- Email: [email protected]
cdb rats
BASIC NUTRITIONAL INVESTIGATION
Effects of Chromium and Copper Depletion on Lymphocyte Reactivity to Mitogens in DiabetesProne BHE/cdb Rats Yeong S. Rhee, PhD, Kim Burnham, PhD, Barbara J. Stoecker, PhD, and Eduralin Lucas, PhD From the Department of Health, Nutrition and Exercise Sciences, North Dakota State University, Fargo, North Dakota, USA; and the Department of Microbiology and Molecular Genetics and the Department of Nutritional Sciences, Oklahoma State University, Stillwater, Oklahoma, USA OBJECTIVE: The purpose of this study was to measure effects of chromium (Cr) and copper (Cu) depletion on lymphocyte reactivity to mitogens in diabetes-prone BHE/cdb rats. METHODS: A 2 ⫻ 2 factorial research design was used, and 40 BHE/cdb rats were fed with Cr- and/or Cu-depleted diets or adequate Cr and/or Cu diets for 21 wk. Cr and Cu concentrations in diets and mineral concentrations of tissues of BHE/cdb rats were measured by using flame and graphite furnace atomic absorption spectrometry. Three glucose tolerance tests were performed to monitor the development of diabetes or glucose intolerance at weeks 12, 18, and 21. Splenocytes (2 ⫻ 106) were incubated with phytohemagglutinin-L (PHA-L), concanavalin A (ConA), and lipopolysaccharides (LPSs), respectively, for 72 h. Four hours before the end of the incubation, splenocytes were pulsed with 3H-thymidine. The 3 H-thymidine uptake by lymphocytes was used to calculate a stimulation index. RESULTS: According to glucose tolerance tests, these rats did not develop diabetes or impaired glucose tolerance throughout the study. Average Cr concentrations were 0.98 to 1.03 mg Cr/kg of diet in adequate Cr diets and 8.2 to 14 g Cr/kg of diet in Cr-depletion diets. Average Cu concentrations were 3.6 to 6.4 mg Cu/kg of diet in adequate Cu diets and 1.1 to 1.3 mg Cu/kg of diet in Cu-depletion diets. Organ weights did not differ significantly among treatment groups at the end of the study. Cr or Cu depletion significantly affected iron, zinc, and magnesium concentrations in the liver. A significant interactive effect of Cr and Cu was observed on lymphocyte proliferation with PHA-L stimulation at 25 g/mL (P ⬍ 0.006). However, there were no significant effects of dietary treatment on lymphocyte proliferation with 10 g/mL of PHA-L, ConA, or LPS stimulations. CONCLUSIONS: When Cr and Cu were adequate in the diets, there was an enhanced effect of Cu or Cr on lymphocyte proliferation. However, when Cr was depleted in the diet, there was a suppressive effect of Cu on lymphocyte proliferation. This result indicates that adequate amounts of Cr and Cu in the diet support the immune system. Nutrition 2004;20:274 –279. ©Elsevier Inc. 2004 KEY WORDS: BHE/cdb rats, chromium, copper, mitogen, lymphocyte
INTRODUCTION Chromium (Cr) and copper (Cu) are essential trace minerals in human and animal nutrition. Many studies have reported on essential functions of Cr and Cu including roles in immune responses of humans and animals. Cr has been shown to have effects on immune functions by enhancing lymphocytic proliferation and blastogenesis with mitogen stimulations1–3 and increasing serum immunoglobulin M and total immunoglobulins4,5 in humans and animals. Chromate (K2CrO4) supplementation in drinking water has been shown to enhance splenocyte proliferation with concanavalin A (ConA) and lipopolysaccharide (LPS) stimulation in rats.1 Moreover, Cr supplementation has been shown to increase serum immunoglobulin M and total immunoglobulins in stressed calves.4,5 Cr supplementation also has been shown to increase mitogen-stimulated blastogenic responses of peripheral blood mononuclear cells in stressed
Correspondence to: Yeong Rhee, PhD, North Dakota State University, Department of Health, Nutrition and Exercise Sciences, 351 EML, Fargo, ND 58105, USA. E-mail: [email protected] Nutrition 20:274 –279, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.
as opposed to non-supplemented control dairy cows.2 In addition, Cr supplementation enhances lymphocytic responses to mitogen in hypercholesterolemic postmenopausal women.3 These studies suggest that Cr has beneficial effects on immune responses. Cu is an essential trace element in the host defense systems of humans and animals. Many studies on the detrimental effects of Cu deficiency on immune functions have been published.6 –10 Cu deficiency reduces T-lymphocytic activities and interleukin-2 production in rats.6,7 Cu deficiency also leads to suppression of the mononuclear cell response to T-cell mitogens and to a decrease in relative numbers of mature T cells in rats.8 The cytotoxicity of killer cells and the mitogen-stimulated proliferation of splenocytes have been found to be reduced and antibody titers to be decreased in Cu-deficient rats.6 Male rats fed a low-Cu diet had decreased ConA-, phytohemagglutinin-L- (PHA-L), and LPS-stimulated mitogen activities of splenocytes as compared with rats fed an adequate Cu diet.9,10 Cu-deficient rats show decreased numbers of T lymphocytes and CD4 and CD8 cells as compared with Cuadequate rats.10 These studies suggest that Cu deficiency can lead to impaired immune responses in rats. Because Cr supplementation has enhanced immune responses in humans and animals, it was assumed that Cr depletion might 0899-9007/04/$30.00 doi:10.1016/j.nut.2003.11.007
Nutrition Volume 20, Number 3, 2004 have detrimental effects on immune responses. In addition, because Cr and Cu have effects on immune responses, it was also assumed that there might be interactions between Cr and Cu in immunity. Therefore, the present study investigated effects of Cr- and/or Cu-depleted diets on the immune function by examining differential cell profiles and T- and/or B-cell proliferation in rats.
MATERIALS AND METHODS Animals Forty inbred BHE/cdb weanling male rats were obtained from the University of Georgia, Athens. Initial body weights were measured before feeding of experimental diets, and rats were randomly assigned into four treatment groups. The rats were housed individually in polystyrene cages with stainless-steel covers and acrylic grating floors. The rats had ad libitum access to experimental diets and deionized water throughout the study. The diets were provided in ceramic food bowls, and water was provided in a glass bottle with a rubber stopper and a stainless-steel sipper tube. The temperature and humidity of the room were controlled and checked by personnel from Laboratory Animal Resources in the College of Veterinary Medicine at Oklahoma State University, Stillwater. The light cycle was controlled to maintain 12 h of daytime and 12 h of night-time. One rat was severely sick with bleeding from the nose and mouth at necropsy, so it was excluded from the data analysis. We did not examine whether the diet that rat was on could have been a contributing factor to the bleeding. The animal protocol was approved by the Institutional Animal Care and Use Committee at Oklahoma State University. At 21 wk rats were anesthetized with ketamine HCl (60 mg/kg of body weight; Mallinckrodt Veterinary, Mundelein, IL, USA) and xylazine (6 mg/kg of body weight; Bayer Company, Shawnee Mission, KS, USA). Rats were exsanguinated by cardiac puncture. Serum and plasma samples were separated and frozen at ⫺20°C for further assays. Spleen samples were collected aseptically for lymphocytic proliferation assays, and other tissues were stored at ⫺20°C until used. Diet Four different diet treatments were applied in a 2 ⫻ 2 factorial design: 1) Cr and Cu depletion (⫺Cr⫺Cu), 2) adequate Cr and Cu depletion (⫹Cr⫺Cu), 3) Cr depletion and adequate Cu (⫺Cr⫹Cu), and 4) adequate Cr and adequate Cu (⫹Cr⫹Cu). A semi-purified diet (Table I) modified from the American Institute of Nutrition (AIN) 93G diet11 was used during the rapid growth phase. For the Cu-depletion treatment, 10% of the AIN-93 Cu recommendation (0.6 mg Cu/kg of diet, as CH2Cu2O5, cupric carbonate) was added to the diet; for the Cr-depletion treatment, no Cr was added to the diet. For the adequate Cr treatment, 0.1466 g Cr/kg of diet as CrCl3 · H2O6 was added to the diet; for the adequate Cu treatment, 6.0 mg Cu/kg of diet as CH2Cu2O5 was added to the diet. After reaching a growth plateau at 16 wk of age, rats were fed high-fat diets (220 g soybean oil/kg of diet; Table I) modified from the AIN-93 M diet11 to induce diabetes in these rats.12,13 Fat content of the diet was increased at the expense of dextrose and casein. In addition, vitamin E (0.6 g/kg of diet) was added to all diets to prevent oxidative stress due to high-fat content in the diet.13 The growth diets consisted of 20% protein (as casein), 62.95% carbohydrate (as cornstarch, dextrose, and sucrose), and 7% fat (as soybean oil), and the maintenance diets consisted of 14% protein, 53.95% carbohydrate, and 22% fat (Table I). Cr and Cu concentrations in diets were measured with a wetand-dry ashing method of Hill et al.14 using flame and graphite
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TABLE I. COMPOSITION OF THE GROWTH AND MAINTENANCE DIETS Growth diet Components Casein Cornstarch Celufil Dextrose Sucrose Vitamin mix* Soybean oil Choline L-cystine Mineral mix† Vitamin E
Maintenance diet
g/kg diet
%
g/kg diet
%
200 150 50 379.48 100 10 70 2.5 3 35 0.6
20 15 5 37.95 10 1 7 0.25 0.3 3.5 0.06
140 150 50 289.5 100 10 220 2.5 3 35 0.6
14 15 5 28.95 10 1 22 0.25 0.3 3.5 0.06
* Vitamin mix formulated to meet the AIN-93G recommendations for growing rats except for a five-fold increase in Vitamin E, or 0.6 g/kg of diet mix. † Mineral mix formulated to meet the AIN-93G recommendations for growing rats except for inadequate levels of chromium (0 g/kg added to mineral mix) and copper (0.03 g/kg added to mineral mix) as specified by the experimental design.
furnace atomic absorption spectrometry (model 5100PC, Perkin Elmer, Norwalk, CT, USA). Glucose Tolerance Test Glucose tolerance tests were performed at weeks 12, 18, and 21 of the experiment to monitor impaired glucose tolerance or diabetes development. Rats were gavaged with glucose solution (50% glucose solution, 1 g of glucose/kg of body weight). Tail blood samples were collected 2 h later, and glucose was measured with an automated glucometer (HemoCue, Ltd., Angelholm, Sweden). All rats were tested for glucose intolerance at week 12, but five to six rats were randomly selected for glucose tolerance at weeks 18 and 21. Differential Cell Profiles Differential cell profiles for BHE/cdb rats were measured with Wright’s stain (Leukostain, Fisher Diagnostic, Kennesaw, GA, USA). The distribution of lymphocytes and neutrophils was measured. Lymphocyte Proliferation Mitogen-stimulated T- and B-cell proliferations using rat splenocytes were measured according to a modified method of Lukasewycz and Prohaska.15 Complete RPMI-1640 (Sigma Chemical Company, St. Louis, MO, USA) culture medium was made by adding 1.0 mM/L of sodium pyruvate, 10% fetal bovine serum, 5 ⫻ 10⫺5 M/L of 2-mercaptoethanol, 2.0 mM/L of L-glutamine, 100 — 000 U/L of penicillin, and 100 mg/L of streptomycin to RPMI-1640 culture medium. PHA-L, ConA, and LPS were purchased from Sigma Chemical Company. The concentrations of 10 g/mL and 25 g/mL of PHA-L or 10 g/mL of ConA for T-cell proliferation and 10 g/mL of LPS for B-cell proliferation were used. Splenocytes were prepared in complete RPMI culture medium. Triplicate samples of splenocytes (2 ⫻ 106) with or without mitogen were pipetted into individual wells of a 96-well microtiter
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Nutrition Volume 20, Number 3, 2004 linear model procedure. Data are presented as mean ⫾ standard error of the mean.
TABLE II. CR AND CU CONCENTRATIONS OF GROWTH AND MAINTENANCE DIETS
RESULTS Group
Cr (g/kg)
Cu (mg/kg)
8.2 1160.4 * 906.6
1.3 1.2 6.4 6.4
11.6 882 16.4 1079.8
1.1 * 3.5 3.6
Growth diet ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Maintenance diet ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu
Cr and Cu Concentrations in Diet Average Cr concentrations were 1.03 mg Cr/kg of growth diet and 0.98 mg Cr/kg of maintenance diet in adequate Cr diets and 8.2 g Cr/kg of growth diet and 14 g Cr/kg of maintenance diet in Cr-depletion diets. Average Cu concentrations were 6.4 mg Cu/kg of growth diet and 3.6 mg Cu/kg of maintenance diet in adequate Cu diets and 1.3 mg Cu/kg of growth diet and 1.1 mg Cu/kg of maintenance diet in Cu-depletion diets (Table II). Weight Gain and Tissue Weight
* The values were below the limit of the detection by the instrument. ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
plate and incubated in a 5% CO2 incubator at 37°C for 72 h. Four hours before the end of the incubation, cells were pulsed with 1.0 Ci of 3H-thymidine. After completion of the incubation, pulsed cells were harvested on glass-fiber filter strips (Cambridge Tech, Cambridge, MA, USA) and dried in the air. Dried sample disks were placed in scintillation vials with scintillation cocktails (Perkin Elmer, Shelton, CT, USA). Then, the 3H-thymidine uptake by lymphocytes was measured by a liquid scintillation counter (Packard Instrument Company), and mean count per minute (cpm) was determined for the triplicate samples. Mean cpm was used to calculate a stimulation index as follows: cpm of stimulated cell cultures/cpm of unstimulated cell cultures. Tissue Mineral Concentrations Mineral concentrations of tissues, including kidney, liver, spleen, and tibia, of BHE/cdb rats were measured with the wet-and-dry ashing method of Hill et al.14 Quintuplet samples were analyzed for tissue mineral concentrations, and the average of the values was used for data analysis. Statistical Analysis Groups were analyzed as a 2 ⫻ 2 factorial design with Statistical Analysis System 8.2 (SAS/STAT, Cary, NC, USA) and the general
Initial weights were similar across the four experimental groups. There were no significant effects of depletion of Cr, Cu, or a combined depletion of Cr and Cu on the weight gain of animals throughout the experiment (data not shown). The change in weight for rats in all test groups was an average of 488 g and was independent of dietary treatment. Liver, kidney, spleen, heart, and thymus were weighed at the end of the experiment (after 21 wk of treatment). Tissue weights were similar across the different diet groups (Table III). Glucose Tolerance Test Fasting serum glucose was within normal ranges for rats after a glucose load and did not differ significantly across treatment groups (Table IV). Differential Cell Profiles Lymphocytes and neutrophils were counted as indicators of health status. The mean percentages of lymphocyte and neutrophil in blood were 84.8% and 14.5%, respectively. Values of these cells were similar across the different diet groups (Table V). Lymphocyte Proliferation Lymphocytic proliferation assays using splenocytes from four rats were not conducted due to the failure of the medium preparation. In addition, some outlying cpm data points (the highest or lowest cpm) from some of the groups were excluded from the data analysis. Therefore, sample numbers ranged from five to nine. The
TABLE III. TISSUE WEIGHTS OF BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK* Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Liver (g)
Kidney (g)
Spleen (g)
Heart (g)
Thymus (g)
14.09 ⫾ 0.42 13.80 ⫾ 0.74 13.90 ⫾ 0.58 14.00 ⫾ 0.41
3.38 ⫾ 0.10 3.15 ⫾ 0.08 3.45 ⫾ 0.23 3.41 ⫾ 0.09
1.26 ⫾ 0.04 1.29 ⫾ 0.03 1.30 ⫾ 0.06 1.28 ⫾ 0.04
0.22 ⫾ 0.03 0.27 ⫾ 0.04 0.27 ⫾ 0.05 0.25 ⫾ 0.04
0.87 0.99 0.73
0.33 0.25 0.50
1.21 ⫾ 0.05 1.10 ⫾ 0.06 1.14 ⫾ 0.04 1.11 ⫾ 0.05 P 0.19 0.56 0.39
0.91 0.75 0.67
0.62 0.62 0.32
* Values are mean ⫾ standard error of the mean (n ⫽ 7–10). ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
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TABLE IV.
TABLE VI.
GLUCOSE TOLERANCE TESTS*
STIMULATION INDICES WITH MITOGEN STIMULATION OF BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK*
Glucose 2 h (mg/dL) Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Test 1
Test 2
Test 3
128 ⫾ 13 137 ⫾ 13 133 ⫾ 12 136 ⫾ 10
121 ⫾ 9 132 ⫾ 5 113 ⫾ 6 119 ⫾ 7 P 0.25 0.18 0.74
115 ⫾ 4 119 ⫾ 3 120 ⫾ 7 128 ⫾ 7
0.62 0.91 0.81
0.24 0.21 0.73
* Values are mean ⫾ standard error of the mean: test 1 (at 12 wk), n ⫽ 9 –10; test 2 (at 18 wk), n ⫽ 5– 6; and test 3 (at 21 wk), n ⫽ 5– 6. ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
level of incorporation of 3H-thymidine in cell cultures without mitogen was not affected by dietary treatment (data not shown). Proliferation of splenocytes was expressed as a mitogenstimulation index according to different mitogens. The mitogenstimulation indices were similar across the different diet groups and mitogen treatments (Table VI). However, there was a significant interactive effect of a combined depletion of Cr and Cu on T-lymphocyte proliferation when using 25 g/mL of PHA-L as a mitogen (P ⬍ 0.006; Table VI). In the ⫺Cr⫹Cu group, lymphocytic proliferation was significantly decreased with 25 g/mL of PHA-L stimulation as compared with the ⫺Cr⫺Cu group (Table VI). In the ⫹Cr⫹Cu group, the stimulation index with 25 g/mL of PHA-L was significantly increased as compared with the ⫹Cr⫺Cu group (only Cr is adequate in the diet) or the ⫺Cr⫹Cu group (only Cu is adequate in the diet; Table VI). Tissue Mineral Concentrations Cr concentrations in kidney, spleen, liver, and tibia samples were not measured. Cu concentrations in kidney and liver were signif-
TABLE V. DISTRIBUTION OF LYMPHOCYTES AND NEUTROPHILS IN BLOOD OF BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK* Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Lymphocytes (%)
Neutrophils (%)
85 ⫾ 3 87 ⫾ 5 87 ⫾ 3 80 ⫾ 5
11 ⫾ 3 14 ⫾ 5 15 ⫾ 2 18 ⫾ 5 P
0.61 0.52 0.33
0.41 0.32 0.90
* Values are mean ⫾ standard error of the mean (n ⫽ 6 –7). ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
PHA-L (10 g/mL)
PHA-L (25 g/mL)
ConA (10 g/mL)
LPS (10 g/mL)
1.13 ⫾ 0.11 1.25 ⫾ 0.31 1.03 ⫾ 0.18 1.08 ⫾ 0.18
3.69 ⫾ 0.69 2.33 ⫾ 0.56 1.80 ⫾ 0.37 4.22 ⫾ 0.78
13.34 ⫾ 1.91 11.61 ⫾ 2.09 12.23 ⫾ 3.05 11.82 ⫾ 2.10
0.69 0.52 0.88
0.39 1.00 ⬍0.006
25.60 ⫾ 10.82 26.29 ⫾ 9.67 17.23 ⫾ 5.72 16.20 ⫾ 5.18 P 0.99 0.29 0.92
0.65 0.85 0.78
* Values are mean ⫾ standard error of the mean (n ⫽ 5–9). ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper; ConA, concanavalin A; LPS, lipopolysaccharide; PHA-L, phytohemagglutin-L
icantly decreased in the Cu-depletion groups as compared with the adequate Cu groups (P ⬍ 0.0001 and P ⬍ 0.02, respectively; Table VII). Cr or Cu depletion significantly affected different mineral concentrations such as iron, zinc, and magnesium in the liver (Table VIII). Cr depletion significantly increased zinc concentration in the liver (P ⬍ 0.03; Table VIII). In addition, Cu depletion significantly increased magnesium and iron concentrations in the liver (P ⬍ 0.01 and P ⬍ 0.02, respectively; Table VIII).
DISCUSSION The primary objective of this study was to investigate the effects of Cr and/or Cu depletion on immune responses in animals with diabetes. For this reason, BHE/cdb rats were selected as an animal model in the current study, because BHE/cdb rats are diabetesprone animals. Also, for this reason, diets were changed to high-fat maintenance diets to induce diabetes in these rats.12,13 These rats have been reported to develop glucose intolerance or diabetes genetically with high-fat diets12,13 and as they age (about 200 to 300 d old) due to genetic abnormalities in the liver.16 However, BHE/cdb rats did not develop or have diabetes as a consequence of high-fat diets in the present study; this was confirmed by glucose tolerance tests at weeks 12, 18, and 21 and by normal glucose, fructosamine, and insulin levels at necropsy. Glucose, fructosamine, and insulin at necropsy were not significantly affected by diet treatment (data not shown). In a study by Kullen and Berdanier,13 male BHE/cdb weanling rats were fed 22% fat diets (21% menhaden oil and 1% corn oil) containing 20.5% sucrose since weaning. These rats showed impaired glucose tolerance between ages 77 and 79 d. In the present study, BHE/cdb male rats were fed 22% fat diets (22% soybean oil) containing 10% sucrose after reaching the growth plateau (16 wk of treatment). Menhaden oil contains more saturated fatty acids and -3 fatty acids (33.6% saturated fatty acids and 21.7% -3 fatty acids) than does soybean oil (14.4% saturated fatty acids and 6.8% -3 fatty acids).17 High fish oil (menhaden oil) in the diets caused increased oxidative stress. This is due to unsaturated fatty acid in the fish oil changing the membrane fatty acid composition.13 The increased oxidative stress might have led to diabetes or impaired glucose tolerance in the study by Kullen and Berdanier,13 but it was not observed in the current study. The different sources of fat and lower sucrose contents in the diets, shorter periods of feeding (about 5 wk) the
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Nutrition Volume 20, Number 3, 2004 TABLE VII.
CU CONCENTRATIONS OF VARIOUS TISSUES FROM BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK* Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Kidney (M/dry wt)
Spleen (M/dry wt)
Liver (M/dry wt)
Tibia (M/dry wt)
0.188 ⫾ 0.004 0.174 ⫾ 0.006 0.264 ⫾ 0.015 0.264 ⫾ 0.013
0.059 ⫾ 0.008 0.051 ⫾ 0.006 0.064 ⫾ 0.006 0.064 ⫾ 0.006
0.118 ⫾ 0.009 0.104 ⫾ 0.006 0.136 ⫾ 0.009 0.128 ⫾ 0.011
0.042 ⫾ 0.001 0.043 ⫾ 0.001 0.046 ⫾ 0.001 0.043 ⫾ 0.002
0.50 ⬍0.0001 0.49
0.57 0.19 0.57
P 0.23 ⬍0.02 0.70
0.40 0.10 0.10
* Values are mean ⫾ SEM, n ⫽ 9 –10. ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper; wt, weight
high-fat diet, and different stages of life for introduction of high-fat diets might have caused a failure in developing impaired glucose tolerance in the present study. In addition, these BHE/cdb rats did not develop diabetes or impaired glucose tolerance despite Cu depletion in the diet. In a study by Weksler-Zangen et al.,18 the high-sucrose (72%) and Cu-deficient (1.2 ppm) diet induced diabetes in Cohen diabetic-sensitive rats. This also supports the idea that the low-sucrose content in the diet was a cause of failure to develop diabetes in these animals. Tissue weights in this study did not increase with Cu deficiency, which contradicts results of some other Cu-deficiency studies. The sizes of organs were changed with chronic dietary Cu deficiency. Cu-deficient rats showed enlarged liver, heart, and spleen as compared with Cu-adequate rats in studies by other investigators.19 –23 The average Cu concentrations in Cu-adequate maintenance diets were lower than the recommended level of 6.0 mg Cu/kg of diet11; the maintenance diets were fed for about 5 wk throughout the study. The Cu concentrations in animal tissues showed that tissue Cu concentrations from the Cu-depleted groups were significantly lower than those from the Cu-adequate groups. However, based on the small decreases in liver Cu, rats fed with Cu-depleted diets were only marginally Cu deficient, and this
TABLE VIII. ZINC, MAGNESIUM, AND IRON CONCENTRATIONS OF THE LIVER FROM BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK*
Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Zinc Magnesium Iron (M/g dry weight) (M/g dry weight) (M/g dry weight) 1.36 ⫾ 0.04 1.27 ⫾ 0.02 1.31 ⫾ 0.04 1.22 ⫾ 0.06 ⬍0.03 0.18 0.93
21.05 ⫾ 0.62 21.78 ⫾ 0.57 20.39 ⫾ 0.82 18.36 ⫾ 1.04 P 0.40 ⬍0.01 0.08
5.20 ⫾ 0.28 5.08 ⫾ 0.20 4.87 ⫾ 0.25 4.03 ⫾ 0.43 0.11 ⬍0.02 0.23
* Values are mean ⫾ standard error of the mean (n ⫽ 9 –10). ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
might be the reason that the expected changes in organ weights were not obtained. The stainless-steel covers and sipper tubes used in this study would have caused Cr contamination in these animals. Cr concentration of water passing through the sipper tube to determine whether Cr leached out of the steel and into the water of the rats was not measured in the current study. In addition, we could not determine whether animals fed the Cr-depleted diets were Cr deficient or animals fed with Cr-adequate diets had adequate Cr status because of non-detectable Cr concentrations in animal tissues. Non-detectable Cr concentrations in animal tissues might be due to increased sample dilution. The liver, kidney, and spleen samples were very hard to dissolve into clear solutions before analysis, so the volumes of the acids and deionized distilled water were increased to obtain clear solutions. This increased sample dilution might have caused undetectable Cr concentrations in liver, kidney, and spleen samples. Cr concentration in the tibia was not measurable due to the interference of calcium in tibia samples. Therefore, it was assumed that there were two different Cr states, Cr depleted and Cr adequate, in these animals based on Cr concentrations in the diets. Cu deficiency affects iron (Fe) metabolism by impairing mobilization and use of Fe.6,24 Thus, we expected to observe decreases in liver Fe concentrations in Cu-depleted groups. However, Cu-depleted diets increased Fe concentrations in the liver as compared with Cr-adequate diets in the present study. A similar observation was reported by Kramer et al.6 who suggested that it was caused by increased use of dietary Fe and liver Fe mobilization to prevent anemia in Cu-deficient rats.6 Cr and Fe interact via binding to transferrin.25 No changes in Fe concentration by Cr depletion indicated that Cr was not low enough to alter liver Fe status in the present study. Moreover, longer duration of Cr and/or Cu depletion (⬎7 mo) might be needed to observe altered liver Fe. In addition, some effects of Cr or Cu depletion on zinc (Zn) or magnesium (Mg) concentrations in the liver were observed in this study. However, these interactions between Cr and Zn or between Cu and Mg need further investigation. The number of neutrophils and lymphocytes are increased during stress or infection status. The normal ranges of neutrophil and lymphocyte of rats are 4.40% to 49.2% and 50.2% to 84.5%, respectively.26 In the present study, the normal or nearly normal values of these cells supported the opinion that these rats were healthy without any infectious diseases at necropsy. Moreover, the results from this study suggested that the level of Cr and/or Cu depletion in the diets may not have been low enough to alter the level of different blood cells and cause metabolic stress. The
Nutrition Volume 20, Number 3, 2004 duration of Cr and/or Cu depletion in the diets also may not have been long enough to alter the level of different blood cells and affect the immune system in these rats. These minerals, when supplied even in low daily dose, may help to maintain rather than to compromise the immune system because it appears that such a long period of depletion is needed to see adverse effects. To analyze the effect of dietary and environmental factors on immunocompetence in vitro, lymphocytic proliferation is used most commonly due to its reliability and technical simplicity.27 To compare dose responses of a selected mitogen-stimulated lymphocytic proliferation, 10 and 25 g/mL of PHA-L were used in these rats. The concentration of 25 g/mL of PHA-L showed the greater proliferation when compared with 10 g/mL of PHA-L in the current study (Table VI). In contrast to our hypothesis, Cr or Cu depletion in the diet did not have any effect on T- and B-lymphocytic proliferation. In addition, the results indicated potential interactions between Cr and Cu. This result also showed that only an adequate content of Cu in the diet has a potentially suppressive effect on T-lymphocytic proliferation if Cr is deficient beyond 21 wk. These results are different from most studies of the effects of Cu deficiency on mitogen responses in animals. Male rats fed a low-Cu diet had decreased ConA- and PHA-stimulated mitogen activities and blastogenesis of splenocytes as compared with rats fed a Cu-adequate diet.9,10,23 In most studies, lymphocytic proliferation was decreased with Cu deficiency. These differences in results from other studies might be due to the differences in the severity of Cu deficiency. The capacity of adequate Cr in the diet to protect against the inhibition of T-cell proliferation in rats by an only adequate content of Cu in the diet correlates with a previous clinical study in our laboratory.3 In the previous study, Cu supplementation blocked enhancement of T-cell proliferation that occurred in hypercholesterolemic postmenopausal women volunteers among those who received only Cr supplementation.3 Although the results these studies do not exactly parallel one another, the interactive effects between dietary Cu and Cr observed in these studies strongly indicate that the effects of these minerals on immunity cannot be studied in isolation. They also support the conventional wisdom of the human health benefits of a balanced diet as opposed to large doses of particular dietary supplements.
SUMMARY Enhanced T-lymphocytic proliferation indicates the beneficial effects of a diet with adequate amounts Cr and Cu on mitogen responses and cell-mediated immune function. The findings from this study indicated that T cells rather than B cells are the primary cell types affected by a diet with adequate amounts of Cr and Cu.
ACKNOWLEDGMENTS The authors acknowledge Dr. C. D. Berdanier for supplying the BHE/cdb rats and providing diet information for this study.
REFERENCES 1. Snyder CA, Valle CD. Immune function assays as indicators of chromate exposure. Environ Health Perspect 1991;92:83
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2. Burton JL, Mallard BA, Mowat DN. Effects of supplemental chromium on immune responses of periparturient and early lactation dairy cows. J Anim Sci 1993;71:1532 3. Rhee Y, Hermann J, Burnham K, Arquitt A, Stoecker B. The effects of chromium and copper supplementation on mitogen stimulated T cell proliferation in hypercholesterolemic postmenopausal women. Clin Exp Immun 2002;127:463 4. Chang X, Mowat DN. Supplemental chromium for stressed and growing feeder calves. J Anim Sci 1992;70:559 5. Moonsie-Shageer M, Mowat DN. Effect of level of supplemental chromium on performance, serum constituents, and immune status of stressed feeder calves. J Anim Sci 1993;71:232 6. Kramer TR, Johnson WT, Briske-Anderson M. Influence of iron and the sex of rats on hematological biochemical and immunological changes during copper deficiency. J Nutr 1988;118:214 7. Davis MA, Johnson WT, Briske-Anderson M, Kramer TR. Lymphoid cell functions during copper deficiency. Nutr Res 1987;7:211 8. Bala S, Failla ML. Copper deficiency reversibly impairs DNA synthesis in activated T lymphocytes by limiting interleukin 2 activity. Proc Natl Acad Sci USA 1992;89:6794 9. Hopkins RG, Failla ML. Chronic intake of a marginally low copper diet impairs in vitro activities of lymphocytes and neutrophils from male rats despite minimal impact on conventional indicators of copper status. J Nutr 1995;125:2658 10. Bala S, Failla ML, Lunney JK. Alterations in splenic lymphoid cell subsets and activation antigens in copper-deficient rats. J Nutr 1991;121:745 11. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition Ad Hoc Writing Committee on the reformulation of the AIN-76 A rodent diet. J Nutr 1993;123: 1939 12. Bue JM, Hausman DB, Berdanier CD. Gestational diabetes in the BHE rat: influence of dietary fat. Am J Obstet Gynecol 1989;161:234 13. Kullen MJ, Berdanier CD. Influence of fish oil feeding on the vitamin E requirement of BHE/cdb rats. Biochem Arch 1992;8:247 14. Hill AD, Patterson KY, Bveillon C, Morris ER. Digestion of biological materials for mineral analysis using a combination of wet and dry ashing. Anal Chem 1986;58:234 15. Lukasewycz OA, Prohaska JR. Lymphocytes from copper-deficient mice exhibit decreased mitogen reactivity. Nutr Res 1983;3:335 16. Berdanier CD. The BHE rat: an animal model for the study of non-insulindependent diabetes mellitus. FASEB J 1991;5:2139 17. Provisional table on the content of omega-3 fatty acids and other fat contents in selected foods (100 grams edible portion). In: Mahan LK, Escott-Stump S, eds. Krause’s food, nutrition, & diet therapy, 9th ed. Philadelphia: WB Saunders, 1996:1100 18. Weksler-Zangen S, Yagil C, Zangen D, Ornoy A, Jacob H, Yagil Y. The newly inbred Cohen diabetic rat. A nonobese normolipidemic genetic model of dietinduced type 2 diabetes expressing sex differences. Diabetes 2001;50:2521 19. Babu U, Failla ML. Respiratory burst and candidacidal activity of peritoneal macrophages are impaired in copper-deficient rats. J Nutr 1990;120:1692 20. Babu U, Failla ML. Copper status and function of neutrophils are reversibly depressed on marginal and severely copper-deficient rats. J Nutr 1990;120:1700 21. Kishimoto S, Iwamoto S, Masutani S, et al. Apoptosis of acinar cells in the pancreas of rats fed on a copper-depleted diet. Exp Toxicol Pathol 1993;45:489 22. Koller LD, Mulhern SA, Frankel NC, Steven MG, Williams JR. Immune dysfunction in rats fed a diet deficient in copper. Am J Clin Nutr 1987;45:997 23. Windhauser MM, Kappel LC, McClure J, Hegsted M. Suboptimal levels of dietary copper vary immunoresponsiveness in rats. Biol Trace Elem Res 1991; 30:205 24. Yu S, West C, Beynen A. Increasing intakes of iron reduce status, absorption and biliary excretion of copper in rats. Brit J Nutr 1994;71:887 25. Hopkins LL Jr, Shwarz K. Chromium (III) binding to serum proteins, specifically siderophilin. Biochim Biophys Acta 1964;90:484 26. Mitruka BM, Rawnsley HM. Clinical, biochemical, and hematological reference values in normal experimental animals and normal humans, 2nd ed. New York: Masson Publishing USA, 1981:64 27. Failla M, Hopkins RG. Copper and immunocompetence. In: Fischer PWF, Labbe MRL, Cockell KA, Gibson RS, eds. Trace elements in man and animals, Vol 9. Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals. Ottawa: NRC Research Press, 1997:425
Effects of Chromium and Copper Depletion on Lymphocyte Reactivity to Mitogens in DiabetesProne BHE/cdb Rats Yeong S. Rhee, PhD, Kim Burnham, PhD, Barbara J. Stoecker, PhD, and Eduralin Lucas, PhD From the Department of Health, Nutrition and Exercise Sciences, North Dakota State University, Fargo, North Dakota, USA; and the Department of Microbiology and Molecular Genetics and the Department of Nutritional Sciences, Oklahoma State University, Stillwater, Oklahoma, USA OBJECTIVE: The purpose of this study was to measure effects of chromium (Cr) and copper (Cu) depletion on lymphocyte reactivity to mitogens in diabetes-prone BHE/cdb rats. METHODS: A 2 ⫻ 2 factorial research design was used, and 40 BHE/cdb rats were fed with Cr- and/or Cu-depleted diets or adequate Cr and/or Cu diets for 21 wk. Cr and Cu concentrations in diets and mineral concentrations of tissues of BHE/cdb rats were measured by using flame and graphite furnace atomic absorption spectrometry. Three glucose tolerance tests were performed to monitor the development of diabetes or glucose intolerance at weeks 12, 18, and 21. Splenocytes (2 ⫻ 106) were incubated with phytohemagglutinin-L (PHA-L), concanavalin A (ConA), and lipopolysaccharides (LPSs), respectively, for 72 h. Four hours before the end of the incubation, splenocytes were pulsed with 3H-thymidine. The 3 H-thymidine uptake by lymphocytes was used to calculate a stimulation index. RESULTS: According to glucose tolerance tests, these rats did not develop diabetes or impaired glucose tolerance throughout the study. Average Cr concentrations were 0.98 to 1.03 mg Cr/kg of diet in adequate Cr diets and 8.2 to 14 g Cr/kg of diet in Cr-depletion diets. Average Cu concentrations were 3.6 to 6.4 mg Cu/kg of diet in adequate Cu diets and 1.1 to 1.3 mg Cu/kg of diet in Cu-depletion diets. Organ weights did not differ significantly among treatment groups at the end of the study. Cr or Cu depletion significantly affected iron, zinc, and magnesium concentrations in the liver. A significant interactive effect of Cr and Cu was observed on lymphocyte proliferation with PHA-L stimulation at 25 g/mL (P ⬍ 0.006). However, there were no significant effects of dietary treatment on lymphocyte proliferation with 10 g/mL of PHA-L, ConA, or LPS stimulations. CONCLUSIONS: When Cr and Cu were adequate in the diets, there was an enhanced effect of Cu or Cr on lymphocyte proliferation. However, when Cr was depleted in the diet, there was a suppressive effect of Cu on lymphocyte proliferation. This result indicates that adequate amounts of Cr and Cu in the diet support the immune system. Nutrition 2004;20:274 –279. ©Elsevier Inc. 2004 KEY WORDS: BHE/cdb rats, chromium, copper, mitogen, lymphocyte
INTRODUCTION Chromium (Cr) and copper (Cu) are essential trace minerals in human and animal nutrition. Many studies have reported on essential functions of Cr and Cu including roles in immune responses of humans and animals. Cr has been shown to have effects on immune functions by enhancing lymphocytic proliferation and blastogenesis with mitogen stimulations1–3 and increasing serum immunoglobulin M and total immunoglobulins4,5 in humans and animals. Chromate (K2CrO4) supplementation in drinking water has been shown to enhance splenocyte proliferation with concanavalin A (ConA) and lipopolysaccharide (LPS) stimulation in rats.1 Moreover, Cr supplementation has been shown to increase serum immunoglobulin M and total immunoglobulins in stressed calves.4,5 Cr supplementation also has been shown to increase mitogen-stimulated blastogenic responses of peripheral blood mononuclear cells in stressed
Correspondence to: Yeong Rhee, PhD, North Dakota State University, Department of Health, Nutrition and Exercise Sciences, 351 EML, Fargo, ND 58105, USA. E-mail: [email protected] Nutrition 20:274 –279, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.
as opposed to non-supplemented control dairy cows.2 In addition, Cr supplementation enhances lymphocytic responses to mitogen in hypercholesterolemic postmenopausal women.3 These studies suggest that Cr has beneficial effects on immune responses. Cu is an essential trace element in the host defense systems of humans and animals. Many studies on the detrimental effects of Cu deficiency on immune functions have been published.6 –10 Cu deficiency reduces T-lymphocytic activities and interleukin-2 production in rats.6,7 Cu deficiency also leads to suppression of the mononuclear cell response to T-cell mitogens and to a decrease in relative numbers of mature T cells in rats.8 The cytotoxicity of killer cells and the mitogen-stimulated proliferation of splenocytes have been found to be reduced and antibody titers to be decreased in Cu-deficient rats.6 Male rats fed a low-Cu diet had decreased ConA-, phytohemagglutinin-L- (PHA-L), and LPS-stimulated mitogen activities of splenocytes as compared with rats fed an adequate Cu diet.9,10 Cu-deficient rats show decreased numbers of T lymphocytes and CD4 and CD8 cells as compared with Cuadequate rats.10 These studies suggest that Cu deficiency can lead to impaired immune responses in rats. Because Cr supplementation has enhanced immune responses in humans and animals, it was assumed that Cr depletion might 0899-9007/04/$30.00 doi:10.1016/j.nut.2003.11.007
Nutrition Volume 20, Number 3, 2004 have detrimental effects on immune responses. In addition, because Cr and Cu have effects on immune responses, it was also assumed that there might be interactions between Cr and Cu in immunity. Therefore, the present study investigated effects of Cr- and/or Cu-depleted diets on the immune function by examining differential cell profiles and T- and/or B-cell proliferation in rats.
MATERIALS AND METHODS Animals Forty inbred BHE/cdb weanling male rats were obtained from the University of Georgia, Athens. Initial body weights were measured before feeding of experimental diets, and rats were randomly assigned into four treatment groups. The rats were housed individually in polystyrene cages with stainless-steel covers and acrylic grating floors. The rats had ad libitum access to experimental diets and deionized water throughout the study. The diets were provided in ceramic food bowls, and water was provided in a glass bottle with a rubber stopper and a stainless-steel sipper tube. The temperature and humidity of the room were controlled and checked by personnel from Laboratory Animal Resources in the College of Veterinary Medicine at Oklahoma State University, Stillwater. The light cycle was controlled to maintain 12 h of daytime and 12 h of night-time. One rat was severely sick with bleeding from the nose and mouth at necropsy, so it was excluded from the data analysis. We did not examine whether the diet that rat was on could have been a contributing factor to the bleeding. The animal protocol was approved by the Institutional Animal Care and Use Committee at Oklahoma State University. At 21 wk rats were anesthetized with ketamine HCl (60 mg/kg of body weight; Mallinckrodt Veterinary, Mundelein, IL, USA) and xylazine (6 mg/kg of body weight; Bayer Company, Shawnee Mission, KS, USA). Rats were exsanguinated by cardiac puncture. Serum and plasma samples were separated and frozen at ⫺20°C for further assays. Spleen samples were collected aseptically for lymphocytic proliferation assays, and other tissues were stored at ⫺20°C until used. Diet Four different diet treatments were applied in a 2 ⫻ 2 factorial design: 1) Cr and Cu depletion (⫺Cr⫺Cu), 2) adequate Cr and Cu depletion (⫹Cr⫺Cu), 3) Cr depletion and adequate Cu (⫺Cr⫹Cu), and 4) adequate Cr and adequate Cu (⫹Cr⫹Cu). A semi-purified diet (Table I) modified from the American Institute of Nutrition (AIN) 93G diet11 was used during the rapid growth phase. For the Cu-depletion treatment, 10% of the AIN-93 Cu recommendation (0.6 mg Cu/kg of diet, as CH2Cu2O5, cupric carbonate) was added to the diet; for the Cr-depletion treatment, no Cr was added to the diet. For the adequate Cr treatment, 0.1466 g Cr/kg of diet as CrCl3 · H2O6 was added to the diet; for the adequate Cu treatment, 6.0 mg Cu/kg of diet as CH2Cu2O5 was added to the diet. After reaching a growth plateau at 16 wk of age, rats were fed high-fat diets (220 g soybean oil/kg of diet; Table I) modified from the AIN-93 M diet11 to induce diabetes in these rats.12,13 Fat content of the diet was increased at the expense of dextrose and casein. In addition, vitamin E (0.6 g/kg of diet) was added to all diets to prevent oxidative stress due to high-fat content in the diet.13 The growth diets consisted of 20% protein (as casein), 62.95% carbohydrate (as cornstarch, dextrose, and sucrose), and 7% fat (as soybean oil), and the maintenance diets consisted of 14% protein, 53.95% carbohydrate, and 22% fat (Table I). Cr and Cu concentrations in diets were measured with a wetand-dry ashing method of Hill et al.14 using flame and graphite
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TABLE I. COMPOSITION OF THE GROWTH AND MAINTENANCE DIETS Growth diet Components Casein Cornstarch Celufil Dextrose Sucrose Vitamin mix* Soybean oil Choline L-cystine Mineral mix† Vitamin E
Maintenance diet
g/kg diet
%
g/kg diet
%
200 150 50 379.48 100 10 70 2.5 3 35 0.6
20 15 5 37.95 10 1 7 0.25 0.3 3.5 0.06
140 150 50 289.5 100 10 220 2.5 3 35 0.6
14 15 5 28.95 10 1 22 0.25 0.3 3.5 0.06
* Vitamin mix formulated to meet the AIN-93G recommendations for growing rats except for a five-fold increase in Vitamin E, or 0.6 g/kg of diet mix. † Mineral mix formulated to meet the AIN-93G recommendations for growing rats except for inadequate levels of chromium (0 g/kg added to mineral mix) and copper (0.03 g/kg added to mineral mix) as specified by the experimental design.
furnace atomic absorption spectrometry (model 5100PC, Perkin Elmer, Norwalk, CT, USA). Glucose Tolerance Test Glucose tolerance tests were performed at weeks 12, 18, and 21 of the experiment to monitor impaired glucose tolerance or diabetes development. Rats were gavaged with glucose solution (50% glucose solution, 1 g of glucose/kg of body weight). Tail blood samples were collected 2 h later, and glucose was measured with an automated glucometer (HemoCue, Ltd., Angelholm, Sweden). All rats were tested for glucose intolerance at week 12, but five to six rats were randomly selected for glucose tolerance at weeks 18 and 21. Differential Cell Profiles Differential cell profiles for BHE/cdb rats were measured with Wright’s stain (Leukostain, Fisher Diagnostic, Kennesaw, GA, USA). The distribution of lymphocytes and neutrophils was measured. Lymphocyte Proliferation Mitogen-stimulated T- and B-cell proliferations using rat splenocytes were measured according to a modified method of Lukasewycz and Prohaska.15 Complete RPMI-1640 (Sigma Chemical Company, St. Louis, MO, USA) culture medium was made by adding 1.0 mM/L of sodium pyruvate, 10% fetal bovine serum, 5 ⫻ 10⫺5 M/L of 2-mercaptoethanol, 2.0 mM/L of L-glutamine, 100 — 000 U/L of penicillin, and 100 mg/L of streptomycin to RPMI-1640 culture medium. PHA-L, ConA, and LPS were purchased from Sigma Chemical Company. The concentrations of 10 g/mL and 25 g/mL of PHA-L or 10 g/mL of ConA for T-cell proliferation and 10 g/mL of LPS for B-cell proliferation were used. Splenocytes were prepared in complete RPMI culture medium. Triplicate samples of splenocytes (2 ⫻ 106) with or without mitogen were pipetted into individual wells of a 96-well microtiter
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Nutrition Volume 20, Number 3, 2004 linear model procedure. Data are presented as mean ⫾ standard error of the mean.
TABLE II. CR AND CU CONCENTRATIONS OF GROWTH AND MAINTENANCE DIETS
RESULTS Group
Cr (g/kg)
Cu (mg/kg)
8.2 1160.4 * 906.6
1.3 1.2 6.4 6.4
11.6 882 16.4 1079.8
1.1 * 3.5 3.6
Growth diet ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Maintenance diet ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu
Cr and Cu Concentrations in Diet Average Cr concentrations were 1.03 mg Cr/kg of growth diet and 0.98 mg Cr/kg of maintenance diet in adequate Cr diets and 8.2 g Cr/kg of growth diet and 14 g Cr/kg of maintenance diet in Cr-depletion diets. Average Cu concentrations were 6.4 mg Cu/kg of growth diet and 3.6 mg Cu/kg of maintenance diet in adequate Cu diets and 1.3 mg Cu/kg of growth diet and 1.1 mg Cu/kg of maintenance diet in Cu-depletion diets (Table II). Weight Gain and Tissue Weight
* The values were below the limit of the detection by the instrument. ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
plate and incubated in a 5% CO2 incubator at 37°C for 72 h. Four hours before the end of the incubation, cells were pulsed with 1.0 Ci of 3H-thymidine. After completion of the incubation, pulsed cells were harvested on glass-fiber filter strips (Cambridge Tech, Cambridge, MA, USA) and dried in the air. Dried sample disks were placed in scintillation vials with scintillation cocktails (Perkin Elmer, Shelton, CT, USA). Then, the 3H-thymidine uptake by lymphocytes was measured by a liquid scintillation counter (Packard Instrument Company), and mean count per minute (cpm) was determined for the triplicate samples. Mean cpm was used to calculate a stimulation index as follows: cpm of stimulated cell cultures/cpm of unstimulated cell cultures. Tissue Mineral Concentrations Mineral concentrations of tissues, including kidney, liver, spleen, and tibia, of BHE/cdb rats were measured with the wet-and-dry ashing method of Hill et al.14 Quintuplet samples were analyzed for tissue mineral concentrations, and the average of the values was used for data analysis. Statistical Analysis Groups were analyzed as a 2 ⫻ 2 factorial design with Statistical Analysis System 8.2 (SAS/STAT, Cary, NC, USA) and the general
Initial weights were similar across the four experimental groups. There were no significant effects of depletion of Cr, Cu, or a combined depletion of Cr and Cu on the weight gain of animals throughout the experiment (data not shown). The change in weight for rats in all test groups was an average of 488 g and was independent of dietary treatment. Liver, kidney, spleen, heart, and thymus were weighed at the end of the experiment (after 21 wk of treatment). Tissue weights were similar across the different diet groups (Table III). Glucose Tolerance Test Fasting serum glucose was within normal ranges for rats after a glucose load and did not differ significantly across treatment groups (Table IV). Differential Cell Profiles Lymphocytes and neutrophils were counted as indicators of health status. The mean percentages of lymphocyte and neutrophil in blood were 84.8% and 14.5%, respectively. Values of these cells were similar across the different diet groups (Table V). Lymphocyte Proliferation Lymphocytic proliferation assays using splenocytes from four rats were not conducted due to the failure of the medium preparation. In addition, some outlying cpm data points (the highest or lowest cpm) from some of the groups were excluded from the data analysis. Therefore, sample numbers ranged from five to nine. The
TABLE III. TISSUE WEIGHTS OF BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK* Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Liver (g)
Kidney (g)
Spleen (g)
Heart (g)
Thymus (g)
14.09 ⫾ 0.42 13.80 ⫾ 0.74 13.90 ⫾ 0.58 14.00 ⫾ 0.41
3.38 ⫾ 0.10 3.15 ⫾ 0.08 3.45 ⫾ 0.23 3.41 ⫾ 0.09
1.26 ⫾ 0.04 1.29 ⫾ 0.03 1.30 ⫾ 0.06 1.28 ⫾ 0.04
0.22 ⫾ 0.03 0.27 ⫾ 0.04 0.27 ⫾ 0.05 0.25 ⫾ 0.04
0.87 0.99 0.73
0.33 0.25 0.50
1.21 ⫾ 0.05 1.10 ⫾ 0.06 1.14 ⫾ 0.04 1.11 ⫾ 0.05 P 0.19 0.56 0.39
0.91 0.75 0.67
0.62 0.62 0.32
* Values are mean ⫾ standard error of the mean (n ⫽ 7–10). ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
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TABLE IV.
TABLE VI.
GLUCOSE TOLERANCE TESTS*
STIMULATION INDICES WITH MITOGEN STIMULATION OF BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK*
Glucose 2 h (mg/dL) Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Test 1
Test 2
Test 3
128 ⫾ 13 137 ⫾ 13 133 ⫾ 12 136 ⫾ 10
121 ⫾ 9 132 ⫾ 5 113 ⫾ 6 119 ⫾ 7 P 0.25 0.18 0.74
115 ⫾ 4 119 ⫾ 3 120 ⫾ 7 128 ⫾ 7
0.62 0.91 0.81
0.24 0.21 0.73
* Values are mean ⫾ standard error of the mean: test 1 (at 12 wk), n ⫽ 9 –10; test 2 (at 18 wk), n ⫽ 5– 6; and test 3 (at 21 wk), n ⫽ 5– 6. ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
level of incorporation of 3H-thymidine in cell cultures without mitogen was not affected by dietary treatment (data not shown). Proliferation of splenocytes was expressed as a mitogenstimulation index according to different mitogens. The mitogenstimulation indices were similar across the different diet groups and mitogen treatments (Table VI). However, there was a significant interactive effect of a combined depletion of Cr and Cu on T-lymphocyte proliferation when using 25 g/mL of PHA-L as a mitogen (P ⬍ 0.006; Table VI). In the ⫺Cr⫹Cu group, lymphocytic proliferation was significantly decreased with 25 g/mL of PHA-L stimulation as compared with the ⫺Cr⫺Cu group (Table VI). In the ⫹Cr⫹Cu group, the stimulation index with 25 g/mL of PHA-L was significantly increased as compared with the ⫹Cr⫺Cu group (only Cr is adequate in the diet) or the ⫺Cr⫹Cu group (only Cu is adequate in the diet; Table VI). Tissue Mineral Concentrations Cr concentrations in kidney, spleen, liver, and tibia samples were not measured. Cu concentrations in kidney and liver were signif-
TABLE V. DISTRIBUTION OF LYMPHOCYTES AND NEUTROPHILS IN BLOOD OF BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK* Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Lymphocytes (%)
Neutrophils (%)
85 ⫾ 3 87 ⫾ 5 87 ⫾ 3 80 ⫾ 5
11 ⫾ 3 14 ⫾ 5 15 ⫾ 2 18 ⫾ 5 P
0.61 0.52 0.33
0.41 0.32 0.90
* Values are mean ⫾ standard error of the mean (n ⫽ 6 –7). ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
PHA-L (10 g/mL)
PHA-L (25 g/mL)
ConA (10 g/mL)
LPS (10 g/mL)
1.13 ⫾ 0.11 1.25 ⫾ 0.31 1.03 ⫾ 0.18 1.08 ⫾ 0.18
3.69 ⫾ 0.69 2.33 ⫾ 0.56 1.80 ⫾ 0.37 4.22 ⫾ 0.78
13.34 ⫾ 1.91 11.61 ⫾ 2.09 12.23 ⫾ 3.05 11.82 ⫾ 2.10
0.69 0.52 0.88
0.39 1.00 ⬍0.006
25.60 ⫾ 10.82 26.29 ⫾ 9.67 17.23 ⫾ 5.72 16.20 ⫾ 5.18 P 0.99 0.29 0.92
0.65 0.85 0.78
* Values are mean ⫾ standard error of the mean (n ⫽ 5–9). ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper; ConA, concanavalin A; LPS, lipopolysaccharide; PHA-L, phytohemagglutin-L
icantly decreased in the Cu-depletion groups as compared with the adequate Cu groups (P ⬍ 0.0001 and P ⬍ 0.02, respectively; Table VII). Cr or Cu depletion significantly affected different mineral concentrations such as iron, zinc, and magnesium in the liver (Table VIII). Cr depletion significantly increased zinc concentration in the liver (P ⬍ 0.03; Table VIII). In addition, Cu depletion significantly increased magnesium and iron concentrations in the liver (P ⬍ 0.01 and P ⬍ 0.02, respectively; Table VIII).
DISCUSSION The primary objective of this study was to investigate the effects of Cr and/or Cu depletion on immune responses in animals with diabetes. For this reason, BHE/cdb rats were selected as an animal model in the current study, because BHE/cdb rats are diabetesprone animals. Also, for this reason, diets were changed to high-fat maintenance diets to induce diabetes in these rats.12,13 These rats have been reported to develop glucose intolerance or diabetes genetically with high-fat diets12,13 and as they age (about 200 to 300 d old) due to genetic abnormalities in the liver.16 However, BHE/cdb rats did not develop or have diabetes as a consequence of high-fat diets in the present study; this was confirmed by glucose tolerance tests at weeks 12, 18, and 21 and by normal glucose, fructosamine, and insulin levels at necropsy. Glucose, fructosamine, and insulin at necropsy were not significantly affected by diet treatment (data not shown). In a study by Kullen and Berdanier,13 male BHE/cdb weanling rats were fed 22% fat diets (21% menhaden oil and 1% corn oil) containing 20.5% sucrose since weaning. These rats showed impaired glucose tolerance between ages 77 and 79 d. In the present study, BHE/cdb male rats were fed 22% fat diets (22% soybean oil) containing 10% sucrose after reaching the growth plateau (16 wk of treatment). Menhaden oil contains more saturated fatty acids and -3 fatty acids (33.6% saturated fatty acids and 21.7% -3 fatty acids) than does soybean oil (14.4% saturated fatty acids and 6.8% -3 fatty acids).17 High fish oil (menhaden oil) in the diets caused increased oxidative stress. This is due to unsaturated fatty acid in the fish oil changing the membrane fatty acid composition.13 The increased oxidative stress might have led to diabetes or impaired glucose tolerance in the study by Kullen and Berdanier,13 but it was not observed in the current study. The different sources of fat and lower sucrose contents in the diets, shorter periods of feeding (about 5 wk) the
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Nutrition Volume 20, Number 3, 2004 TABLE VII.
CU CONCENTRATIONS OF VARIOUS TISSUES FROM BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK* Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Kidney (M/dry wt)
Spleen (M/dry wt)
Liver (M/dry wt)
Tibia (M/dry wt)
0.188 ⫾ 0.004 0.174 ⫾ 0.006 0.264 ⫾ 0.015 0.264 ⫾ 0.013
0.059 ⫾ 0.008 0.051 ⫾ 0.006 0.064 ⫾ 0.006 0.064 ⫾ 0.006
0.118 ⫾ 0.009 0.104 ⫾ 0.006 0.136 ⫾ 0.009 0.128 ⫾ 0.011
0.042 ⫾ 0.001 0.043 ⫾ 0.001 0.046 ⫾ 0.001 0.043 ⫾ 0.002
0.50 ⬍0.0001 0.49
0.57 0.19 0.57
P 0.23 ⬍0.02 0.70
0.40 0.10 0.10
* Values are mean ⫾ SEM, n ⫽ 9 –10. ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper; wt, weight
high-fat diet, and different stages of life for introduction of high-fat diets might have caused a failure in developing impaired glucose tolerance in the present study. In addition, these BHE/cdb rats did not develop diabetes or impaired glucose tolerance despite Cu depletion in the diet. In a study by Weksler-Zangen et al.,18 the high-sucrose (72%) and Cu-deficient (1.2 ppm) diet induced diabetes in Cohen diabetic-sensitive rats. This also supports the idea that the low-sucrose content in the diet was a cause of failure to develop diabetes in these animals. Tissue weights in this study did not increase with Cu deficiency, which contradicts results of some other Cu-deficiency studies. The sizes of organs were changed with chronic dietary Cu deficiency. Cu-deficient rats showed enlarged liver, heart, and spleen as compared with Cu-adequate rats in studies by other investigators.19 –23 The average Cu concentrations in Cu-adequate maintenance diets were lower than the recommended level of 6.0 mg Cu/kg of diet11; the maintenance diets were fed for about 5 wk throughout the study. The Cu concentrations in animal tissues showed that tissue Cu concentrations from the Cu-depleted groups were significantly lower than those from the Cu-adequate groups. However, based on the small decreases in liver Cu, rats fed with Cu-depleted diets were only marginally Cu deficient, and this
TABLE VIII. ZINC, MAGNESIUM, AND IRON CONCENTRATIONS OF THE LIVER FROM BHE/CDB RATS DEPLETED OF CR, CU, OR BOTH FOR 21 WK*
Group ⫺Cr⫺Cu ⫹Cr⫺Cu ⫺Cr⫹Cu ⫹Cr⫹Cu Factors Cr Cu Cr ⫻ Cu
Zinc Magnesium Iron (M/g dry weight) (M/g dry weight) (M/g dry weight) 1.36 ⫾ 0.04 1.27 ⫾ 0.02 1.31 ⫾ 0.04 1.22 ⫾ 0.06 ⬍0.03 0.18 0.93
21.05 ⫾ 0.62 21.78 ⫾ 0.57 20.39 ⫾ 0.82 18.36 ⫾ 1.04 P 0.40 ⬍0.01 0.08
5.20 ⫾ 0.28 5.08 ⫾ 0.20 4.87 ⫾ 0.25 4.03 ⫾ 0.43 0.11 ⬍0.02 0.23
* Values are mean ⫾ standard error of the mean (n ⫽ 9 –10). ⫺Cr⫺Cu, chromium and copper depletion; ⫹Cr⫺Cu, adequate chromium and copper depletion; ⫺Cr⫹Cu, chromium depletion and adequate copper; ⫹Cr⫹Cu, adequate chromium and adequate copper
might be the reason that the expected changes in organ weights were not obtained. The stainless-steel covers and sipper tubes used in this study would have caused Cr contamination in these animals. Cr concentration of water passing through the sipper tube to determine whether Cr leached out of the steel and into the water of the rats was not measured in the current study. In addition, we could not determine whether animals fed the Cr-depleted diets were Cr deficient or animals fed with Cr-adequate diets had adequate Cr status because of non-detectable Cr concentrations in animal tissues. Non-detectable Cr concentrations in animal tissues might be due to increased sample dilution. The liver, kidney, and spleen samples were very hard to dissolve into clear solutions before analysis, so the volumes of the acids and deionized distilled water were increased to obtain clear solutions. This increased sample dilution might have caused undetectable Cr concentrations in liver, kidney, and spleen samples. Cr concentration in the tibia was not measurable due to the interference of calcium in tibia samples. Therefore, it was assumed that there were two different Cr states, Cr depleted and Cr adequate, in these animals based on Cr concentrations in the diets. Cu deficiency affects iron (Fe) metabolism by impairing mobilization and use of Fe.6,24 Thus, we expected to observe decreases in liver Fe concentrations in Cu-depleted groups. However, Cu-depleted diets increased Fe concentrations in the liver as compared with Cr-adequate diets in the present study. A similar observation was reported by Kramer et al.6 who suggested that it was caused by increased use of dietary Fe and liver Fe mobilization to prevent anemia in Cu-deficient rats.6 Cr and Fe interact via binding to transferrin.25 No changes in Fe concentration by Cr depletion indicated that Cr was not low enough to alter liver Fe status in the present study. Moreover, longer duration of Cr and/or Cu depletion (⬎7 mo) might be needed to observe altered liver Fe. In addition, some effects of Cr or Cu depletion on zinc (Zn) or magnesium (Mg) concentrations in the liver were observed in this study. However, these interactions between Cr and Zn or between Cu and Mg need further investigation. The number of neutrophils and lymphocytes are increased during stress or infection status. The normal ranges of neutrophil and lymphocyte of rats are 4.40% to 49.2% and 50.2% to 84.5%, respectively.26 In the present study, the normal or nearly normal values of these cells supported the opinion that these rats were healthy without any infectious diseases at necropsy. Moreover, the results from this study suggested that the level of Cr and/or Cu depletion in the diets may not have been low enough to alter the level of different blood cells and cause metabolic stress. The
Nutrition Volume 20, Number 3, 2004 duration of Cr and/or Cu depletion in the diets also may not have been long enough to alter the level of different blood cells and affect the immune system in these rats. These minerals, when supplied even in low daily dose, may help to maintain rather than to compromise the immune system because it appears that such a long period of depletion is needed to see adverse effects. To analyze the effect of dietary and environmental factors on immunocompetence in vitro, lymphocytic proliferation is used most commonly due to its reliability and technical simplicity.27 To compare dose responses of a selected mitogen-stimulated lymphocytic proliferation, 10 and 25 g/mL of PHA-L were used in these rats. The concentration of 25 g/mL of PHA-L showed the greater proliferation when compared with 10 g/mL of PHA-L in the current study (Table VI). In contrast to our hypothesis, Cr or Cu depletion in the diet did not have any effect on T- and B-lymphocytic proliferation. In addition, the results indicated potential interactions between Cr and Cu. This result also showed that only an adequate content of Cu in the diet has a potentially suppressive effect on T-lymphocytic proliferation if Cr is deficient beyond 21 wk. These results are different from most studies of the effects of Cu deficiency on mitogen responses in animals. Male rats fed a low-Cu diet had decreased ConA- and PHA-stimulated mitogen activities and blastogenesis of splenocytes as compared with rats fed a Cu-adequate diet.9,10,23 In most studies, lymphocytic proliferation was decreased with Cu deficiency. These differences in results from other studies might be due to the differences in the severity of Cu deficiency. The capacity of adequate Cr in the diet to protect against the inhibition of T-cell proliferation in rats by an only adequate content of Cu in the diet correlates with a previous clinical study in our laboratory.3 In the previous study, Cu supplementation blocked enhancement of T-cell proliferation that occurred in hypercholesterolemic postmenopausal women volunteers among those who received only Cr supplementation.3 Although the results these studies do not exactly parallel one another, the interactive effects between dietary Cu and Cr observed in these studies strongly indicate that the effects of these minerals on immunity cannot be studied in isolation. They also support the conventional wisdom of the human health benefits of a balanced diet as opposed to large doses of particular dietary supplements.
SUMMARY Enhanced T-lymphocytic proliferation indicates the beneficial effects of a diet with adequate amounts Cr and Cu on mitogen responses and cell-mediated immune function. The findings from this study indicated that T cells rather than B cells are the primary cell types affected by a diet with adequate amounts of Cr and Cu.
ACKNOWLEDGMENTS The authors acknowledge Dr. C. D. Berdanier for supplying the BHE/cdb rats and providing diet information for this study.
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