc was excreted in the feces and was not absorbed. Those steers dosed orally with zinc 6~ excreted 72.2 and 65.8 percent in the feces and 0.24 and 0.39 percent in the urine on the high-zinc and control rations, respectively. Following intravenous administration, 20.6 and 0.25 percent of the dose was excreted in the feces and urine, respectively, with no differences attributable to dietary zinc level noted. The rate of disappearance of zinc 65 from the blood following intravenous administration resulted in the attainment of equilibrium between blood and soft tissue in 10 hours. The partition between plasma and cells presented as a function of time indicated that equilibrium between the two fractions occurred at about 6 hours, after which activity in the red cells predominated. The accumulation of retained radiozinc was, in general, highest in the soft tissue, the pancreas, liver, pituitary, kidney and adrenals having highest concentration. Turnover rate was highest for the liver and lowest for the incisor teeth. The accumulation and turnover of zinc in the various bones paralleled their metabolic activity. L i t e r a t u r e Cited Berfenstam, R. 1952. Studies on carbonic anhydrase activity in children. Acta paediat. 41 :32. Bertrand, G. and R. C. Bhattacherjee. 1934. The combined action of zinc and vitamins in the feeding of animals. Compt. Rend. Acad. 198:1823. Ebaugh, F. G. and J. F. Ross. 1952. Radioactive zinc metabolism in patients with neoplastic diseases. Fed. Proc. 11:413. Gray, L. F. and G. H. Ellis. 1950. Some interrelationships of copper, molybdenum, zinc, and lead in the nutrition of the rat. Jour. Nutr. 40:441.

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Hansard, Sam L., C. L. Comar and M. P. Plumlee. 1951. Radioisotope Procedures with farm animals. I. Nucleonics 9:13. Hansard, Sam. L., C. L. Comar and M. P. Plumlee. 1951. Radioisotope l~rocedures with farm animals. II Nucleonics 9:38. Hansard, Sam L., W. O. Butler, C. L. Comar and C. S. Hobbs. 1953. Blood volume of farm animals. JOUP.NAL OF A17IiVIALSCIENCE 12:402. Heath, J. C. and J. Liquier-Milward. 1950. Distribution and function of zinc in normal and malignant tissues. I. Uptake and distribution of radioactive zinc 6~. Biochem. Biophys. Acta 5:404. Keilin, D. and T. Mann. 1939. Carbonic anbydrase. Nature 144:442. Lowry, J. R., R. R. Baldwin, and R. V. Harrington. 1954. Uptake of radiozinc by normal and diabetic rat pancreas. Science 119:219. McCance, R. A. and E. M. Widdowson. 1942. The absorption and excretion of zinc. Biochem. Jour. 36:692. Menzel, R. G. and M. L. Jackson. 1951. Determination of copper and zinc in soils or plants. Anal. Chem. 23:1861. Montgomery, M. L., G. E. Sheline and I. L. Chaikoff. 1943. Elimination of administered zinc in pancreatic juice, duodenal juice and bile of the dog as measured by its radioactive isotope (Zn65). J. Exp. Med. 78:151. Scott, D. A. and A. M. Fisher. 1938. The pancreas and liver of normal and zincfed cats. Am. Jour. Physiol. 121:253. Sheline, G. E., I. L. Chaikoff, H. G. Jones, and M. L. Montgomery. 1943. Studies on the metabolism of zinc with the aid of its radioactive isotope. I. Excretion of administered zinc in urine and feces. Jour. Biol. Chem. 147:409. Sheline, G. E., I. L. Chaikoff, H. B. Jones and M. L. Montgomery. 1943. Studies on the metabolism of zinc with the aid of its radioactive isotope. II. The distribution of administered radioactive zinc in the tissue of mice and dogs. Jour. Biol. Chem. 149:139. Stirn, F. E., C. A. Elvehjem and E. B. Hart. 1935. The indispensability of zinc in the nutrition of the rat. Jour. Biol. Chem. 109:347. Sutton, W. R. and V. E. Nelson. 1937. Studies on zinc. Proc. Soc. Exp. Biol. Med. 36:211. Todd, W. R., C. A. Elvehjem and E. B. Hart. 1934. Zinc in the nutrition of the rat. Am. Jour. Physiol. 107:146. Tupper, R., R. W. E. Watts and A. Wormal. 1952. Some experiments on the zinc in carbonic anhydrase. Biochem. Jour. 50:429. Vallee, B. L. and J. G. Gibson. 1948. The zinc content of normal human whole blood, plasma, leucocytes and erythrocytes. Jour. Biol. Chem. 176:446.

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