BIOLOGICAL TRACE ELEMENT RESEARCH 4, 125-143 (1982)
Interactions Among Nickel, Copper, and Iron in Rats Liver and Plasma Content of Lipids and Trace Elements FORREST H . N I E L S E N , * THOMAS J. ZIMMERMAN, A N D TERRENCE R . S H U L E R
United States Department of Agriculture, Agriculture Research Service, Grand Forks Human Nutrition Research Center, and Department of Biochemistry, University of North Dakota, Grand Forks, North Dakota 58202 Accepted February 15, 1982
Abstract In two fully crossed, three-way, two by three by three, factorially arranged experiments, female weanling rats were fed a basal diet supplemented with iron at 15 and 45 Ixg/g, nickel at 0, 5, and 50 Ixg/gand copper at 0, 0.5, and 5 Ixg/g(Expt. 1) or 0, 0.25, and 12 ixg/g (Expt. 2). Expt. 1 was terminated at 11 weeks, and Expt. 2 at 8 weeks because, at those times, some rats fed no supplemental copper and the high level of nickel began to lose weight, or die from heart rupture. The experiments showed that nickel interacted with copper and this interaction was influenced by dietary iron. If copper deficiency was neither very severe or mild, copper deficiency signs of elevated levels of total lipids and lipid phosphorus in liver and plasma, and cholesterol in plasma, were made more severe by supplemental dietary nickel. Rats in which nickel supplementation exacerbated copper deficiency did not exhibit a depressed level of copper in liver and plasma. Also, although iron deprivation enhanced the interaction between nickel and copper, iron deprivation did not significantly depress the level of copper in liver and plasma. The findings confirmed that, in rats, a complex relationship exists between nickel, copper, and iron, thus indicating that both the iron and copper status of experimental animals must be controlled before data about nickel nutriture and metabolism can be compared among studies. 9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163~-984/82/6900-0125 $03.80
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126
NIELSEN, ZIMMERMAN, AND SHULER
Index Entries: Nickel, interaction with copper and iron; copper, interaction with nickel and iron; iron, interaction with nickel and copper; nickel-copper-iron interactions; liver, lipids and trace elements in; plasma, lipids and trace elements in; lipids, effect of trace elements on liver and plasma contents of.
Introduction In a previous report, we demonstrated a complex relationship between nickel, copper, and iron (1). Nickel interacted with copper and this interaction was influenced by dietary iron. Signs of copper deficiency were made more severe by nickel supplementation, provided copper deprivation was neither very severe nor mild. Iron deprivation accentuated the antagonism between nickel and copper. Signs of copper deficiency made more severe by nickel supplementation were depressed weight gain, hematocrit, hemoglobin and plasma alkaline phosphatase activity, and elevated heart wt/body wt, kidney wt/body wt, and liver wt/body wt ratios. This report is an extension of the preceding studies. We were interested in ascertaining whether the complex relationship between copper, nickel, and iron affected, in addition to the above parameters, the plasma and liver content of total lipids, lipid phosphorus, cholesterol, copper, iron, manganese, nickel, and zinc.
Materials and Methods Female weanling Sprague-Dawley rats (Gibco Animal Resources Laboratory, Madison, WI)* were weighed individually upon arrival and housed three per allplastic cage measuring 50 • 24 • 16 cm and located inside a laminar flow rack (Lab Products, Carfield, NJ). The rats were assigned to groups of six in a fully crossed, three-factor, two by three by three experiment, using a completely randomized factorial arrangement of treatments. The levels of copper, iron, and nickel supplemented to the basal diet were the variables. In Expt. 1, the basal diet was supplemented with copper at 0, 0.5, and 5 p~g/g; iron, 15 and 45 ~xg/g; and nickel, 0, 5, and 50 Ixg/g. In Expt. 2, the basal diet was supplemented with copper at 0, 0.25, and 12 Ixg/g; iron, 15 and 45 p,g/g; and nickel, 0, 5, and 50 fxg/g. Nickel was supplemented as NiC12"3H20 (Ultrapure grade, Alfa Inorganics, Beverly, MA). Copper was CuSO4"5H20 (Puratronic grade, Alfa Inorganics, Beverly, MA). Iron was a mixture of 60% Fe2(SO4)3"nH20 and 40% FeSO4-nH20 that was prepared from iron sponge (Puratronic grade, Alfa Inorganics, Beverly, MA) and sulfuric acid (Ultrex grade, J. T. Baker Chemical Co., Phillipsburg, NJ). *Mention of a trademark or proprietaryproduct does not constitute a guarantee or warranty of the product by the US Departmentof Agricultureand does not imply its approvalto the exclusion of other products that may also be suitable.
NI, Cu, FE INTERACTIONS
127
The rats had access to deionized water (Super Q System, Millipore Corp., Bedford, MA) in plastic cups. Fresh food in plastic cups was provided each day. Plastic equipment and cleaning procedures were described (2-4). Absorbent paper under the cages caught droppings and was changed every other day. Room temperature was maintained at 23~ Room lighting was controlled automatically to provide 12 h of light and 12 h of darkness. Animals were weighed weekly. The diets were mixed 3 days before the start of each experiment and about biweekly, thereafter. They were stored at - 16~ in tightly capped plastic containers. The air-dried basal diets with copper omitted (5) contained, per gram, about 16 ng of nickel, 2.3 ~g of iron, and 0.47 p~gof copper in Expt. 1; and 20 ng of nickel, 1.3 Ixg of iron, and 0.39 p~g of copper in Expt. 2 as determined by atomic absorption spectrometric methods (5). The rats were fed their respective diets for eleven weeks in Expt. 1 and 8 weeks in Expt. 2. The experiments were terminated at that time because some rats fed no supplemental copper and 50 p,g of nickel/g of diet began to lose weight and die from heart rupture. The rats were weighed, then decapitated subsequent to ether anesthesia and cardiac exsanguination with a heparin-coated needle and syringe. The liver was removed, blotted dry, and stored at - 16~ in plastic bags for later analysis. The plasma was separated from the heart blood by centrifugation, and stored at -16~ in plastic tubes for later analysis. Lipids were extracted from plasma not used for the trace element analyses by the method of Allen and Klevay (6). The plasma content of lipid phosphorus was determined by the method of Taussky and Shorr (7), while total lipids were measured by gravimetric analysis. Total lipids and lipid phosphorus in 1-2 g portions of liver were determined by published methods (8). Cholesterol in the dried liver lipid extracts reconstituted in 50 p,L of isopropyl alcohol, and plasma cholesterol, were each determined by enzymatic assay (Cholesterol Reagent Set, Boehringer Mannheim Biochemical, Indianapolis, IN). In preparation for trace element analysis, 1 mL of plasma was transferred to a 12 x 100 mm borosilicate glass culture tube ("Kimax," Kimble Division, OwensIllinois, Toledo, OH) to which 0.6 mL of nitric acid (double distilled in Vycor, G. Frederick Smith, Columbus, OH) was added. The tubes were tightly capped with Teflon-lined screw caps and placed into an 80~ water bath for 16 h. After digestion, the samples were stored briefly in 12 x 75 mm polyethylene tubes until analysis. Duplicate tubes of a control serum (Wellcontrol| One BCOL Quality Control Serum, Burroughs Wellcome Co., Greenville, NY), nickel standard (50 p,g/L), and blanks were subjected to the same procedures used for the plasma samples. The portions of the livers not used for lipid analyses were freeze-dried and prepared for trace element analysis by the following methods. The livers were pulverized in polyethylene bags. In Expt. 1, 1 g portions of the pulverized, freeze-dried liver were dry-ashed by a described method (5). Samples were quantitatively transferred to 5 mL volumetric flasks and brought to volume with deionized water
128
NIELSEN, ZIMMERMAN, AND SHULER
(Super Q System). In Expt. 2, 1 g portions of the freeze-dried liver were placed into a 50 mL Pyrex edenmeyer flask to which 10 mL of concentrated, doubledistilled in Vycor, nitric acid was added. The flasks were covered and allowed to digest overnight at room temperature, then placed at low heat on a hot plate until dry. Next, 5 mL of Concentrated, double-distilled in Vycor, nitric acid was added to the flasks and the samples were placed at low heat on a hot plate until nearly dry. The last step was repeated, then, to the samples, 5 mL of concentrated, doubledistilled in Vycor, nitric acid and 10 mL of 30% H202 (Baker Analyzed, J. T. Baker Co., Phillipsburg, NJ) were carefully added so that foaming over did not occur. The samples were placed at low heat on a hot plate until 1-2 mL remained, cooled, and diluted to 5 mL with deionized water (Super Q System). The copper, iron, manganese, and zinc in plasma and liver were determined by atomic absorption spectrophotometry (9). A described method (5) was modified in the following manner for the plasma nickel determinations. A 50-1xL sample was mixed with 50 I~L of concentrated ammonium hydroxide (Instra-Analyzed, J. T. Baker Co., Phillipsburg, NJ) inside the pyrolytically coated graphite tube. The lamp current was 20 mA and the recorder range was 10 mV full scale. The electrothermal atomizer was programmed for the following cycle: drying, ramped for 30 s from 25 to 125~ and maintained for 80 s; ashing, ramped for 80 s from 125 to 1000~ and maintained for 10 s; atomization at 2650~ for 8 s. The system was purged with argon at a flow rate of 25 cc/min. The reliability of the analytical procedure was ascertained by using orchard leaves (SRM-1571, National Bureau of Standards, Washington, D.C.) certified to contain 1.3 --- 0.2 I~g of nickel/g. The value we found, 1.22 - 0.16 txg of nickel/g, compared favorably with the certified value. Data were treated by three-way analysis of variance (10).
Results The first sign of an interaction between nickel and copper occurred during the last week of each experiment. In Expt. 1, three of 12 rats fed the diet containing no additional copper began to lose weight when the diet contained 50 ~xg of nickel/g; two in the group fed 15 I~g of iron/g and one in the group fed 45 I~g of iron/g of diet. In Expt. 2, six of 12 rats, three in each group described above, began to lose weight and two died (one from each group) from heart rupture before the experiment was terminated. No deaths or weight losses were seen in severely copper-deficient rats fed the dietary nickel supplements of 0 and 5 I~g/g. The effects of copper, iron, nickel, and their interactions on weight, hematocrit, hemoglobin, plasma alkaline phosphatase activity, and heart, kidney, liver, and spleen size were described (1). It was found that in Expt. 2 the low iron diet apparently depressed hemoglobin levels more markedly than in Expt. 1, especially when the diet was low in copper. Thus, some of the interaction effects were slightly different in the two experiments. Those differences continued on to the parameters examined here.
NI, Cu, FE INTERACTIONS
129
Tables 1 and 2 show that copper deprivation elevated total lipids, lipid phosphorus and cholesterol in plasma. In Expt. 1, nickel supplementation exacerbated those apparent signs of copper deficiency. Thus, for all three parameters in Expt. 1, the interaction between nickel and copper was significant. In Expt. 2, where the severely copper-deficient rats fed inadequate iron were very anemic, nickel supplementation did not further elevate the levels of total lipids, lipid phosphorus, or cholesterol in plasma. Only plasma cholesterol was affected by an interaction between nickel and copper, and this effect was less marked than in Expt. 1. However, nickel supplementation apparently elevated plasma lipid phosphorus in copper-deprived rats supplemented with 45, but not 15, I~g of iron/g of diet. Thus, with lipid phosphorus, the nickel-copper-iron interaction was significant. The levels of various lipid fractions in liver was not determined in Expt. 1. However, the plasma findings prompted us to determine those levels in Expt. 2. Unlike plasma, the liver total lipids were unaffected by copper deprivation, but were af-
TABLE 1A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Plasma Lipids, Phospholipids, and Cholesterol (Experiment 1) Plasmab Treatment'a ~g/g Cu Fe Ni 0.0 0.0 0.0 0.5 0.5 0.5 5.0 5.0 5.0 0.0 0.0 0.0 0.5 0.5 0.5 5.0 5.0 5.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
Total lipids, c mg/mL
Lipid phosphorus, mg/lO0 mL
Cholesterol, rag/100 mL
4.58 6.00 7.04 2.63 3.88 3.58 3.58 2.92 3.08 5.05 3.88 8.04 3.46 3.54 2.96 3.42 2.17 2.13
6.79 9.18 9.46 5.40 5.81 5.62 5.38 5.16 5.48 6.82 6.44 10.84 5.43 5.43 5.52 5.00 5.03 5.05
119 163 167 94 100 104 89 96 93 119 113 195 99 94 101 86 90 95
aAmountsof Ni (nickel chloride), Fe (mixtureof 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplementedto the diet. bFresh weight basis. CEachvalue represents the mean of 6 rats.
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NIELSEN, ZIMMERMAN, AND SHULER
TABLE IB Summary of Significant Effects in Plasma: P Values Obtained by Analysis of Variance Ni
0
5
.~
Plasma total lipids a
all
0.0 0.5 5.0
50
4.80 3.04 3.50 3.75
4.94 3.71 2,54 3.73
7.54 3.27 2.60 4,47
5.79 3.34 2.88
Plasma lipid phosphorus b
0.0 0.5 5.0
6.80 5.41 5.19 5.77
7.81 5,62 5.10 6.17
10.15 5.57 5.25 7.04
8.30 5.53 5.18
Plasma cholesterol c
0.0 0.5 5.0
119 96 88 100
138 97 93 110
181 103 94 128
147 99 91
"Copper effect, 0.0001. Nickel--copper,0.005~ bCopper effect, 0.0001. Nickel effect, 0.01. Nickelcopper, 0.005. CCopper effect, 0.0001. Nickel effect, 0,003. Nickelcopper, 0.01. fected by an interaction between nickel and copper (Table 3). That interaction was apparently the result of nickel supplementation slightly elevating the total lipid content in liver from severely copper-deficient, but not in copper-supplemented, rats. Similar to plasma, liver lipid phosphorus was elevated by copper deprivation. High levels of nickel supplementation apparently exacerbated slightly that sign of copper deprivation in severely copper-deficient rats. On the other hand, in coppersupplemented rats, nickel supplementation slightly depressed the lipid phosphorus content in liver; thus, the interaction between nickel and copper was significant. In contrast to the plasma findings, liver cholesterol was depressed by severe copper deficiency. This depression was not exacerbated, but slightly alleviated, by nickel supplementation. On the other hand, liver cholesterol levels were apparently higher in nickel-deprived than nickel-supplemented rats fed 0.25 I~g of Cu. Because the effect of dietary nickel was different at different levels of copper supplementation, the interaction between copper and nickel was significant. Tables 4 and 5 show that copper deprivation depressed copper and zinc, and elevated iron (Expt. 2) and manganese contents in the liver. The liver contents of copper (Expt. 2), manganese and zinc were higher, and, iron lower in rats fed 15 ~g
NI, Cu, FE INTERACTIONS
131
TABLE 2A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Plasma Lipids, Phospholipids, and Cholesterol (Experiment 2) Plasmab Treatmentf ixg/g Cu Fe Ni 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
Total lipids, c mg/mL
Lipid phosphorus, nag/100 mL
Cholesterol, mg/100 mL
4.76 3.92 4.07 3.52 3.64 3.54 3.56 3.17 3.52 4.00 4.37 4.75 3.54 3.98 4.17 3.37 3.19 2.79
7.15 6.36 6.43 5.36 5.99 6.00 5.88 5.26 5.99 6.32 7.17 7.39 5.73 6.24 6.54 5.44 5.27 4.50
128 106 119 94 99 104 105 87 94 119 122 130 99 107 117 101 95 78
aAmounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bFresh weight basis. tEach value represents the mean of 6 rats.
than 45 p~g of iron/g of diet. Although the effects of dietary iron and copper were statistically similar in both experiments, there were differences in the extent of the effects. In severely copper-deficient rats fed 15 ~g of iron/g of diet, the liver content of copper was apparently lower in Expt. 2 than in Expt. 1. Also, in copperadequate rats fed 15 txg of iron/g of diet, the liver content of iron was lower and copper was higher, in Expt. 2 than in Expt. 1. The apparent difference in copper and iron status in those groups between the two experiments probably explains the inconsistency of the effects of various interactions between the dietary variables on the copper and iron contents in the liver. In Expt. 2, dietary nickel significantly affected the copper content of liver, with most of the effect occurring in the copperadequate rats fed 15 ~xg of iron/g of diet (Table 5). In that group, nickel supplementation partially prevented the elevation of the copper caused by iron deprivation. The findings with the copper-adequate group fed 15 ~g of iron/g of diet explain the
132
NIELSEN, ZIMMERMAN, AND SHULER
TABLE 2B Summary of Significant Effects of Nickel: P Values Obtained by Analysis of Variance Cu
0
0.25
12
Plasma t o m l l ~ i d s ~
Ni Cu 0.0 0.25 12.0
4.29
3.73
3.26
0
5
50
Plasma choWsteror
124 85 103 108
114 103 91 102
125 110 85 106
120 103 93
Plasma lipid phosphorus, r Fe = 15
0.0 0.25 12.0 .~
7.15 5.36 5.88 6.13
6.36 5.99 5.26 5.87
6.43 6.00 5.99 6.12
6.66 5.87 5.71
Plasma lipid phosphorus, c Fe = 45
0.0 0.25 12.0 Cu Fe 15 45 ,i
6.32 5.73 5.44 5.85
7.17 6.24 5.27 6.23
7.39 6.54 4.50 5.99
0
0.25
12
6.91 6.17 5.05
Plasma lipid phosphorus a
6.66 6.91 6.78
5.78 6.17 5.98
5.71 5.05 5.39
6.04 6.03
aCopper effect, 0.0001. bCopper effect, 0.0001. Nickel--copper, 0.04. CNickel-copper-iron, 0.03. aCopper effect, 0.0001. Copper-iron, 0.01. significant effect on liver copper content by interactions between nickel and copper, and nickel, copper, and iron. In Expt. 1, the feeding of a supplemental 15 p~g of iron/g of diet apparently did not produce an iron deficiency severe enough to elevate copper in the liver of copper-adequate rats. Thus, except for copper, no other dietary variable, nor interaction; affected the copper content of the liver. In Expt. 2, the effects of dietary nickel, and an interaction between nickel and copper on the iron content in liver were significant because nickel deprivation depressed the iron content in liver, and this effect was more marked in the copper-deficient rats. The basis for the significant interaction between nickel and copper in Expt. 1
NI, Co, FE INTERACTIONS
133
TABLE 3A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Liver Lipids, Phospholipids, and Cholesterol (Experiment 2) Liverb Treatment'a Ixg/g Cu Fe Ni 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
Total lipids, c %
Lipid phosphorus, mg/g
Cholesterol, mg/g
6.97 7.14 7.58 7.53 7.41 7.37 7.32 6.98 6.96 6.92 6.94 7.39 7.18 6.81 6.71 7.04 6.87 7.14
1.323 1.312 1.373 1.278 1.286 !.248 1.313 1.170 1.197 1.258 1.273 1.358 1.245 1.227 1.187 1.215 1.183 1.187
2.50 2.83 2.71 3.23 3.13 2.80 3.27 3.06 3.07 2.72 2.76 3.03 3.16 2.98 2.92 3.03 3.13 3.28
aAmounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bFresh weight basis. tEach value represents the mean of 6 rats. was similar to that for Expt. 2. In Expt. 1, nickel supplementation elevated the manganese content in liver, and this effect was more marked in copper-deficient rats. That finding explains the significant effect of dietary nickel, and an interaction between nickel and copper. Neither dietary nickel, nor any interaction between dietary variables, affected the zinc content in liver. In Expt. 2, the plasma content of copper, iron, nickel, and zinc was determined. Nickel deprivation depressed the nickel content of plasma (Table 6). Copper deficiency also depressed the nickel content of plasma, and this effect was most marked in rats fed 50 Ixg of nickel/g of diet. Thus, with plasma nickel, both the dietary copper effect, and the interaction between copper and nickel, were significant. Copper deprivation depressed the contents of copper and iron in plasma. Because nickel deprivation depressed the copper level in plasma levels only in rats fed adequate copper, the interaction between nickel and copper was significant. Plasma iron was also significantly affected by an interaction between nickel and
134
NIELSEN, ZIMMERMAN,AND SHULER TABLE 3B Summary of Significant Effects in Liver: P Values Obtained by Analysis of Variance Fe 15
Ni
45
Cu
0
5
50
7.04 7.11 6.93 7.03
7.50 7.04 7.04 7.18
7.14 7.16 7.05
1.292 1.256 1.777 1.242
1.367 1.218 1.193 1.255
1.313 1.244 1.212
2.79 3.05 3.09 2.98
2.84 2.86 3.16 2.96
2.74 3.03 3.14
Liver total lipids a
7.24
6.98
0.0 0.25 12.0 k
6.94 7.35 7.18 7.15
Liver lipid phosphorus b
1.278
1.233
0.0 0.25 12.0 k
1.290 1.262 1.264 1.272
Liver cholesterol C
0.0 0.25 12.0
2.61 3.20 3.15 2.98
~lron effect, 0.01. NickeI--copper,0.05. blron effect, 0.03. Copper effect, 0.0001. Nickel-Copper, 0.05. CCopper effect, 0.0001. Nickel-copper, 0.004. copper, as reflected by the finding that nickel deprivation depressed the iron content of plasma in copper-adequate rats, but not in rats fed 0.25 p~g of copper/g of diet. The zinc content was depressed in plasma of severely copper-deficient rats fed the low level of iron. Nickel deprivation depressed the zinc content in plasma in severely copper-deficient rats fed 15 Ixg, but not in severely copper-deficient rats fed 45 Ixg, of iron/g of diet. This explains the significant interaction between nickel, iron, and copper.
Discussion In a previous report (1), we showed that the weight gain, hematocrit, hemoglobin, plasma alkaline phosphatase activity, and organ weight findings were similar for these two experiments, except for when the diet contained no supplemental copper and 15 ~g of iron/g. We suggested that the rats fed that diet were more copper deficient in Expt. 2 than in Expt. 1. The liver copper and iron findings here confirm that suggestion. In copper deficiency, the liver content of copper is depressed, and iron is elevated. Those signs of copper deficiency were apparently more severe in
NI, Cu, FE INTERACTIONS
135
TABLE 4A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Liver Content of Copper, Iron, Manganese, and Zinc (Experiment 1) Liver, b Ixg/g
Treatment, a Ixg/g Cu
Fe
Ni
Copper~
Iron
Manganese
Zinc
0.0 0.0 0.0 0.5 0.5 0.5 5.0 5.0 5.0 0.0 0.0 0.0 0.5 0.5 0.5 5.0 5.0 5.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
2.043 2.25 2.01 7.08 6.65 6.68 13.69 13.67 13.25 1.78 2.05 2.06 6.78 6.98 6.50 13.62 12.93 13.23
300 540 592 514 393 454 440 457 273 533 809 506 603 700 709 604 504 542
7.81 9.99 8.71 7.07 7.88 7.04 7.11 7.74 7.85 7.25 9.30 8.02 6.59 6.40 6.46 6.47 6.43 6.57
66 74 72 74 77 75 72 74 76 66 67 72 71 73 72 73 74 72
aAmounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bDry weight basis. CEach value represents the mean of 6 rats. Expt. 2 than in Expt. 1 when the rats were fed no supplemental copper and inadequate iron. The basal diet in Expt. 2 contained less copper (0.39 txg/g) than in Expt. 1 (0.47 txg/g). Also, the diets supplemented with 15 ixg of iron in Expt. 2 contained slightly less iron by analysis (15.7 ~g/g) than in Expt. 1 (18.3 ~xg/g). That dietary iron difference probably was significant because in the rats fed adequate copper and 15 p~g of iron/g of diet, the liver iron content was apparently lower in Expt. 2 than in Expt. 1. Because of the apparent differences in iron and copper status of rats, it was not surprising to see some statistical differences between the two experiments, especially since the findings clearly demonstrate a complex relationship between nickel, iron and copper. The changes in the parameters described in the results and following discussion were assumed to be caused by specific changes in dietary metal content, and not by inappetence, for the following reasons. Growth was not markedly affected by any dietary treatment except severe copper deficiency (1). Also, copper apparently specifically affects lipid metabolism (11) and the trace element content in tissue
136
NIELSEN, ZIMMERMAN, AND SHULER
TABLE 4B Summary of Significant Effects in Liver: P Values Obtained by Analysis of Variance Fe Cu
15
Ni 45
.~
Cu
0
Liver zinc a 0.0
0.5 5.0
71 75 74 73
68 72 73 71
5
50
Liver copper b
69 74 74
0.0 0.5 5.0 k
1.92 6.93 13.65 7.66
2.15 6.80 13.30 7.43
2.03 6.59 13.24 7.28
2.04 6.77 13.26
406 559 522 489
674 547 478 569 15e
549 581 395 512
547 562 466
540 393 457 463
592 454 273 440
477 454 390
809 700 504 681
506 709 542 588
621 671 553
9.68 7.14 7.08 7.92
8.37 6.75 7.27 7.47
8.53 6.91 7.04
L i v e r iron ~ ee
15
45
440
617
0.0 0.5 5.0
Fe =
0.0 0.5 5.0
300 514 440 418 Fe =
0.0 0.5 5.0 .~
533 603 604 583
45 e
Liver manganese f
7.91
7.02
0.0 0.5 5.0
7.56 6.83 6.79 7.05
~Copper effect, 0.006. Iron effect, 0.05. bCopper effect, 0.0001. qron effect, 0.0001. aNickel-copper, 0.02. eNickel-copper-iron, 0.03. Ylron effect, 0.0001. Copper effect, 0.0001. Nickel effect, 0.006. Nickel-copper, 0.03.
NI, Cu, FE INTERACTIONS
137
TABLE 5A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Liver Content of Copper, Iron, Manganese, and Zinc (Experiment 2) Liver, b Ixg/g
Treatment a Ixg/g Cu
Fe
Ni
Copperr
Iron
Manganese
Zinc
0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
1.34 1.48 1.49 2.83 3.17 2.62 26.49 21.52 18.60 2.04 2.13 1.90 4.51 3.76 3.85 15.14 14.96 14.74
538 571 633 406 457 558 128 124 138 689 862 971 919 1194 968 600 618 564
9.79 8.12 10.49 7.78 7.87 6.94 7.93 7.15 7.24 8.69 9.03 8.60 6.32 6.51 6.68 5.82 5.67 5.65
75 83 79 82 82 79 89 82 83 74 74 77 79 75 78 81 81 84
"Amounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bDry weight basis. tEach value represents the mean of 6 rats.
(12). Food consumption was not markedly affected by any treatment except when the high level of nickel was fed to severely copper-deficient rats. Those rats, as a group, consumed less feed the last week in both experiments. This difference in food consumption was probably caused by those rats that had begun to lose weight. Fortunately, those rats gave values, for the various parameters examined, similar to those that did not lose weight. Of course, those rats that died were not used for any analysis. Like the previous findings (1), the plasma lipid fractions findings in Expt. 1 suggest an antagonistic relationship between nickel and copper. The signs of copper deficiency of elevated total lipids, lipid phosphorus, and cholesterol in plasma were made more severe by nickel supplementation. On the other hand, in Expt. 2, where the copper plus iron deprivation was very severe, 5 or 50/~g of nickeI/g of diet did not consistently antagonize copper deficiency signs. Nickel supplementation of rats fed diets containing 0 copper and 15
138
NIELSEN, ZIMMERMAN, AND SHULER
p~g of iron/g did not make more severe the copper deficiency signs of elevated total lipids, lipid phosphorus, and cholesterol in plasma. However, in these rats, the elevated total lipids and lipid phosphorus in liver were exacerbated slightly by nickel supplementation. Also, if the copper, or copper plus iron, deprivation was mild, 5 or 50 ~xg of nickel/g of diet did not antagonize copper deficiency-induced lipid changes in liver and plasma to any great extent. This was shown by the findings from rats fed the dietary supplement of 0.5 Ixg of copper. In Expt. 2, rats fed 45 ~g of iron/g of diet and no supplemental copper apparently were the only rats in the proper copper deficiency state to show a consistent antagonism between nickel and copper. In these rats, nickel supplementation, especially the 50 ixg/g of diet supplement, seemed to exacerbate the copper deficiency-induced elevation of plasma total lipids, lipid phosphorus, and cholesterol, and liver total lipids and lipid phosphorus. This effect of nickel supplementation is easily seen in the summary of significant effects on plasma lipid phosphorus shown in Table 2. TABLE 5B Summary of Significant Effects in Liver: P Values Obtained by Analysis of Variance Liver copper
Fea Cu 0.0 0.25 12.0
Cu 0.0 0.25 12.0
15
45
Nib k
Cu 0.0 0.25 12.0
1.44 2.03 1.72 2.87 4.04 3.46 22.20 14.94 18.57 8.84 7.10 Ni, c Fe = 15 0
5
50
1.34
1.48
1.49
1.44
2.83 26.49 10.22
3.17 21.52 8.72
2.62 18.60 7.57
2.87 22.20
0
5
50
.t
1.69 1.80 1.68 1.72 3.67 3.47 3.23 3.46 20.81 28.24 16.67 18.57 8.72 7.83 7.35 Ni, c Fe = 45
Cu
0
5
0.0 0.25 12.0
2.04 4.51 15.14 7.23
2.13 3.76 14.96 6.95
50 1.90 2.03 3.85 4.04 14.74 14.94 7.12
Liver iron
Ni e
Fea Cu 0.0 0.25 12.0
15
45
581 474 130 395
842 1023 594 811
704 732 362
Cu
0
5
50
0.0 0.25 12.0
607 639 364 532
716 792 371 622
787 763 351 629
704 732 362
(continued)
N1, Cu, FE INTERACTIONS
139 Table 5B (continued) Liver manganese Cue
Fd
Cu 0.0 0.25 12.0
t5
45
0.0
0.25
8.15
6.94 Ni, h Fe = 15
9.15
7.03 6.58 Ni, h Fe = 45
0
5
50
k
Cu
0
5
50
9.79 7.78 7.93 8.50
8.12 7.87 7.15 7.71
10.49 6.94 7.24 8.22
9.47 7.53 7.44
0.0 0.25 12.0
8.69 6.32 5.82 6.84
9.03 6.51 5.67 7.10
8.60 6.68 5.65 6.88
12.0
8.79 6.50 5.71
Liver Zinc Fei Cu
15
45
0.0 0.25 12.0
79 81 85 82
75 77 82 78
77 79 83
~Copper effect, 0.0001. Iron effect, 0.0001. Copper-iron, 0.0001. bCopper effect, 0.0001. Nickel effect, 0.03. Nickel-copper, 0.03. CCopper-nickel-iron, 0.03. dCopper effect, 0.0001. Iron effect, 0.0001. Copper-iron, 0.000t. ~Copper effect, 0.0001. Nickel effect, 0.004. Nickel-copper, 0.04. flron effect, 0.0001. gCopper effect, 0.0001. hNickel-copper-iron, 0.03. /Copper effect, 0.003. Iron effect, 0.02.
Apparently, demonstration of an antagonism between copper and nickel depends upon the dietary levels of these elements used. The antagonism can be accentuated by altering dietary iron. Thus, in factorially arranged experiments, by carefully choosing the dietary levels of the three elements, it should be possible to show a nickel-copper interaction, a nickel-copper-iron interaction, or no interaction. Some indication of the nature of the antagonism between nickel and copper can be obtained from the liver and plasma trace element findings. Nickel supplementation did not depress the level of copper in plasma and liver of copper-deficient rats. This indicates that nickel did not exacerbate copper deficiency signs by interfering with the delivery of copper to the biological fluid or tissue in which the lipid changes occurred. However, nickel and copper can have similar physical and chemical properties (13) so that isomorphous replacement of copper by nickel may occur at various functional sites. Thus, a possible explanation for the antagonism is
140
NIELSEN, ZIMMERMAN, AND SHULER
TABLE 6A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Plasma Content of Copper, Iron, Nickel, and Zinc (Experiment 2) Plasmab Cu 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0
Treatment, ~ Ixg/g Fe Ni 15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
Copper, C Ixg/mL 0.05 0.06 0.04 0.07 0.06 0.06 1.35 1.46 1.50 0.06 0.05 0.03 0.07 0.07 0.06 1.24 1.50 1.50
Iron, Nickel, I x g / m L ng/mL 0.58 0.64 0.99 0.83 0.75 0.38 1.68 2.49 3.30 1.27 1.92 1.45 3.10 2.33 2.45 4.17 4.58 6.96
3.6 5.6 10.4 6.5 10.0 31.7 4.4 8.7 67.3 5.5 7.2 11.3 7.6 10.2 44.0 4.0 7.5 51.2
Zinc, ixg/mL 0.81 1.03 1.07 1.53 1.32 1.26 1.27 1.45 1.25 1.31 1.33 1.10 1.39 1.28 1.22 1.15 1.21 1.16
~Amounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bFresh weight basis. tEach value represents the mean of 6 rats.
that upon replacing copper, nickel cannot perform, or less efficiently performs, the functions of copper, resulting in less copper function at various physiological sites and a more severe copper deficiency. On the other hand, under some conditions, nickel may spare copper for some vital functions, or be able to perform some copper functions efficiently enough to alleviate certain copper deficiency signs. Spears et al. (14) found that nickel supplementation alleviated the copper deficiency signs of anemia and depressed growth in rats. The basal diet used by Spears et al. (14) contained, per gram, 0.1 Ixg of nickel, 0.95 ~g of Cu, and adequate iron. A nickel supplement of 20 I~g/g of diet elevated hematocrits in the copper-deficient male rats from 38.1 to 41.5% and 28-day weight gains from 141.1 to 160.9 g. Spears et al. (14)suggested that nickel may substitute for copper at certain biological sites, thus preferentially sparing copper for vital functions. The mechanism through which iron influenced the interaction between nickel and copper is unclear. A possible explanation could be that the influence was the
NI, Cu, FE INTERACTIONS
14l
TABLE 6B Summary of Significant Effects in Plasma: P Values Obtained by Analysis of Variance Plasma copper
Plasma nickel
Ni a
Ni b
Cu
0
5
50
.~
Cu
0
5
50
Yc
0.0 0.25 12.0 .~
0.05 0.07 1.30 0.47
0.05 0.06 1.48 0.54
0.03 0.06 1.50 0.52
0.05 0.07 1.42
0.0 0.25 12.0 k
4.5 7.1 4.2 5.3
6.4 10.1 8. I 8.2
10.9 37.8 60.0 38.8
6.8 17.2 23.5
Plasma iron Ni c Cu 0.0 0.25 12.0
Fe d
0
5
50
.~
Cu
15
45
0.93 1.97 2.93 1.94
1.28 1.54 3.54 2.12
1.22 1.32 4.96 2.54
1.14 1.62 3.77
0.0 0.25 12.0 Yc
0.72 0.65 2.49 1.30
1.55 2.64 5.13 3.11
1.14 1.62 3.77
Plasma zinc Ni t Cu 0.0 0.25 12.0 .~
Cu 0.0 0.25 12.0 k
0 1,06 1.46 1.21 1.24 Ni g,
5
Fd 50
1.18 1.08 1.30 1.24 1.33 1.20 1.27 1.18 Fe = 15
.~
Cu
1.11 1.25 1.34
0.0 0.25 12.0 .~
15
45
0.96 1,27 1.37 1.30 1.33 1.17 1.22 1.25 Ni g,Fe = 45
.~ 1.11 1.25 1.34
0
5
50
~
Cu
0
5
50
.t
0.81 1.53 1.27 1.20
1.03 1.32 1.45 1.27
1.07 !.26 1.25 1.20
0.96 1.37 1.33
0.0 0.25 12.0 Yc
1.31 1.39 1.15 1.28
1.33 1.28 1.21 1.27
1.10 1.22 1.16 1.16
1.27 1.30 1.17
"Copper effect, 0.0001. Nickel effect, 0.003. Nickel-copper, 0.0001. bCopper effect, 0.0001. Nickel effect, 0.0001. Nickel--copper, 0.0001. CCopper effect, 0.0001. Nickel effect, 0.02. Nickel-copper, 0.0001. dCopper effect, 0.0001. Iron effect, 0.0001. Copper-iron, 0.0003. eCopper effect, 0.0001. Nickel-copper, 0.02. fCopper-iron, 0.0001. gNickel-copper-iron, 0.04.
142
NIELSEN, ZIMMERMAN, AND SHULER
result of iron deprivation affecting copper metabolism. In Expt. 2, the copper levels in liver tended to be lower in iron-inadequate than iron-adequate rats fed copper-deficient diets. On the other hand, when dietary copper was adequate, iron deprivation significantly elevated the level of copper in the liver. Although nickel, or iron, deprivation did not markedly alter the levels of copper in plasma and liver, the converse was found. Copper deprivation depressed the level of iron and nickel in plasma and elevated the level of iron in liver. Apparently, the lack of copper prevents the efficient utilization of iron, and the iron not utilized is stored in liver (12). Whether this effect of copper is true for nickel is not discernible from the data presented here. Nonetheless, because copper does affect nickel levels in plasma, dietary copper probably can affect the appearance and geverity of the signs of nickel deprivation. We have suggested elsewhere (5) that the iron status of experimental animals must be controlled before data about nickel nutriture and metabolism can be compared among studies. Perhaps the same can be said about the copper status. The findings clearly demonstrate that the optimal requirements for all trace elements, including those present in very small amounts and recently recognized as essential, must be determined if trace element imbalances and undesirable interactions, which impair health, are to be prevented. In this study, the finding that elevated dietary nickel can exacerbate copper deficiency may have nutritional implications because of the recent concern over the possible inadequacy of copper in the diet (15).
Summary In rats, a complex relationship exists between nickel, copper, and iron. When rats were in the appropriate copper deficiency state (neither very severe or mild), copper deficiency signs of elevated levels of total lipids and lipid phosphorus in liver and plasma, and cholesterol in plasma, were made more severe by supplemental dietary nickel. The findings suggested that nickel replaced copper at various functional sites, resulting in less copper function at various physiological sites and a more severe copper deficiency.
Acknowledgments The authors thank Sawaluk Taing for helping with the care of the animals, and Barry Shull for his assistance with the statistical analysis of the data.
References 1. F. H. Nielsen and T. J. Zimmerman, Biol. Trace Element Res. 3, 83 (1981). 2. F. H. Nielsen, T. J. Zimmerman, M. E. Collings, and D. R. Myron, J. Nutr. 109, 1623 (1979).
NI, Cu, FE INTERACTIONS
143
3. F. H. Nielsen, D. R. Myron, S. H. Givand, and D. A. Ollerich, J. Nutr. 105, 1607 (1975). 4. F. H. Nielsen and B. Bailey, Lab. Anita. Sci. 29, 502 (1979). 5. F. H. Nielsen, T. R. Shuler, T. J. Zimmerman, M. E. Collings, and E. O. Uthus, Biol. Trace Element Res. 1, 325 (1979). 6. K. G. D. Allen and L. M. Klevay, Life Sci. 22, 1691 (1978). 7. H. H. Taussky and E. Shorr, J. Biol. Chem. 202, 675 (1953). 8. F. H. Nielsen, D. R. Myron, S. H. Givand, T. J. Zimmerman, and D. A. Ollerich, J. Nutr. 105, 1620 (1975). 9. Analytical Methods for Atomic Absorption Spectrophotometry, Perkin-Elmer Corp., Norwalk, CT, 1976. 10. H. Scheff6, The Analysis of Variance, Wiley, New York, 1959, pp. 90-137. 11. L. M. Klevay, Am. J. Clin. Nutr. 26, 1060 (1973). 12. J. L. Evans and P. A. Abraham, J. Nutr. 103, 196 (t973). 13. P. D. Whanger, Toxicol. Appl. Pharmacol. 25, 323 (1973). 14. J. W. Spears, E. E. Hatfield, and R. M. Forbes, Proc. Soc. Exp. Biol. Med. 156, 140 (1977). 15. L. M. Klevay, Ann. NYAcad. Sci. 355, 140 (1980).
Interactions Among Nickel, Copper, and Iron in Rats Liver and Plasma Content of Lipids and Trace Elements FORREST H . N I E L S E N , * THOMAS J. ZIMMERMAN, A N D TERRENCE R . S H U L E R
United States Department of Agriculture, Agriculture Research Service, Grand Forks Human Nutrition Research Center, and Department of Biochemistry, University of North Dakota, Grand Forks, North Dakota 58202 Accepted February 15, 1982
Abstract In two fully crossed, three-way, two by three by three, factorially arranged experiments, female weanling rats were fed a basal diet supplemented with iron at 15 and 45 Ixg/g, nickel at 0, 5, and 50 Ixg/gand copper at 0, 0.5, and 5 Ixg/g(Expt. 1) or 0, 0.25, and 12 ixg/g (Expt. 2). Expt. 1 was terminated at 11 weeks, and Expt. 2 at 8 weeks because, at those times, some rats fed no supplemental copper and the high level of nickel began to lose weight, or die from heart rupture. The experiments showed that nickel interacted with copper and this interaction was influenced by dietary iron. If copper deficiency was neither very severe or mild, copper deficiency signs of elevated levels of total lipids and lipid phosphorus in liver and plasma, and cholesterol in plasma, were made more severe by supplemental dietary nickel. Rats in which nickel supplementation exacerbated copper deficiency did not exhibit a depressed level of copper in liver and plasma. Also, although iron deprivation enhanced the interaction between nickel and copper, iron deprivation did not significantly depress the level of copper in liver and plasma. The findings confirmed that, in rats, a complex relationship exists between nickel, copper, and iron, thus indicating that both the iron and copper status of experimental animals must be controlled before data about nickel nutriture and metabolism can be compared among studies. 9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163~-984/82/6900-0125 $03.80
125
126
NIELSEN, ZIMMERMAN, AND SHULER
Index Entries: Nickel, interaction with copper and iron; copper, interaction with nickel and iron; iron, interaction with nickel and copper; nickel-copper-iron interactions; liver, lipids and trace elements in; plasma, lipids and trace elements in; lipids, effect of trace elements on liver and plasma contents of.
Introduction In a previous report, we demonstrated a complex relationship between nickel, copper, and iron (1). Nickel interacted with copper and this interaction was influenced by dietary iron. Signs of copper deficiency were made more severe by nickel supplementation, provided copper deprivation was neither very severe nor mild. Iron deprivation accentuated the antagonism between nickel and copper. Signs of copper deficiency made more severe by nickel supplementation were depressed weight gain, hematocrit, hemoglobin and plasma alkaline phosphatase activity, and elevated heart wt/body wt, kidney wt/body wt, and liver wt/body wt ratios. This report is an extension of the preceding studies. We were interested in ascertaining whether the complex relationship between copper, nickel, and iron affected, in addition to the above parameters, the plasma and liver content of total lipids, lipid phosphorus, cholesterol, copper, iron, manganese, nickel, and zinc.
Materials and Methods Female weanling Sprague-Dawley rats (Gibco Animal Resources Laboratory, Madison, WI)* were weighed individually upon arrival and housed three per allplastic cage measuring 50 • 24 • 16 cm and located inside a laminar flow rack (Lab Products, Carfield, NJ). The rats were assigned to groups of six in a fully crossed, three-factor, two by three by three experiment, using a completely randomized factorial arrangement of treatments. The levels of copper, iron, and nickel supplemented to the basal diet were the variables. In Expt. 1, the basal diet was supplemented with copper at 0, 0.5, and 5 p~g/g; iron, 15 and 45 ~xg/g; and nickel, 0, 5, and 50 Ixg/g. In Expt. 2, the basal diet was supplemented with copper at 0, 0.25, and 12 Ixg/g; iron, 15 and 45 p,g/g; and nickel, 0, 5, and 50 fxg/g. Nickel was supplemented as NiC12"3H20 (Ultrapure grade, Alfa Inorganics, Beverly, MA). Copper was CuSO4"5H20 (Puratronic grade, Alfa Inorganics, Beverly, MA). Iron was a mixture of 60% Fe2(SO4)3"nH20 and 40% FeSO4-nH20 that was prepared from iron sponge (Puratronic grade, Alfa Inorganics, Beverly, MA) and sulfuric acid (Ultrex grade, J. T. Baker Chemical Co., Phillipsburg, NJ). *Mention of a trademark or proprietaryproduct does not constitute a guarantee or warranty of the product by the US Departmentof Agricultureand does not imply its approvalto the exclusion of other products that may also be suitable.
NI, Cu, FE INTERACTIONS
127
The rats had access to deionized water (Super Q System, Millipore Corp., Bedford, MA) in plastic cups. Fresh food in plastic cups was provided each day. Plastic equipment and cleaning procedures were described (2-4). Absorbent paper under the cages caught droppings and was changed every other day. Room temperature was maintained at 23~ Room lighting was controlled automatically to provide 12 h of light and 12 h of darkness. Animals were weighed weekly. The diets were mixed 3 days before the start of each experiment and about biweekly, thereafter. They were stored at - 16~ in tightly capped plastic containers. The air-dried basal diets with copper omitted (5) contained, per gram, about 16 ng of nickel, 2.3 ~g of iron, and 0.47 p~gof copper in Expt. 1; and 20 ng of nickel, 1.3 Ixg of iron, and 0.39 p~g of copper in Expt. 2 as determined by atomic absorption spectrometric methods (5). The rats were fed their respective diets for eleven weeks in Expt. 1 and 8 weeks in Expt. 2. The experiments were terminated at that time because some rats fed no supplemental copper and 50 p,g of nickel/g of diet began to lose weight and die from heart rupture. The rats were weighed, then decapitated subsequent to ether anesthesia and cardiac exsanguination with a heparin-coated needle and syringe. The liver was removed, blotted dry, and stored at - 16~ in plastic bags for later analysis. The plasma was separated from the heart blood by centrifugation, and stored at -16~ in plastic tubes for later analysis. Lipids were extracted from plasma not used for the trace element analyses by the method of Allen and Klevay (6). The plasma content of lipid phosphorus was determined by the method of Taussky and Shorr (7), while total lipids were measured by gravimetric analysis. Total lipids and lipid phosphorus in 1-2 g portions of liver were determined by published methods (8). Cholesterol in the dried liver lipid extracts reconstituted in 50 p,L of isopropyl alcohol, and plasma cholesterol, were each determined by enzymatic assay (Cholesterol Reagent Set, Boehringer Mannheim Biochemical, Indianapolis, IN). In preparation for trace element analysis, 1 mL of plasma was transferred to a 12 x 100 mm borosilicate glass culture tube ("Kimax," Kimble Division, OwensIllinois, Toledo, OH) to which 0.6 mL of nitric acid (double distilled in Vycor, G. Frederick Smith, Columbus, OH) was added. The tubes were tightly capped with Teflon-lined screw caps and placed into an 80~ water bath for 16 h. After digestion, the samples were stored briefly in 12 x 75 mm polyethylene tubes until analysis. Duplicate tubes of a control serum (Wellcontrol| One BCOL Quality Control Serum, Burroughs Wellcome Co., Greenville, NY), nickel standard (50 p,g/L), and blanks were subjected to the same procedures used for the plasma samples. The portions of the livers not used for lipid analyses were freeze-dried and prepared for trace element analysis by the following methods. The livers were pulverized in polyethylene bags. In Expt. 1, 1 g portions of the pulverized, freeze-dried liver were dry-ashed by a described method (5). Samples were quantitatively transferred to 5 mL volumetric flasks and brought to volume with deionized water
128
NIELSEN, ZIMMERMAN, AND SHULER
(Super Q System). In Expt. 2, 1 g portions of the freeze-dried liver were placed into a 50 mL Pyrex edenmeyer flask to which 10 mL of concentrated, doubledistilled in Vycor, nitric acid was added. The flasks were covered and allowed to digest overnight at room temperature, then placed at low heat on a hot plate until dry. Next, 5 mL of Concentrated, double-distilled in Vycor, nitric acid was added to the flasks and the samples were placed at low heat on a hot plate until nearly dry. The last step was repeated, then, to the samples, 5 mL of concentrated, doubledistilled in Vycor, nitric acid and 10 mL of 30% H202 (Baker Analyzed, J. T. Baker Co., Phillipsburg, NJ) were carefully added so that foaming over did not occur. The samples were placed at low heat on a hot plate until 1-2 mL remained, cooled, and diluted to 5 mL with deionized water (Super Q System). The copper, iron, manganese, and zinc in plasma and liver were determined by atomic absorption spectrophotometry (9). A described method (5) was modified in the following manner for the plasma nickel determinations. A 50-1xL sample was mixed with 50 I~L of concentrated ammonium hydroxide (Instra-Analyzed, J. T. Baker Co., Phillipsburg, NJ) inside the pyrolytically coated graphite tube. The lamp current was 20 mA and the recorder range was 10 mV full scale. The electrothermal atomizer was programmed for the following cycle: drying, ramped for 30 s from 25 to 125~ and maintained for 80 s; ashing, ramped for 80 s from 125 to 1000~ and maintained for 10 s; atomization at 2650~ for 8 s. The system was purged with argon at a flow rate of 25 cc/min. The reliability of the analytical procedure was ascertained by using orchard leaves (SRM-1571, National Bureau of Standards, Washington, D.C.) certified to contain 1.3 --- 0.2 I~g of nickel/g. The value we found, 1.22 - 0.16 txg of nickel/g, compared favorably with the certified value. Data were treated by three-way analysis of variance (10).
Results The first sign of an interaction between nickel and copper occurred during the last week of each experiment. In Expt. 1, three of 12 rats fed the diet containing no additional copper began to lose weight when the diet contained 50 ~xg of nickel/g; two in the group fed 15 I~g of iron/g and one in the group fed 45 I~g of iron/g of diet. In Expt. 2, six of 12 rats, three in each group described above, began to lose weight and two died (one from each group) from heart rupture before the experiment was terminated. No deaths or weight losses were seen in severely copper-deficient rats fed the dietary nickel supplements of 0 and 5 I~g/g. The effects of copper, iron, nickel, and their interactions on weight, hematocrit, hemoglobin, plasma alkaline phosphatase activity, and heart, kidney, liver, and spleen size were described (1). It was found that in Expt. 2 the low iron diet apparently depressed hemoglobin levels more markedly than in Expt. 1, especially when the diet was low in copper. Thus, some of the interaction effects were slightly different in the two experiments. Those differences continued on to the parameters examined here.
NI, Cu, FE INTERACTIONS
129
Tables 1 and 2 show that copper deprivation elevated total lipids, lipid phosphorus and cholesterol in plasma. In Expt. 1, nickel supplementation exacerbated those apparent signs of copper deficiency. Thus, for all three parameters in Expt. 1, the interaction between nickel and copper was significant. In Expt. 2, where the severely copper-deficient rats fed inadequate iron were very anemic, nickel supplementation did not further elevate the levels of total lipids, lipid phosphorus, or cholesterol in plasma. Only plasma cholesterol was affected by an interaction between nickel and copper, and this effect was less marked than in Expt. 1. However, nickel supplementation apparently elevated plasma lipid phosphorus in copper-deprived rats supplemented with 45, but not 15, I~g of iron/g of diet. Thus, with lipid phosphorus, the nickel-copper-iron interaction was significant. The levels of various lipid fractions in liver was not determined in Expt. 1. However, the plasma findings prompted us to determine those levels in Expt. 2. Unlike plasma, the liver total lipids were unaffected by copper deprivation, but were af-
TABLE 1A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Plasma Lipids, Phospholipids, and Cholesterol (Experiment 1) Plasmab Treatment'a ~g/g Cu Fe Ni 0.0 0.0 0.0 0.5 0.5 0.5 5.0 5.0 5.0 0.0 0.0 0.0 0.5 0.5 0.5 5.0 5.0 5.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
Total lipids, c mg/mL
Lipid phosphorus, mg/lO0 mL
Cholesterol, rag/100 mL
4.58 6.00 7.04 2.63 3.88 3.58 3.58 2.92 3.08 5.05 3.88 8.04 3.46 3.54 2.96 3.42 2.17 2.13
6.79 9.18 9.46 5.40 5.81 5.62 5.38 5.16 5.48 6.82 6.44 10.84 5.43 5.43 5.52 5.00 5.03 5.05
119 163 167 94 100 104 89 96 93 119 113 195 99 94 101 86 90 95
aAmountsof Ni (nickel chloride), Fe (mixtureof 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplementedto the diet. bFresh weight basis. CEachvalue represents the mean of 6 rats.
130
NIELSEN, ZIMMERMAN, AND SHULER
TABLE IB Summary of Significant Effects in Plasma: P Values Obtained by Analysis of Variance Ni
0
5
.~
Plasma total lipids a
all
0.0 0.5 5.0
50
4.80 3.04 3.50 3.75
4.94 3.71 2,54 3.73
7.54 3.27 2.60 4,47
5.79 3.34 2.88
Plasma lipid phosphorus b
0.0 0.5 5.0
6.80 5.41 5.19 5.77
7.81 5,62 5.10 6.17
10.15 5.57 5.25 7.04
8.30 5.53 5.18
Plasma cholesterol c
0.0 0.5 5.0
119 96 88 100
138 97 93 110
181 103 94 128
147 99 91
"Copper effect, 0.0001. Nickel--copper,0.005~ bCopper effect, 0.0001. Nickel effect, 0.01. Nickelcopper, 0.005. CCopper effect, 0.0001. Nickel effect, 0,003. Nickelcopper, 0.01. fected by an interaction between nickel and copper (Table 3). That interaction was apparently the result of nickel supplementation slightly elevating the total lipid content in liver from severely copper-deficient, but not in copper-supplemented, rats. Similar to plasma, liver lipid phosphorus was elevated by copper deprivation. High levels of nickel supplementation apparently exacerbated slightly that sign of copper deprivation in severely copper-deficient rats. On the other hand, in coppersupplemented rats, nickel supplementation slightly depressed the lipid phosphorus content in liver; thus, the interaction between nickel and copper was significant. In contrast to the plasma findings, liver cholesterol was depressed by severe copper deficiency. This depression was not exacerbated, but slightly alleviated, by nickel supplementation. On the other hand, liver cholesterol levels were apparently higher in nickel-deprived than nickel-supplemented rats fed 0.25 I~g of Cu. Because the effect of dietary nickel was different at different levels of copper supplementation, the interaction between copper and nickel was significant. Tables 4 and 5 show that copper deprivation depressed copper and zinc, and elevated iron (Expt. 2) and manganese contents in the liver. The liver contents of copper (Expt. 2), manganese and zinc were higher, and, iron lower in rats fed 15 ~g
NI, Cu, FE INTERACTIONS
131
TABLE 2A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Plasma Lipids, Phospholipids, and Cholesterol (Experiment 2) Plasmab Treatmentf ixg/g Cu Fe Ni 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
Total lipids, c mg/mL
Lipid phosphorus, nag/100 mL
Cholesterol, mg/100 mL
4.76 3.92 4.07 3.52 3.64 3.54 3.56 3.17 3.52 4.00 4.37 4.75 3.54 3.98 4.17 3.37 3.19 2.79
7.15 6.36 6.43 5.36 5.99 6.00 5.88 5.26 5.99 6.32 7.17 7.39 5.73 6.24 6.54 5.44 5.27 4.50
128 106 119 94 99 104 105 87 94 119 122 130 99 107 117 101 95 78
aAmounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bFresh weight basis. tEach value represents the mean of 6 rats.
than 45 p~g of iron/g of diet. Although the effects of dietary iron and copper were statistically similar in both experiments, there were differences in the extent of the effects. In severely copper-deficient rats fed 15 ~g of iron/g of diet, the liver content of copper was apparently lower in Expt. 2 than in Expt. 1. Also, in copperadequate rats fed 15 txg of iron/g of diet, the liver content of iron was lower and copper was higher, in Expt. 2 than in Expt. 1. The apparent difference in copper and iron status in those groups between the two experiments probably explains the inconsistency of the effects of various interactions between the dietary variables on the copper and iron contents in the liver. In Expt. 2, dietary nickel significantly affected the copper content of liver, with most of the effect occurring in the copperadequate rats fed 15 ~xg of iron/g of diet (Table 5). In that group, nickel supplementation partially prevented the elevation of the copper caused by iron deprivation. The findings with the copper-adequate group fed 15 ~g of iron/g of diet explain the
132
NIELSEN, ZIMMERMAN, AND SHULER
TABLE 2B Summary of Significant Effects of Nickel: P Values Obtained by Analysis of Variance Cu
0
0.25
12
Plasma t o m l l ~ i d s ~
Ni Cu 0.0 0.25 12.0
4.29
3.73
3.26
0
5
50
Plasma choWsteror
124 85 103 108
114 103 91 102
125 110 85 106
120 103 93
Plasma lipid phosphorus, r Fe = 15
0.0 0.25 12.0 .~
7.15 5.36 5.88 6.13
6.36 5.99 5.26 5.87
6.43 6.00 5.99 6.12
6.66 5.87 5.71
Plasma lipid phosphorus, c Fe = 45
0.0 0.25 12.0 Cu Fe 15 45 ,i
6.32 5.73 5.44 5.85
7.17 6.24 5.27 6.23
7.39 6.54 4.50 5.99
0
0.25
12
6.91 6.17 5.05
Plasma lipid phosphorus a
6.66 6.91 6.78
5.78 6.17 5.98
5.71 5.05 5.39
6.04 6.03
aCopper effect, 0.0001. bCopper effect, 0.0001. Nickel--copper, 0.04. CNickel-copper-iron, 0.03. aCopper effect, 0.0001. Copper-iron, 0.01. significant effect on liver copper content by interactions between nickel and copper, and nickel, copper, and iron. In Expt. 1, the feeding of a supplemental 15 p~g of iron/g of diet apparently did not produce an iron deficiency severe enough to elevate copper in the liver of copper-adequate rats. Thus, except for copper, no other dietary variable, nor interaction; affected the copper content of the liver. In Expt. 2, the effects of dietary nickel, and an interaction between nickel and copper on the iron content in liver were significant because nickel deprivation depressed the iron content in liver, and this effect was more marked in the copper-deficient rats. The basis for the significant interaction between nickel and copper in Expt. 1
NI, Co, FE INTERACTIONS
133
TABLE 3A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Liver Lipids, Phospholipids, and Cholesterol (Experiment 2) Liverb Treatment'a Ixg/g Cu Fe Ni 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
Total lipids, c %
Lipid phosphorus, mg/g
Cholesterol, mg/g
6.97 7.14 7.58 7.53 7.41 7.37 7.32 6.98 6.96 6.92 6.94 7.39 7.18 6.81 6.71 7.04 6.87 7.14
1.323 1.312 1.373 1.278 1.286 !.248 1.313 1.170 1.197 1.258 1.273 1.358 1.245 1.227 1.187 1.215 1.183 1.187
2.50 2.83 2.71 3.23 3.13 2.80 3.27 3.06 3.07 2.72 2.76 3.03 3.16 2.98 2.92 3.03 3.13 3.28
aAmounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bFresh weight basis. tEach value represents the mean of 6 rats. was similar to that for Expt. 2. In Expt. 1, nickel supplementation elevated the manganese content in liver, and this effect was more marked in copper-deficient rats. That finding explains the significant effect of dietary nickel, and an interaction between nickel and copper. Neither dietary nickel, nor any interaction between dietary variables, affected the zinc content in liver. In Expt. 2, the plasma content of copper, iron, nickel, and zinc was determined. Nickel deprivation depressed the nickel content of plasma (Table 6). Copper deficiency also depressed the nickel content of plasma, and this effect was most marked in rats fed 50 Ixg of nickel/g of diet. Thus, with plasma nickel, both the dietary copper effect, and the interaction between copper and nickel, were significant. Copper deprivation depressed the contents of copper and iron in plasma. Because nickel deprivation depressed the copper level in plasma levels only in rats fed adequate copper, the interaction between nickel and copper was significant. Plasma iron was also significantly affected by an interaction between nickel and
134
NIELSEN, ZIMMERMAN,AND SHULER TABLE 3B Summary of Significant Effects in Liver: P Values Obtained by Analysis of Variance Fe 15
Ni
45
Cu
0
5
50
7.04 7.11 6.93 7.03
7.50 7.04 7.04 7.18
7.14 7.16 7.05
1.292 1.256 1.777 1.242
1.367 1.218 1.193 1.255
1.313 1.244 1.212
2.79 3.05 3.09 2.98
2.84 2.86 3.16 2.96
2.74 3.03 3.14
Liver total lipids a
7.24
6.98
0.0 0.25 12.0 k
6.94 7.35 7.18 7.15
Liver lipid phosphorus b
1.278
1.233
0.0 0.25 12.0 k
1.290 1.262 1.264 1.272
Liver cholesterol C
0.0 0.25 12.0
2.61 3.20 3.15 2.98
~lron effect, 0.01. NickeI--copper,0.05. blron effect, 0.03. Copper effect, 0.0001. Nickel-Copper, 0.05. CCopper effect, 0.0001. Nickel-copper, 0.004. copper, as reflected by the finding that nickel deprivation depressed the iron content of plasma in copper-adequate rats, but not in rats fed 0.25 p~g of copper/g of diet. The zinc content was depressed in plasma of severely copper-deficient rats fed the low level of iron. Nickel deprivation depressed the zinc content in plasma in severely copper-deficient rats fed 15 Ixg, but not in severely copper-deficient rats fed 45 Ixg, of iron/g of diet. This explains the significant interaction between nickel, iron, and copper.
Discussion In a previous report (1), we showed that the weight gain, hematocrit, hemoglobin, plasma alkaline phosphatase activity, and organ weight findings were similar for these two experiments, except for when the diet contained no supplemental copper and 15 ~g of iron/g. We suggested that the rats fed that diet were more copper deficient in Expt. 2 than in Expt. 1. The liver copper and iron findings here confirm that suggestion. In copper deficiency, the liver content of copper is depressed, and iron is elevated. Those signs of copper deficiency were apparently more severe in
NI, Cu, FE INTERACTIONS
135
TABLE 4A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Liver Content of Copper, Iron, Manganese, and Zinc (Experiment 1) Liver, b Ixg/g
Treatment, a Ixg/g Cu
Fe
Ni
Copper~
Iron
Manganese
Zinc
0.0 0.0 0.0 0.5 0.5 0.5 5.0 5.0 5.0 0.0 0.0 0.0 0.5 0.5 0.5 5.0 5.0 5.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
2.043 2.25 2.01 7.08 6.65 6.68 13.69 13.67 13.25 1.78 2.05 2.06 6.78 6.98 6.50 13.62 12.93 13.23
300 540 592 514 393 454 440 457 273 533 809 506 603 700 709 604 504 542
7.81 9.99 8.71 7.07 7.88 7.04 7.11 7.74 7.85 7.25 9.30 8.02 6.59 6.40 6.46 6.47 6.43 6.57
66 74 72 74 77 75 72 74 76 66 67 72 71 73 72 73 74 72
aAmounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bDry weight basis. CEach value represents the mean of 6 rats. Expt. 2 than in Expt. 1 when the rats were fed no supplemental copper and inadequate iron. The basal diet in Expt. 2 contained less copper (0.39 txg/g) than in Expt. 1 (0.47 txg/g). Also, the diets supplemented with 15 ixg of iron in Expt. 2 contained slightly less iron by analysis (15.7 ~g/g) than in Expt. 1 (18.3 ~xg/g). That dietary iron difference probably was significant because in the rats fed adequate copper and 15 p~g of iron/g of diet, the liver iron content was apparently lower in Expt. 2 than in Expt. 1. Because of the apparent differences in iron and copper status of rats, it was not surprising to see some statistical differences between the two experiments, especially since the findings clearly demonstrate a complex relationship between nickel, iron and copper. The changes in the parameters described in the results and following discussion were assumed to be caused by specific changes in dietary metal content, and not by inappetence, for the following reasons. Growth was not markedly affected by any dietary treatment except severe copper deficiency (1). Also, copper apparently specifically affects lipid metabolism (11) and the trace element content in tissue
136
NIELSEN, ZIMMERMAN, AND SHULER
TABLE 4B Summary of Significant Effects in Liver: P Values Obtained by Analysis of Variance Fe Cu
15
Ni 45
.~
Cu
0
Liver zinc a 0.0
0.5 5.0
71 75 74 73
68 72 73 71
5
50
Liver copper b
69 74 74
0.0 0.5 5.0 k
1.92 6.93 13.65 7.66
2.15 6.80 13.30 7.43
2.03 6.59 13.24 7.28
2.04 6.77 13.26
406 559 522 489
674 547 478 569 15e
549 581 395 512
547 562 466
540 393 457 463
592 454 273 440
477 454 390
809 700 504 681
506 709 542 588
621 671 553
9.68 7.14 7.08 7.92
8.37 6.75 7.27 7.47
8.53 6.91 7.04
L i v e r iron ~ ee
15
45
440
617
0.0 0.5 5.0
Fe =
0.0 0.5 5.0
300 514 440 418 Fe =
0.0 0.5 5.0 .~
533 603 604 583
45 e
Liver manganese f
7.91
7.02
0.0 0.5 5.0
7.56 6.83 6.79 7.05
~Copper effect, 0.006. Iron effect, 0.05. bCopper effect, 0.0001. qron effect, 0.0001. aNickel-copper, 0.02. eNickel-copper-iron, 0.03. Ylron effect, 0.0001. Copper effect, 0.0001. Nickel effect, 0.006. Nickel-copper, 0.03.
NI, Cu, FE INTERACTIONS
137
TABLE 5A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Liver Content of Copper, Iron, Manganese, and Zinc (Experiment 2) Liver, b Ixg/g
Treatment a Ixg/g Cu
Fe
Ni
Copperr
Iron
Manganese
Zinc
0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0
15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
1.34 1.48 1.49 2.83 3.17 2.62 26.49 21.52 18.60 2.04 2.13 1.90 4.51 3.76 3.85 15.14 14.96 14.74
538 571 633 406 457 558 128 124 138 689 862 971 919 1194 968 600 618 564
9.79 8.12 10.49 7.78 7.87 6.94 7.93 7.15 7.24 8.69 9.03 8.60 6.32 6.51 6.68 5.82 5.67 5.65
75 83 79 82 82 79 89 82 83 74 74 77 79 75 78 81 81 84
"Amounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bDry weight basis. tEach value represents the mean of 6 rats.
(12). Food consumption was not markedly affected by any treatment except when the high level of nickel was fed to severely copper-deficient rats. Those rats, as a group, consumed less feed the last week in both experiments. This difference in food consumption was probably caused by those rats that had begun to lose weight. Fortunately, those rats gave values, for the various parameters examined, similar to those that did not lose weight. Of course, those rats that died were not used for any analysis. Like the previous findings (1), the plasma lipid fractions findings in Expt. 1 suggest an antagonistic relationship between nickel and copper. The signs of copper deficiency of elevated total lipids, lipid phosphorus, and cholesterol in plasma were made more severe by nickel supplementation. On the other hand, in Expt. 2, where the copper plus iron deprivation was very severe, 5 or 50/~g of nickeI/g of diet did not consistently antagonize copper deficiency signs. Nickel supplementation of rats fed diets containing 0 copper and 15
138
NIELSEN, ZIMMERMAN, AND SHULER
p~g of iron/g did not make more severe the copper deficiency signs of elevated total lipids, lipid phosphorus, and cholesterol in plasma. However, in these rats, the elevated total lipids and lipid phosphorus in liver were exacerbated slightly by nickel supplementation. Also, if the copper, or copper plus iron, deprivation was mild, 5 or 50 ~xg of nickel/g of diet did not antagonize copper deficiency-induced lipid changes in liver and plasma to any great extent. This was shown by the findings from rats fed the dietary supplement of 0.5 Ixg of copper. In Expt. 2, rats fed 45 ~g of iron/g of diet and no supplemental copper apparently were the only rats in the proper copper deficiency state to show a consistent antagonism between nickel and copper. In these rats, nickel supplementation, especially the 50 ixg/g of diet supplement, seemed to exacerbate the copper deficiency-induced elevation of plasma total lipids, lipid phosphorus, and cholesterol, and liver total lipids and lipid phosphorus. This effect of nickel supplementation is easily seen in the summary of significant effects on plasma lipid phosphorus shown in Table 2. TABLE 5B Summary of Significant Effects in Liver: P Values Obtained by Analysis of Variance Liver copper
Fea Cu 0.0 0.25 12.0
Cu 0.0 0.25 12.0
15
45
Nib k
Cu 0.0 0.25 12.0
1.44 2.03 1.72 2.87 4.04 3.46 22.20 14.94 18.57 8.84 7.10 Ni, c Fe = 15 0
5
50
1.34
1.48
1.49
1.44
2.83 26.49 10.22
3.17 21.52 8.72
2.62 18.60 7.57
2.87 22.20
0
5
50
.t
1.69 1.80 1.68 1.72 3.67 3.47 3.23 3.46 20.81 28.24 16.67 18.57 8.72 7.83 7.35 Ni, c Fe = 45
Cu
0
5
0.0 0.25 12.0
2.04 4.51 15.14 7.23
2.13 3.76 14.96 6.95
50 1.90 2.03 3.85 4.04 14.74 14.94 7.12
Liver iron
Ni e
Fea Cu 0.0 0.25 12.0
15
45
581 474 130 395
842 1023 594 811
704 732 362
Cu
0
5
50
0.0 0.25 12.0
607 639 364 532
716 792 371 622
787 763 351 629
704 732 362
(continued)
N1, Cu, FE INTERACTIONS
139 Table 5B (continued) Liver manganese Cue
Fd
Cu 0.0 0.25 12.0
t5
45
0.0
0.25
8.15
6.94 Ni, h Fe = 15
9.15
7.03 6.58 Ni, h Fe = 45
0
5
50
k
Cu
0
5
50
9.79 7.78 7.93 8.50
8.12 7.87 7.15 7.71
10.49 6.94 7.24 8.22
9.47 7.53 7.44
0.0 0.25 12.0
8.69 6.32 5.82 6.84
9.03 6.51 5.67 7.10
8.60 6.68 5.65 6.88
12.0
8.79 6.50 5.71
Liver Zinc Fei Cu
15
45
0.0 0.25 12.0
79 81 85 82
75 77 82 78
77 79 83
~Copper effect, 0.0001. Iron effect, 0.0001. Copper-iron, 0.0001. bCopper effect, 0.0001. Nickel effect, 0.03. Nickel-copper, 0.03. CCopper-nickel-iron, 0.03. dCopper effect, 0.0001. Iron effect, 0.0001. Copper-iron, 0.000t. ~Copper effect, 0.0001. Nickel effect, 0.004. Nickel-copper, 0.04. flron effect, 0.0001. gCopper effect, 0.0001. hNickel-copper-iron, 0.03. /Copper effect, 0.003. Iron effect, 0.02.
Apparently, demonstration of an antagonism between copper and nickel depends upon the dietary levels of these elements used. The antagonism can be accentuated by altering dietary iron. Thus, in factorially arranged experiments, by carefully choosing the dietary levels of the three elements, it should be possible to show a nickel-copper interaction, a nickel-copper-iron interaction, or no interaction. Some indication of the nature of the antagonism between nickel and copper can be obtained from the liver and plasma trace element findings. Nickel supplementation did not depress the level of copper in plasma and liver of copper-deficient rats. This indicates that nickel did not exacerbate copper deficiency signs by interfering with the delivery of copper to the biological fluid or tissue in which the lipid changes occurred. However, nickel and copper can have similar physical and chemical properties (13) so that isomorphous replacement of copper by nickel may occur at various functional sites. Thus, a possible explanation for the antagonism is
140
NIELSEN, ZIMMERMAN, AND SHULER
TABLE 6A Effect in Rats of Copper, Iron, Nickel, and Their Interaction on Plasma Content of Copper, Iron, Nickel, and Zinc (Experiment 2) Plasmab Cu 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0 0.0 0.0 0.0 0.25 0.25 0.25 12.0 12.0 12.0
Treatment, ~ Ixg/g Fe Ni 15 15 15 15 15 15 15 15 15 45 45 45 45 45 45 45 45 45
0 5 50 0 5 50 0 5 50 0 5 50 0 5 50 0 5 50
Copper, C Ixg/mL 0.05 0.06 0.04 0.07 0.06 0.06 1.35 1.46 1.50 0.06 0.05 0.03 0.07 0.07 0.06 1.24 1.50 1.50
Iron, Nickel, I x g / m L ng/mL 0.58 0.64 0.99 0.83 0.75 0.38 1.68 2.49 3.30 1.27 1.92 1.45 3.10 2.33 2.45 4.17 4.58 6.96
3.6 5.6 10.4 6.5 10.0 31.7 4.4 8.7 67.3 5.5 7.2 11.3 7.6 10.2 44.0 4.0 7.5 51.2
Zinc, ixg/mL 0.81 1.03 1.07 1.53 1.32 1.26 1.27 1.45 1.25 1.31 1.33 1.10 1.39 1.28 1.22 1.15 1.21 1.16
~Amounts of Ni (nickel chloride), Fe (mixture of 40% ferrous sulfate and 60% ferric sulfate), and Cu (copper sulfate) supplemented to the diet. bFresh weight basis. tEach value represents the mean of 6 rats.
that upon replacing copper, nickel cannot perform, or less efficiently performs, the functions of copper, resulting in less copper function at various physiological sites and a more severe copper deficiency. On the other hand, under some conditions, nickel may spare copper for some vital functions, or be able to perform some copper functions efficiently enough to alleviate certain copper deficiency signs. Spears et al. (14) found that nickel supplementation alleviated the copper deficiency signs of anemia and depressed growth in rats. The basal diet used by Spears et al. (14) contained, per gram, 0.1 Ixg of nickel, 0.95 ~g of Cu, and adequate iron. A nickel supplement of 20 I~g/g of diet elevated hematocrits in the copper-deficient male rats from 38.1 to 41.5% and 28-day weight gains from 141.1 to 160.9 g. Spears et al. (14)suggested that nickel may substitute for copper at certain biological sites, thus preferentially sparing copper for vital functions. The mechanism through which iron influenced the interaction between nickel and copper is unclear. A possible explanation could be that the influence was the
NI, Cu, FE INTERACTIONS
14l
TABLE 6B Summary of Significant Effects in Plasma: P Values Obtained by Analysis of Variance Plasma copper
Plasma nickel
Ni a
Ni b
Cu
0
5
50
.~
Cu
0
5
50
Yc
0.0 0.25 12.0 .~
0.05 0.07 1.30 0.47
0.05 0.06 1.48 0.54
0.03 0.06 1.50 0.52
0.05 0.07 1.42
0.0 0.25 12.0 k
4.5 7.1 4.2 5.3
6.4 10.1 8. I 8.2
10.9 37.8 60.0 38.8
6.8 17.2 23.5
Plasma iron Ni c Cu 0.0 0.25 12.0
Fe d
0
5
50
.~
Cu
15
45
0.93 1.97 2.93 1.94
1.28 1.54 3.54 2.12
1.22 1.32 4.96 2.54
1.14 1.62 3.77
0.0 0.25 12.0 Yc
0.72 0.65 2.49 1.30
1.55 2.64 5.13 3.11
1.14 1.62 3.77
Plasma zinc Ni t Cu 0.0 0.25 12.0 .~
Cu 0.0 0.25 12.0 k
0 1,06 1.46 1.21 1.24 Ni g,
5
Fd 50
1.18 1.08 1.30 1.24 1.33 1.20 1.27 1.18 Fe = 15
.~
Cu
1.11 1.25 1.34
0.0 0.25 12.0 .~
15
45
0.96 1,27 1.37 1.30 1.33 1.17 1.22 1.25 Ni g,Fe = 45
.~ 1.11 1.25 1.34
0
5
50
~
Cu
0
5
50
.t
0.81 1.53 1.27 1.20
1.03 1.32 1.45 1.27
1.07 !.26 1.25 1.20
0.96 1.37 1.33
0.0 0.25 12.0 Yc
1.31 1.39 1.15 1.28
1.33 1.28 1.21 1.27
1.10 1.22 1.16 1.16
1.27 1.30 1.17
"Copper effect, 0.0001. Nickel effect, 0.003. Nickel-copper, 0.0001. bCopper effect, 0.0001. Nickel effect, 0.0001. Nickel--copper, 0.0001. CCopper effect, 0.0001. Nickel effect, 0.02. Nickel-copper, 0.0001. dCopper effect, 0.0001. Iron effect, 0.0001. Copper-iron, 0.0003. eCopper effect, 0.0001. Nickel-copper, 0.02. fCopper-iron, 0.0001. gNickel-copper-iron, 0.04.
142
NIELSEN, ZIMMERMAN, AND SHULER
result of iron deprivation affecting copper metabolism. In Expt. 2, the copper levels in liver tended to be lower in iron-inadequate than iron-adequate rats fed copper-deficient diets. On the other hand, when dietary copper was adequate, iron deprivation significantly elevated the level of copper in the liver. Although nickel, or iron, deprivation did not markedly alter the levels of copper in plasma and liver, the converse was found. Copper deprivation depressed the level of iron and nickel in plasma and elevated the level of iron in liver. Apparently, the lack of copper prevents the efficient utilization of iron, and the iron not utilized is stored in liver (12). Whether this effect of copper is true for nickel is not discernible from the data presented here. Nonetheless, because copper does affect nickel levels in plasma, dietary copper probably can affect the appearance and geverity of the signs of nickel deprivation. We have suggested elsewhere (5) that the iron status of experimental animals must be controlled before data about nickel nutriture and metabolism can be compared among studies. Perhaps the same can be said about the copper status. The findings clearly demonstrate that the optimal requirements for all trace elements, including those present in very small amounts and recently recognized as essential, must be determined if trace element imbalances and undesirable interactions, which impair health, are to be prevented. In this study, the finding that elevated dietary nickel can exacerbate copper deficiency may have nutritional implications because of the recent concern over the possible inadequacy of copper in the diet (15).
Summary In rats, a complex relationship exists between nickel, copper, and iron. When rats were in the appropriate copper deficiency state (neither very severe or mild), copper deficiency signs of elevated levels of total lipids and lipid phosphorus in liver and plasma, and cholesterol in plasma, were made more severe by supplemental dietary nickel. The findings suggested that nickel replaced copper at various functional sites, resulting in less copper function at various physiological sites and a more severe copper deficiency.
Acknowledgments The authors thank Sawaluk Taing for helping with the care of the animals, and Barry Shull for his assistance with the statistical analysis of the data.
References 1. F. H. Nielsen and T. J. Zimmerman, Biol. Trace Element Res. 3, 83 (1981). 2. F. H. Nielsen, T. J. Zimmerman, M. E. Collings, and D. R. Myron, J. Nutr. 109, 1623 (1979).
NI, Cu, FE INTERACTIONS
143
3. F. H. Nielsen, D. R. Myron, S. H. Givand, and D. A. Ollerich, J. Nutr. 105, 1607 (1975). 4. F. H. Nielsen and B. Bailey, Lab. Anita. Sci. 29, 502 (1979). 5. F. H. Nielsen, T. R. Shuler, T. J. Zimmerman, M. E. Collings, and E. O. Uthus, Biol. Trace Element Res. 1, 325 (1979). 6. K. G. D. Allen and L. M. Klevay, Life Sci. 22, 1691 (1978). 7. H. H. Taussky and E. Shorr, J. Biol. Chem. 202, 675 (1953). 8. F. H. Nielsen, D. R. Myron, S. H. Givand, T. J. Zimmerman, and D. A. Ollerich, J. Nutr. 105, 1620 (1975). 9. Analytical Methods for Atomic Absorption Spectrophotometry, Perkin-Elmer Corp., Norwalk, CT, 1976. 10. H. Scheff6, The Analysis of Variance, Wiley, New York, 1959, pp. 90-137. 11. L. M. Klevay, Am. J. Clin. Nutr. 26, 1060 (1973). 12. J. L. Evans and P. A. Abraham, J. Nutr. 103, 196 (t973). 13. P. D. Whanger, Toxicol. Appl. Pharmacol. 25, 323 (1973). 14. J. W. Spears, E. E. Hatfield, and R. M. Forbes, Proc. Soc. Exp. Biol. Med. 156, 140 (1977). 15. L. M. Klevay, Ann. NYAcad. Sci. 355, 140 (1980).
