Effects of Dietary Zinc upon Tissue Zinc and Percent Unsaturated Plasma-Zinc Binding Capacity I R. L. K I N C A I D and J. D. C R O N R A T H Washington State University Putlman 99164
ABSTRACT
Calves were fed 520 ppm zinc for 21 days, then placed on a low zinc diet (20 ppm zinc) for 35 days to determine the biological availability of zinc reserves of tissue. Concentrations of zinc in liver and kidney were elevated greatly by feeding high dietary zinc for 21 days and continued to increase with time. On the low zinc diet, the tissue burden of zinc decreased within 35 days to those comparable to calves initially fed low zinc. Likewise, both zinc in plasma and percent capacity o f unsaturated plasma for binding zinc were near negative controls 21 days after removal of calves from high zinc diets. Tissue stores of zinc will not maintain adequate zinc in blood for extended periods, and hence, they probably will not sustain optimal feed intakes and growth rates of calves on lowintakes of zinc. INTRODUCTION
Zinc is a required trace element for domestic livestock, and signs of a zinc deficiency, such as reduced growth, impaired sexual development, and parakeratosis, are documented (16, 18, 19, 21). In a recent survey of forages grown in Washington, the average concentration of zinc was approximately 20 ppm, with a range of 14 ppm to 28 ppm (12). These concentrations are below the dietary requirement of 40 ppm zinc for dairy cattle (17). Thus, zinc is a potential, practical problem for ruminants grazing forages in Washington. One of the first signs of zinc deficiency in an animal is reduction of feed intake (5, 6). However, moderate feed reductions, which are
Received September 25, 1978. Scientific Paper No. 5200. College of Agriculture Research Center, Washington State University. Project No. 0408. 1979 J Dairy Sci 62:572-576
difficult to detect under field conditions, may be indicative of many disorders in cattle. Blood data are of limited usefulness in detecting marginal zinc deficiencies because of the wide fluctuations of zinc in plasma. Many factors can affect the concentration of zinc in blood besides the dietary zinc intake; among these are fasting, infections, and stresses (4, 15, 22, 23, 24). Zinc concentrations in hair sometimes are used as an indicator of the zinc state of an animal (2). However, concentrations of zinc in hair do not appear to be sensitive enough of dietary amount to be a suitable indicator of zinc status. Another problem is the concentration of zinc in hair tells past intakes of zinc rather than current intake. Recent work by Evans (8) indicates zinc is absorbed from the basolateral membrane of the intestinal tract by apotransferrin. Apparently, apotransferrin has specific binding sites on its molecule for zinc. In the portal system, transferrin transports zinc to the liver where zinc is transferred to albumin. However, some zinc undoubtedly remains associated with transferrin. Thus, the relative number of free binding sites on transferrin in peripheral blood may be an indicator of the nutritional zinc status of an animal. Zinc has accumulated in large amounts in tissues of calves in response to high zinc intakes (13). Apparently, this effect is specific to age and species, not occurring in rats, chicks, or cows (1, 13, 14). However, feeding trials of calves were confined to 21 days, and no indications were made that further accumulation of zinc in tissues would occur if feeding trials were longer than 21 days. Likewise, the biological availability to the calf of this elevated burden of tissue zinc is not known. If this zinc is available for metabolism and released slowly from the tissues, the tissue burden of zinc could be a means of biological supplementation of zinc to calves on low-zinc diets. Accordingly, the objective was to evaluate the biological availability of zinc stores in tissue
572
ZINC RESERVES IN CALVES TABLE 1. Composition of basal diet. a
TABLE 2. Concentration of zinc in tissues of calves fed 0 and 500 ppm zinc for 21 days.
Ingredient
(kg/100 kg diet)
Barley Alfalfa Milk, dried whole Molasses Urea Dicalcium phosphate NaC1 Terramycin Vitamin A premix b Vitamin D premixC
67.825 10 10 8 1 2 1 .1 .05 .025
aThe diet, by analysis, contained 20 ppm Zn, 7 ppm Cu, and 250 ppm Fe (dry matter). bvitamin A premix contained 5,000,000 USP units/kg. CVitamin D premix contained 11,000,000 USP units/kg.
to calves on a low zinc diet. Zinc depletion from tissue and measure of blood were indexes of the nutritional state of zinc. MATERIALS AND METHODS
Twenty male early-weaned Holstein calves were in this experiment. These calves were 5 w old when placed on their respective diets. A practical ration formulated to contain inadequate zinc was fed (Table 1). Zinc was supplemented as ZnSO4. Heparinized samples of plasma were taken weekly from calves for determination of zinc in plasma and the percent capacity of unsaturated plasma for binding zinc. The procedure for the
2 ml add 3 ral ol , l S HC1
Plasma ]
2 ml add 2 ml 7nC12 s o l u t i o n (500 p l Z n / 1 0 0 ,hi)
l D~,termine Z i nc vtmcentration
;o** add 200 rag MgCO 3 | ~ mix 4x o v e r 30 rain. period
Amounts of added dietary zinc 500 SE
Tissue
0
Liver Kidney Heart Bonec Muscled
130a 68 a 64 59 78
Zn, ppm dry matter 307b 183b 65 64 69
30 47 9 24 2
a'bvalues not followed in the same horizontal line by the same letter are different at 5%. CCoccyx. dsemitendinosus.
percent binding-capacity is in Figure 1. Direct dilution of plasma with .1N HC1 was more suitable to determine zinc concentrations in our laboratory than wet-ashing. Differences in aspiration rates due to the relative viscosity between the plasma solution and the standard solutions made with .1N HC1 were only about 2%; this was insignificant. In the second part of the procedure magnesium carbonate aided removal of excess zinc not bound to protein. In blank samples, no measurable zinc remained in solution after addition of magnesium carbonate. The magnesium carbonate and excess zinc were discarded as a precipitate. The percent binding capacity is found by subtracting the amount of zinc in 1 ml of plasma from the amount of zinc in the spiked sample and dividing this by the a m o u n t of zinc in the spiked sample. This tells us the relative ability of the plasma to take up additional zinc, hence, is an indication of the number of free binding sites. Tissue samples were dry ashed, and zinc determinations were by atomic absorption spectrophotometry.
RESULTS AND DISCUSSION
Centrifuge
precipitate (d i s c a l'(t )
573
add 3 ml
,IN HCI
Detorttline
Zinc
concentra t ion
FIG. 1. Procedure for the percent capacity of unsaturated plasma for binding zinc.
The concentration of zinc in the liver and kidney increased significantly with elevated zinc intakes for 21 days (Table 2). Zinc of bone, heart, and muscle was not affected by feeding 520 ppm for 21 days. Further increases in zinc of the kidney, liver, Journal of Dairy Science Vol. 62, No. 4, 1979
574
KINCAID AND CRONRATH
TABLE 3. Effects of zinc depletion upon the concentration of zinc in tissues of calves previously fed 500 ppm added zinc. Amount of added dietary zinc Tissue
0 ppm for 56 days
Liver Kidney Heart Bone c Muscled
89 a 52a 63 36 59
500 ppm for 56 days
500 ppm for 21 days; 0 ppm for 35 days
SE
74a 66 a 66 42 71
36 84 27 27 27
Zn, ppm dry matter 479 b
391 b 56 80 65
a'bvalues not followed in the same horizontal line by the same letter are different at 5%. CCoccyx. dsemitendinosus.
and bone occurred with time of feed 520 ppm zinc (Table 3). When calves were fed 520 ppm zinc for 21 days, then placed on a low zinc diet (20 ppm zinc) for 35 days, large reductions in concentrations of zinc of kidney, liver, and bone occurred. Effects were similar when total organ zinc (in mg) was calculated: e.g., in the liver there were 86 mg of zinc in calves fed 520 ppm zinc for 21 days, 220 mg zinc in calves fed 520 ppm zinc for 56 days, and 36 mg zinc in calves fed 520 ppm zinc for the first 21 days, and 20 ppm zinc for the remaining 35 days. Zinc concentrations in plasma were slightly lower in calves fed 20 ppm zinc compared to 70 ppm zinc (Table 4). Zinc increased significantly (P
ABSTRACT
Calves were fed 520 ppm zinc for 21 days, then placed on a low zinc diet (20 ppm zinc) for 35 days to determine the biological availability of zinc reserves of tissue. Concentrations of zinc in liver and kidney were elevated greatly by feeding high dietary zinc for 21 days and continued to increase with time. On the low zinc diet, the tissue burden of zinc decreased within 35 days to those comparable to calves initially fed low zinc. Likewise, both zinc in plasma and percent capacity o f unsaturated plasma for binding zinc were near negative controls 21 days after removal of calves from high zinc diets. Tissue stores of zinc will not maintain adequate zinc in blood for extended periods, and hence, they probably will not sustain optimal feed intakes and growth rates of calves on lowintakes of zinc. INTRODUCTION
Zinc is a required trace element for domestic livestock, and signs of a zinc deficiency, such as reduced growth, impaired sexual development, and parakeratosis, are documented (16, 18, 19, 21). In a recent survey of forages grown in Washington, the average concentration of zinc was approximately 20 ppm, with a range of 14 ppm to 28 ppm (12). These concentrations are below the dietary requirement of 40 ppm zinc for dairy cattle (17). Thus, zinc is a potential, practical problem for ruminants grazing forages in Washington. One of the first signs of zinc deficiency in an animal is reduction of feed intake (5, 6). However, moderate feed reductions, which are
Received September 25, 1978. Scientific Paper No. 5200. College of Agriculture Research Center, Washington State University. Project No. 0408. 1979 J Dairy Sci 62:572-576
difficult to detect under field conditions, may be indicative of many disorders in cattle. Blood data are of limited usefulness in detecting marginal zinc deficiencies because of the wide fluctuations of zinc in plasma. Many factors can affect the concentration of zinc in blood besides the dietary zinc intake; among these are fasting, infections, and stresses (4, 15, 22, 23, 24). Zinc concentrations in hair sometimes are used as an indicator of the zinc state of an animal (2). However, concentrations of zinc in hair do not appear to be sensitive enough of dietary amount to be a suitable indicator of zinc status. Another problem is the concentration of zinc in hair tells past intakes of zinc rather than current intake. Recent work by Evans (8) indicates zinc is absorbed from the basolateral membrane of the intestinal tract by apotransferrin. Apparently, apotransferrin has specific binding sites on its molecule for zinc. In the portal system, transferrin transports zinc to the liver where zinc is transferred to albumin. However, some zinc undoubtedly remains associated with transferrin. Thus, the relative number of free binding sites on transferrin in peripheral blood may be an indicator of the nutritional zinc status of an animal. Zinc has accumulated in large amounts in tissues of calves in response to high zinc intakes (13). Apparently, this effect is specific to age and species, not occurring in rats, chicks, or cows (1, 13, 14). However, feeding trials of calves were confined to 21 days, and no indications were made that further accumulation of zinc in tissues would occur if feeding trials were longer than 21 days. Likewise, the biological availability to the calf of this elevated burden of tissue zinc is not known. If this zinc is available for metabolism and released slowly from the tissues, the tissue burden of zinc could be a means of biological supplementation of zinc to calves on low-zinc diets. Accordingly, the objective was to evaluate the biological availability of zinc stores in tissue
572
ZINC RESERVES IN CALVES TABLE 1. Composition of basal diet. a
TABLE 2. Concentration of zinc in tissues of calves fed 0 and 500 ppm zinc for 21 days.
Ingredient
(kg/100 kg diet)
Barley Alfalfa Milk, dried whole Molasses Urea Dicalcium phosphate NaC1 Terramycin Vitamin A premix b Vitamin D premixC
67.825 10 10 8 1 2 1 .1 .05 .025
aThe diet, by analysis, contained 20 ppm Zn, 7 ppm Cu, and 250 ppm Fe (dry matter). bvitamin A premix contained 5,000,000 USP units/kg. CVitamin D premix contained 11,000,000 USP units/kg.
to calves on a low zinc diet. Zinc depletion from tissue and measure of blood were indexes of the nutritional state of zinc. MATERIALS AND METHODS
Twenty male early-weaned Holstein calves were in this experiment. These calves were 5 w old when placed on their respective diets. A practical ration formulated to contain inadequate zinc was fed (Table 1). Zinc was supplemented as ZnSO4. Heparinized samples of plasma were taken weekly from calves for determination of zinc in plasma and the percent capacity of unsaturated plasma for binding zinc. The procedure for the
2 ml add 3 ral ol , l S HC1
Plasma ]
2 ml add 2 ml 7nC12 s o l u t i o n (500 p l Z n / 1 0 0 ,hi)
l D~,termine Z i nc vtmcentration
;o** add 200 rag MgCO 3 | ~ mix 4x o v e r 30 rain. period
Amounts of added dietary zinc 500 SE
Tissue
0
Liver Kidney Heart Bonec Muscled
130a 68 a 64 59 78
Zn, ppm dry matter 307b 183b 65 64 69
30 47 9 24 2
a'bvalues not followed in the same horizontal line by the same letter are different at 5%. CCoccyx. dsemitendinosus.
percent binding-capacity is in Figure 1. Direct dilution of plasma with .1N HC1 was more suitable to determine zinc concentrations in our laboratory than wet-ashing. Differences in aspiration rates due to the relative viscosity between the plasma solution and the standard solutions made with .1N HC1 were only about 2%; this was insignificant. In the second part of the procedure magnesium carbonate aided removal of excess zinc not bound to protein. In blank samples, no measurable zinc remained in solution after addition of magnesium carbonate. The magnesium carbonate and excess zinc were discarded as a precipitate. The percent binding capacity is found by subtracting the amount of zinc in 1 ml of plasma from the amount of zinc in the spiked sample and dividing this by the a m o u n t of zinc in the spiked sample. This tells us the relative ability of the plasma to take up additional zinc, hence, is an indication of the number of free binding sites. Tissue samples were dry ashed, and zinc determinations were by atomic absorption spectrophotometry.
RESULTS AND DISCUSSION
Centrifuge
precipitate (d i s c a l'(t )
573
add 3 ml
,IN HCI
Detorttline
Zinc
concentra t ion
FIG. 1. Procedure for the percent capacity of unsaturated plasma for binding zinc.
The concentration of zinc in the liver and kidney increased significantly with elevated zinc intakes for 21 days (Table 2). Zinc of bone, heart, and muscle was not affected by feeding 520 ppm for 21 days. Further increases in zinc of the kidney, liver, Journal of Dairy Science Vol. 62, No. 4, 1979
574
KINCAID AND CRONRATH
TABLE 3. Effects of zinc depletion upon the concentration of zinc in tissues of calves previously fed 500 ppm added zinc. Amount of added dietary zinc Tissue
0 ppm for 56 days
Liver Kidney Heart Bone c Muscled
89 a 52a 63 36 59
500 ppm for 56 days
500 ppm for 21 days; 0 ppm for 35 days
SE
74a 66 a 66 42 71
36 84 27 27 27
Zn, ppm dry matter 479 b
391 b 56 80 65
a'bvalues not followed in the same horizontal line by the same letter are different at 5%. CCoccyx. dsemitendinosus.
and bone occurred with time of feed 520 ppm zinc (Table 3). When calves were fed 520 ppm zinc for 21 days, then placed on a low zinc diet (20 ppm zinc) for 35 days, large reductions in concentrations of zinc of kidney, liver, and bone occurred. Effects were similar when total organ zinc (in mg) was calculated: e.g., in the liver there were 86 mg of zinc in calves fed 520 ppm zinc for 21 days, 220 mg zinc in calves fed 520 ppm zinc for 56 days, and 36 mg zinc in calves fed 520 ppm zinc for the first 21 days, and 20 ppm zinc for the remaining 35 days. Zinc concentrations in plasma were slightly lower in calves fed 20 ppm zinc compared to 70 ppm zinc (Table 4). Zinc increased significantly (P
