Abstract
The purpose of this study was to analyse effects of chromium and/or copper supplementation on immune function in hypercholesterolaemic postmenopausal women. A 2 × 2 factorial research design was used and 40 subjects were supplemented with 0·394 g lactose, 200 μg Cr, 3·0 mg Cu, or 200 μg Cr and 3·0 mg Cu/d for 12 weeks. A significant interactive effect of Cr and Cu supplementation on lymphocyte proliferation was observed with ConA 50 μg/ml stimulation. After 12 weeks of supplementation, ConA-stimulated (50 μg/ml) lymphocyte proliferation was significantly lower when Cu was added to the Cr supplementation group. Moreover, ConA-stimulated (100 μg/ml) lymphocyte proliferation was significantly lower in the Cu supplementation group compared to the Cr supplementation group after 12 weeks of supplementation. These results suggest that Cu blocks enhancement of lymphocyte proliferation by Cr supplementation and that Cu supplementation has potential suppressive effects on the immune function in these subjects.
Keywords: chromium, copper, postmenopausal women, mitogen, lymphocyte
Introduction
Environment as well as nutrition influences the immune response. A proper immune response requires adequate trace metal nutriture [1]. The effects of a general nutritional deficiency state or the effects of a single nutrient deficiency over a certain period have been investigated in relation to immune function in humans and animals [2]. A well-balanced nutritional intake has multiple important roles in immune function in all age groups of the population [3]. The host defense system of humans, including the number of lymphocytes, mature T cells, and helper T cells, decreases with ageing [4]. Improved nutritional intake has the potential to increase immune function among the aged [4,5].
No studies about the effects of chromium (Cr) supplementation on immune function in humans or rats have been reported. However, Cr supplementation increases serum IgM, total immunoglobulins and antibody titres to human erythrocytes and decreases serum cortisol in stressed calves. Increased serum immunoglobulins and reduced serum cortisol may impove immune function [6,7]. Chromium supplementation has also been shown to increase mitogen-stimulated blastogenic responses of peripheral blood mononuclear cells (PBMNC) compared with unsupplemented controls in stressed dairy cows [8].
Copper (Cu) is an essential trace element for the host defense system of humans and animals. Copper deficiency increases susceptibility to pathogens in mice and leads to reduced T-lymphocyte activation and interleukin 2 production in rats [9–11]. In the offspring of C58 mice fed Cu-deficient diets, the number of antibody producing cells following a sheep erythrocyte injection was significantly decreased. Also, reactivity to both T cell and B cell mitogens and mixed lymphocyte reactions were abnormal in the Cu-deficient offspring of C58 mice [12–14]. Rats fed a low Cu diet had decreased ConA- and PHA-stimulated mitogen activity of splenocytes compared to rats fed an adequate Cu diet [10,15]. In humans, one month of copper sulphate supplementation significantly increased phagocytic indices in subjects with severe or marginal Cu deficiency [16]. In addition, T and B lymphocyte responses to mitogen stimulation were impaired in subjects with Cu deficiency and in subjects with a low Cu diet [17,18]. Cr and Cu supplementation play roles in immune function by increasing mitogen-stimulated immune responses or immunoglobulins. In this study, the effects of Cr and/or Cu supplementation on the immune function in hypercholesterolaemic postmenopausal women were investigated. The results of this study suggest that Cr and/or Cu supplementation in these subjects may affect lymphocyte proliferation and basophil levels.
Materials and methods
Subjects
Forty subjects were recruited in and around Stillwater, Oklahoma. Postmenopausal women, including those with surgically induced menopause, with blood cholesterol concentration >200 mg/dl and not taking lipid lowering medications or hormone replacement therapy were qualified as subjects for the study. The subjects were defined as postmenopausal based on health questionnaires. Subjects were requested not to change medication intake or eating patterns. Dietary intake was obtained using 7-day food frequency forms and analysed using the Food Processor software [19]. The protocol was approved by the Institutional Review Board (IRB) for human subjects at Oklahoma State University.
Treatment groups
A 2 × 2 factorial design was applied in this study. Ten subjects were randomly assigned into each treatment group: (1) placebo (− Cr – Cu); (2) chromium supplemented (+ Cr – Cu); (3) copper supplemented (− Cr + Cu); and (4) chromium and copper supplemented (+ Cr + Cu) groups. Subjects were supplemented with either 0·394 g lactose as placebo, 200 μg Cr, 3·0 mg Cu, or 200 μg Cr and 3·0 mg Cu/d for 12 weeks. Subjects took one capsule at breakfast and one capsule at dinner. Each month, a new bottle of supplements was delivered to subjects and the leftovers from the previous bottle were collected to check for compliance. Treatments were double blinded throughout the study.
Blood collection
Subjects were screened for total cholesterol before starting the study. Fasting blood was collected two more times, i.e. at the beginning (presupplementation) and the end (postsupplementation) of the study. Blood was collected between 7.30 a.m. and 9.00 a.m. in heparin-coated and EDTA-coated syringes, kept at room temperature, and whole blood cell proliferation assays were initiated within 2 h of the first blood draw.
Differential cell profiles
Cell profiles were measured using a VEGA haematology autoanalyser (ABX, Montpellier, Cedex, France). The distribution of lymphocytes, monocytes, neutrophils, basophils and eosinophils was measured in this study.
Mitogenic proliferative responsiveness
Mitogen-stimulated T cell proliferation using human whole blood cell cultures was measured following the method of Kramer and Burri [20]. Complete RPMI (CRPMI)-1640 (Sigma Chem. Co., St. Louis, MO) culture medium was made by adding 2·0 mmol/l of l-glutamine, 100 000 U/l of penicillin, and 100 mg/l of streptomycin to RPMI-1640 culture medium. Phytohaemagglutinin-L (PHA-L) and concanavalin A (ConA) were purchased from Sigma Chemical Co. Four hundred μl of heparinized blood were diluted with 1200 μl of CRPMI in 4·0 ml polystyrene tubes. Subsequently, 50 μl of diluted blood and 50 μl of CRPMI (unstimulated), 50 μl of PHA-L, or 50 μl of ConA were added to each set of triplicate culture wells in 96-well tissue culture plates, followed by the addition of 100 μl of CRPMI-1640. The cell cultures were incubated in a 5% CO2 incubator at 37°C for 96 h. Eighteen hours before the termination of incubation, 1·0 μCi methyl-3H-thymidine was added to each culture well. The 3H-thymidine uptake by lymphocytes was used to calculate a stimulation index as follows: cpm of stimulated cell cultures/cpm of unstimulated cell cultures.
Statistical analysis
Groups were analysed as a 2 × 2 factorial design using the Statistical Analysis System, version 6.11 [21] and the general linear model procedure. Data are presented as mean ± s.e.m.
Results
A total of 11 subjects were excluded from the data analysis: five subjects were diabetic, three subjects dropped out after the baseline sample collection, one subject was found to be on oestrogen, one subject stopped taking supplement capsules towards the end of the study (60 capsules leftover, 36% of total capsules), and one subject was not able to have blood drawn at the baseline due to dehydration. Data from a total of 29 subjects were analysed.
Subjects
The mean age of subjects was 59 years with a range of 38–81 years. There were no significant age differences among supplement groups (Table 1). In addition, there were no significant differences in body mass index (BMI) at baseline or after 12 weeks of supplementation among supplement groups (Table 1). The average BMI of subjects was 31 kg/m2. The average kCal intake of these subjects was 1824 kCal (Table 1) and average percentage of kCal provided by carbohydrate, protein and fat was 48%, 17% and 33%, respectively, at baseline and after 12 weeks of supplementation in these subjects (data not shown). There were no significant differences in carbohydrate, protein or fat intake among supplement groups (data not shown). The average dietary Cu intake was 1·0 mg/d and there were no significant differences in dietary Cu intake among treatment groups (data not shown).
Table 1.
Age, body mass index (BMI), and kCal intake at baseline and after 12 weeks of supplementation with Cr, Cu or both Cr and Cu*
| BMI (kg/m2) | kCal intake | ||||||
|---|---|---|---|---|---|---|---|
| Group† | Age (year) | Baseline | 12 weeks | Change‡ | Baseline | 12 weeks | Change‡ |
| − Cr −Cu | 57 ± 4 | 28·0 ± 1·5 | 28·7 ± 1·4 | 0·6 ± 0·3 | 1859 ± 224 | 1653 ± 234 | − 206 ± 183 |
| + Cr −Cu | 59 ± 5 | 32·0 ± 2·4 | 30·4 ± 1·0 | − 1·6 ± 2·4 | 1574 ± 135 | 1633 ± 207 | 59 ± 175 |
| − Cr +Cu | 59 ± 2 | 31·6 ± 2·6 | 31·8 ± 2·6 | 0·2 ± 0·2 | 1632 ± 169 | 1651 ± 64 | 19 ± 197 |
| + Cr +Cu | 58 ± 5 | 30·9 ± 1·6 | 31·1 ± 1·5 | 0·3 ± 0·3 | 2327 ± 254 | 2102 ± 162 | − 225 ± 212 |
| − Cr | 58 ± 2 | 29·8 ± 1·5 | 30·2 ± 1·5 | 0·4 ± 0·2 | 1746 ± 139 | 1652 ± 117 | − 94 ± 133 |
| + Cr | 58 ± 3 | 31·5 ± 1·5 | 30·7 ± 0·8 | − 0·7 ± 1·3 | 1922 ± 171 | 1849 ± 145 | − 72 ± 136 |
| − Cu | 58 ± 3 | 30·0 ± 1·4 | 29·5 ± 0·9 | − 0·4 ± 1·1 | 1726 ± 136 | 1644 ± 152 | − 82 ± 128 |
| + Cu | 59 ± 3 | 31·3 ± 1·6 | 31·5 ± 1·6 | 0·2 ± 0·1 | 1930 ± 169 | 1844 ± 97 | − 86 ± 143 |
| Factors | P-values | ||||||
| Cr | 0·94 | 0·46 | 0·77 | 0·37 | 0·31 | 0·25 | 0·96 |
| Cu | 0·93 | 0·58 | 0·30 | 0·56 | 0·20 | 0·21 | 0·88 |
| Cr × Cu | 0·75 | 0·30 | 0·53 | 0·34 | < 0·02 | 0·21 | 0·20 |
Values are mean ± s.e.m., n = 7–8.
− Cr –Cu, placebo group; +Cr –Cu, chromium-supplemented group; –Cr +Cu, copper-supplemented group; +Cr +Cu, chromium and copper supplemented group.
Change, difference in values between baseline and the end of the study (after 12 weeks of supplementation).
No subjects reported having infectious disease or taking antibiotics throughout the study and this was checked using health questionnaires. In addition, there were no subjects who reported taking additional Cr or Cu supplement (not supplemented by researchers) during the study. Four subjects reported taking vitamin or mineral supplement such as vitamin E, calcium, niacin, potassium, vitamin B12 and folic acid during the study. The effects of these vitamin or mineral supplements on Cr and/or Cu supplementation as well as on analysis variables were not determined.
Differential cell profiles
Differential cell profiles were measured as indicators of health status. The changes in lymphocytes, monocytes, neutrophils and eosinophils after 12 weeks of supplementation were not significantly different among supplement groups (data not shown). However, basophils were significantly increased with 12 weeks of Cu supplementation (P < 0·003) compared to groups not supplemented with Cu (Table 2).
Table 2.
The distribution of basophils (%) in blood at baseline and after 12 weeks of supplementation with Cr, Cu or both Cr and Cu*
| Basophils | |||
|---|---|---|---|
| Group† | Baseline | 12 weeks | Change‡ |
| − Cr −Cu | 1·1 ± 0·1 | 0·9 ± 0·1 | − 0·2 ± 0·1 |
| + Cr −Cu | 1·0 ± 0·1 | 0·9 ± 0·1 | − 0·1 ± 0·1 |
| − Cr +Cu | 0·8 ± 0·2 | 1·1 ± 0·1 | 0·2 ± 0·1 |
| + Cr +Cu | 1·0 ± 0·1 | 1·1 ± 0·2 | 0·1 ± 0·2 |
| − Cr | 0·9 ± 0·1 | 1·0 ± 0·1 | 0·1 ± 0·1 |
| + Cr | 1·0 ± 0·1 | 1·0 ± 0·1 | 0·0 ± 0·1 |
| − Cu | 1·0 ± 0·1 | 0·9 ± 0·1 | − 0·1 ± 0·1 |
| + Cu | 0·9 ± 0·1 | 1·1 ± 0·1 | 0·2 ± 0·1 |
| Factors | P-values | ||
| Cr | 0·74 | 1·00 | 0·92 |
| Cu | 0·23 | 0·10 | x003C; 0·003 |
| Cr × Cu | 0·34 | 1·00 | 0·45 |
Values are mean ± s.e.m., n = 6–8.
See Table 1 for description of groups.
Change, difference in values between baseline and the end of the study (after 12 weeks of supplementation).
Mitogenic proliferative responsiveness
Lymphocyte proliferation was measured as an indicator of immune function. There were significant differences in the stimulation index at baseline in the Cr supplement groups with varying concentrations of PHA-L or ConA stimulation (Tables 3 and 4). In addition, Cr supplementation had the greatest increase in stimulation index with either PHA-L or ConA stimulation among supplementation groups after 12 weeks of supplementation as compared to baseline (Tables 3 and 4). There were no significant differences in stimulation index with PHA-L 40 μg/ml or PHA-L 80 μg/ml stimulation among the treatment groups after 12 weeks of supplementation (Table 3). A significant interactive effect of Cr and Cu was observed on lymphocyte proliferation with ConA stimulation (50 μg/ml) after 12 weeks of supplementation (P < 0·05, Fig. 1,Table 4). In Cr supplemented groups, when Cu was additionally supplemented, the stimulation index after 12 weeks was significantly lower with ConA stimulation (50 μg/ml) compared to the group supplemented with Cr alone (Fig. 1,Table 4). After 12 weeks of Cu supplementation, lymphocyte proliferation was decreased to a small degree with ConA stimulation (100 μg/ml) compared to baseline; however, the difference was not significant (Fig. 2,Table 4). In addition, the stimulation index with ConA (100 μg/ml) was significantly lower (P < 0·02, Fig. 2,Table 4) following 12 weeks of Cu supplementation compared to Cr supplementation.
Table 3.
Lymphocyte proliferation (stimulation index) with PHA-L stimulation from whole blood cell cultures at baseline and after 12 weeks of supplementation with Cr, Cu or both Cr and Cu*
| PHA-L (40 μg/ml) | PHA-L (80 μg/ml) | |||||
|---|---|---|---|---|---|---|
| Group† | Baseline | 12 weeks | Change‡ | Baseline | 12 weeks | Change‡ |
| − Cr −Cu | 211·7 ± 38·1 | 303·5 ± 101·3 | 91·9 ± 86·1 | 233·2 ± 41·4 | 335·4 ± 107·6 | 102·2 ± 88·0 |
| + Cr −Cu | 92·6 ± 22·4 | 319·3 ± 73·3 | 226·7 ± 85·5 | 108·7 ± 31·8 | 321·6 ± 74·9 | 203·2 ± 94·4 |
| − Cr +Cu | 184·5 ± 32·2 | 213·2 ± 49·4 | 28·7 ± 66·4 | 211·1 ± 37·9 | 216·5 ± 35·4 | 5·5 ± 57·5 |
| + Cr +Cu | 149·9 ± 37·4 | 174·1 ± 59·9 | 24·2 ± 73·8 | 165·0 ± 42·9 | 199·9 ± 56·8 | 34·9 ± 67·1 |
| − Cr | 197·2 ± 24·1 | 255·3 ± 53·4 | 58·2 ± 52·2 | 221·4 ± 27·1 | 272·0 ± 53·9 | 50·6 ± 51·0 |
| + Cr | 119·1 ± 21·7 | 258·8 ± 52·1 | 133·9 ± 65·2 | 134·7 ± 26·3 | 266·3 ± 49·8 | 114·7 ± 64·7 |
| − Cu | 152·1 ± 26·9 | 311·4 ± 60·1 | 159·3 ± 61·2 | 171·0 ± 30·4 | 329·0 ± 64·8 | 148·8 ± 63·3 |
| + Cu | 169·7 ± 23·9 | 198·1 ± 37·0 | 19·2 ± 47·9 | 191·3 ± 28·0 | 210·1 ± 29·5 | 6·7 ± 42·0 |
| Factors | P-values | |||||
| Cr | < 0·03 | 0·88 | 0·50 | < 0·04 | 0·84 | 0·52 |
| Cu | 0·65 | 0·13 | 0·09 | 0·66 | 0·13 | 0·08 |
| Cr × Cu | 0·21 | 0·72 | 0·33 | 0·32 | 0·99 | 0·54 |
Values are mean ± s.e.m., n = 7–8.
See Table 1 for description of groups.
Change, difference in values between baseline and the end of the study (after 12 weeks of supplementation).
Table 4.
Lymphocyte proliferation (stimulation index) with ConA stimulation from whole blood cell cultures at baseline and after 12 weeks of supplementation with Cr, Cu or both Cr and Cu*
| ConA (50 μg/ml) | ConA (100 μg/ml) | |||||
|---|---|---|---|---|---|---|
| Group† | Baseline | 12 weeks | Change‡ | Baseline | 12 weeks | Change‡ |
| − Cr −Cu | 167·1 ± 12·2 | 167·9 ± 39·1 | 0·8 ± 39·4 | 180·3 ± 18·2 | 211·2 ± 49·0 | 30·8 ± 51·3 |
| + Cr −Cu | 93·1 ± 20·0 | 246·5 ± 32·5 | 153·4 ± 40·7 | 105·8 ± 24·4 | 279·5 ± 35·5 | 173·7 ± 47·4 |
| − Cr +Cu | 153·1 ± 21·1 | 175·9 ± 36·9 | 22·9 ± 41·8 | 178·8 ± 21·3 | 166·5 ± 35·3 | − 12·3 ± 40·4 |
| + Cr +Cu | 110·7 ± 27·8 | 119·1 ± 23·4 | 8·4 ± 33·7 | 150·9 ± 39·6 | 134·6 ± 30·5 | − 16·3 ± 49·8 |
| − Cr | 159·6 ± 12·3 | 172·2 ± 25·9 | 12·6 ± 28·0 | 179·5 ± 13·7 | 188·8 ± 29·7 | 7·1 ± 32·0 |
| + Cr | 101·2 ± 16·2 | 187·7 ± 27·0 | 86·5 ± 33·2 | 126·6 ± 22·4 | 212·7 ± 30·9 | 86·0 ± 42·8 |
| − Cu | 130·1 ± 15·2 | 207·2 ± 26·8 | 77·1 ± 34·5 | 143·1 ± 17·9 | 245·4 ± 30·6 | 102·3 ± 39·0 |
| + Cu | 134·9 ± 17·3 | 151·6 ± 23·9 | 16·7 ± 27·0 | 166·9 ± 20·3 | 151·8 ± 23·1 | − 16·5 ± 30·3 |
| Factors | P-values | |||||
| Cr | < 0·01 | 0·76 | 0·10 | 0·06 | 0·64 | 0·14 |
| Cu | 0·93 | 0·10 | 0·14 | 0·41 | < 0·02 | < 0·02 |
| Cr × Cu | 0·45 | 0·06 | < 0·05 | 0·38 | 0·21 | 0·15 |
Values are mean ± s.e.m., n = 7–8.
See Table 1 for description of groups.
Change, difference in values between baseline and the end of the study (after 12 weeks of supplementation).
Fig. 1.
Lymphocyte proliferation with ConA (50 g/ml) stimulation from whole blood cell cultures at baseline (□) and after 12 weeks of supplementation (■) with Cr, Cu or both Cr and Cu (n = 7–8). See Table 1 for abbreviations. *Significantly different from baseline for this treatment group (P < 0·05). When comparing treatment groups at baseline (A, B or AB), values with different capital letters are significantly different (P < 0·05). When comparing treatment groups after 12 weeks of supplementation (a, b or ab), values with different lower case letters are significantly different (P < 0·05).
Fig. 2.
Lymphocyte proliferation with ConA (100 g/ml) stimulation from whole blood cell cultures at baseline (□) and after 12 weeks of supplementation (■) with Cr, Cu or both Cr and Cu (n = 7–8). See Table 1 for abbreviations. *Significantly different from baseline for this treatment group (P < 0·05). When comparing treatment groups at baseline (A, B or AB), values with different capital letters are significantly different (P < 0·05). When comparing treatment groups after 12 weeks of supplementation (a, b or ab), values with different lower case letters are significantly different (P < 0·05).
Discussion
Originally, this study was designed to investigate the effect of Cr and/or Cu supplementation on blood lipid parameters in postmenopausal women with high blood cholesterol. Cr and/or Cu were found to have effects on lipid parameters in ovariectomized rats in a previous study in our laboratory. Therefore, postmenopausal women with high cholesterol who are not taking any lipid lowering medication or hormone replacement therapy were included in this study. Blood glucose was not considered in the subject inclusion criteria. However, people with diabetes tend to have impaired immune function compared with people without diabetes [22–24]. Therefore, subjects with diabetes were excluded from the data analysis in the present study.
Oestrogen affects cell differentiation in the thymus and bone marrow. Oestrogen treatment in ovariectomized rats suppresses B cell differentiation. The number of bone marrow cells and the level of B lymphocyte differentiation significantly increases following oestrogen deficiency in ovariectomized rats [25]. Moreover, increased oestrogen levels during pregnancy causes thymic atrophy. Thus, it alters the number and subset composition of thymus lymphocytes [26]. After 1 month of ethinyl oestradiol treatment, the mixed lymphocyte reaction was significantly decreased compared to before treatment in postmenopausal women [27]. These studies suggest that oestrogen deficiency in postmenopausal women might have beneficial effects on immune responses.
However, there are no studies about the effects of mineral supplementation on postmenopausal women not receiving oestrogen replacement therapy. Although previous studies have investigated the effects of oestrogen therapy on immune function in postmenopausal women [27–29], no subjects analysed in this study were taking oestrogen.
The average BMI of subjects in this study indicates that many subjects were obese. A BMI > 30 kg/m2 is considered as obesity [30]. The average reported energy intake of subjects was less than the recommended energy allowances. The recommended energy allowance for females over 51 years is 1900 kCal [31]. In spite of a lower kCal intake, these subjects were classified as obese. It is common that obese people underestimate their intake when self-reporting their dietary intake. People report about 50% of their actual kCal intake and they report foods that they believe they should have consumed instead of those they actually consumed [32,33]. In addition, the errors might have occurred in the food intake recall estimates. Subjects were taught about completion of food frequency forms using food models. These subjects might have reported a lower kCal intake than the actual intake because subjects had to depend on their memories for completion of food frequency forms for seven days. These factors might have created errors in dietary intake assessment.
The percentage of carbohydrate intake of these subjects is lower than the US dietary goals (58%) and the percentage of protein and fat higher (12% protein and 30% fat) [34]. In the present study, the supplemental level of Cr, Cu or combined Cr and Cu was based on estimated safe and adequate daily dietary intakes (ESADDI). The ESADDI of Cr is 50–200 μg/d [31]. However, the dietary Cr intake was not analysed in the present study due to limitations of the Food Processor software. The average dietary Cu intake (1·0 mg/d) was lower than the current ESADDI for Cu (1·5–3·0 mg/d) and higher than the average intake by females (0·93 mg/d) in the US [31,35,36].
Basophil numbers are rapidly increased during allergic conditions and parasitic infection. The distribution of basophils at baseline (0·9%) and after 12 weeks of Cu supplementation (1·1%) was within a normal or close to a normal range (< 1%), so this significant change in the basophils with Cu supplementation may or may not have any clinical importance.
However, a significant increase in basophil numbers might indicate that there were unknown infections or allergic reactions in the Cu-supplemented groups. It was not clear whether the increased basophil numbers might be due to allergic conditions, infectious conditions, or 12 weeks of Cu supplementation in this study.
To analyse the effect of dietary and environmental factors on immunocompetence in vitro, the lymphocyte proliferation assay is most commonly used due to its reliability and technical simplicity [37]. In the current study, whole blood cell cultures were used for the lymphocyte proliferation measurement. Whole blood cell cultures have beneficial effects in this assay compared to lymphocyte cultures separated from whole blood by a density gradient. Whole blood cell cultures prevent changes in leucocyte populations and other mediators in cell cultures [38,39] which would further affect the immune response to PHA or ConA stimulation [40]; moreover, the lymphocyte proliferation assay using whole blood cell cultures is simpler and easier compared to using separated lymphocyte cultures [38,39].
To the best of our knowledge, this is the first report on the effects of Cr supplementation on lymphocyte proliferation in human subjects. Chromium as Cr(H2O)6·Cl3 in the cell culture medium does not affect either human peripheral blood lymphocyte proliferation with PHA stimulation or cell morphology [41,42]. However, these studies involved investigation of the toxicity of Cr in vitro rather than the effects of Cr supplementation on immune function in vivo and are not relevant to the current study. Therefore, the effects of Cr supplementation on immune function in cows are discussed due to their relevance.
In the current study, the greatest increases in lymphocyte proliferation after 12 weeks compared to baseline were obtained with Cr supplementation and indicate that Cr supplementation might have potential beneficial effects on immune response. Similar beneficial effects were observed in Burton et al. study [8]. Chromium-supplemented diets fed to cows resulted in increased antiovalbumin and ConA-stimulated responses [8]. However, in the current study, the Cr supplement group showed lower lymphocyte proliferation with varying concentrations of PHA-L or ConA stimulation at baseline compared to the other groups. The lower lymphocyte proliferation at baseline might have affected the increase in lymphocyte proliferation following 12 weeks of Cr supplementation. The lower proliferation at baseline in the Cr supplement group could not be explained in this study: there were no significant differences in subject number, age, BMI or dietary intakes.
Unlike supplementation with Cr alone which significantly increased the stimulation index, the combination of Cr and Cu supplementation failed to increase the index. Thus, the presence of Cu prevented the enhancing effect of Cr on lymphocyte proliferation. As a result, the stimulation index in the Cr and Cu supplementation group was significantly lower than in the Cr supplementation group. Also, a small decrease in lymphocyte proliferation after 12 weeks of Cu supplementation as compared to baseline and a significantly lower lymphocyte proliferation with ConA stimulation (100 μg/ml) after 12 weeks of Cu supplementation compared to Cr supplementation indicate the potential immunosuppressive effect of Cu in these subjects. This result contradicts the results of some studies on the effects of Cu on immune responses. In most animal studies, lymphocyte proliferation decreased with Cu deficiency [15,43,44]. Furthermore, Prohaska and Lukasewycz [45] found changes in the overall incorporation of thymidine into DNA following stimulation by ConA, PHA or LPS in C58 mice fed a Cu-deficient diet. In humans, serum immunoglobulin levels were normal in subjects with Cu deficiency or in subjects with a genetic Cu deficiency such as Menkes kinky-hair [16,46]. In addition, the response of T and B lymphocytes to mitogen stimulation is normal in subjects with Menkes kinky-hair [46]. In contrast, PBMNC proliferation induced by PHA, ConA and pokeweed mitogens (PWM) is lower after a low Cu diet (0·38 mg Cu/d) compared to diet with 0·66 mg Cu/d or 2·49 mg Cu/d in young healthy nonsmoking men [18]. Also, PHA- and ConA-stimulated lymphocyte proliferation using whole blood culture is significantly decreased in subjects with a low plasma Cu level compared to normal subjects [17]. The different results from these studies might be affected by the Cu status of subjects. The results in the present study indicate that the Cu supplementation level (3·0 mg Cu/d) used might be at greater than optimal concentrations for promoting a human immune response.
In conclusion, the results of this study suggest that Cr and Cu have interactive functions in lymphocyte proliferation. A potential immunosuppressive and preventive interaction of Cr and/or Cu supplementation on lymphocyte proliferation was observed in these subjects. However, the nature of these interactive effects between Cr and Cu require further investigation.
Acknowledgments
We would like to acknowledge Oklahoma Center for the Advancement of Science and Technology for funding this study.
References
- 1.Chandra RK, Newberne PM. Interactions of nutrition, infection and immune response in animals. In: Chandra RK, Newberne PM, editors. Nutrition, immunity and infection, mechanisms of interactions. New York: Plenum Press; 1977. pp. 127–80. [Google Scholar]
- 2.Harbige LS. Nutrition and immunity with emphasis on infection and autoimmune disease. Nutrition Health. 1996;10:285–312. doi: 10.1177/026010609601000401. [DOI] [PubMed] [Google Scholar]
- 3.Chandra RK. Graying of the immune system: can nutrient supplements improve immunity in the elderly? JAMA. 1997;277:1398–9. [PubMed] [Google Scholar]
- 4.Kawakami K, Kadota J, Iida K, et al. Reduced immune function and malnutrition in the elderly. Tohoku J Exp Med. 1999;187:157–71. doi: 10.1620/tjem.187.157. [DOI] [PubMed] [Google Scholar]
- 5.Hulsewe KW, van Acker BA, von Meyenfeldt MF, et al. Nutritional depletion and dietary manipulation: effects on the immune response. World J Surg. 1999;23:536–44. doi: 10.1007/pl00012344. [DOI] [PubMed] [Google Scholar]
- 6.Chang X, Mowat DN. Supplemental chromium for stressed and growing feeder calves. J Anim Sci. 1992;70:559–65. doi: 10.2527/1992.702559x. [DOI] [PubMed] [Google Scholar]
- 7.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;71:232–8. doi: 10.2527/1993.711232x. [DOI] [PubMed] [Google Scholar]
- 8.Burton JL, Mallard BA, Mowat DN. Effects of supplemental chromium on immune responses of periparturient and early lactation dairy cows. J Anim Sci. 1993;71:1532–9. doi: 10.2527/1993.7161532x. [DOI] [PubMed] [Google Scholar]
- 9.Jones DG, Suttle NF. The effect of copper deficiency on the resistance of mice to infection with Pasteurella haemolytica. J Comp Pathol. 1983;93:143–9. doi: 10.1016/0021-9975(83)90052-x. [DOI] [PubMed] [Google Scholar]
- 10.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;118:214–21. doi: 10.1093/jn/118.2.214. [DOI] [PubMed] [Google Scholar]
- 11.Davis MA, Johnson WT, Briske-Anderson M, et al. Lymphoid cell functions during copper deficiency. Nutr Res. 1987;7:211–22. [Google Scholar]
- 12.Prohaska JR, Lukasewycz OA. Copper-deficiency suppresses the immune response of mice. Science. 1981;213:559–61. doi: 10.1126/science.7244654. [DOI] [PubMed] [Google Scholar]
- 13.Lukasewycz OA, Prohaska JR, Meyer SG, et al. Alterations in lymphocyte subpopulations in copper-deficient mice. Infection Immunity. 1985;48:644–7. doi: 10.1128/iai.48.3.644-647.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Lucasewycz OA, Kolquist KL, Prohaska JR. Splenocytes from copper-deficient mice are low responders and weak stimulators in mixed lymphocyte reactions. Nutr Res. 1987;7:43–52. [Google Scholar]
- 15.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;125:2658–68. doi: 10.1093/jn/125.10.2658. [DOI] [PubMed] [Google Scholar]
- 16.Heresi G, Castillo-Duran C, Munoz C, et al. Phagocytosis and immunoglobulin levels in hypocupremic infants. Nutr Res. 1985;5:1327–34. [Google Scholar]
- 17.Smith D, Hopkins RG, Kutlar A, et al. Diagnosis and treatment of copper deficiency in adult humans. FASEB J. 1994;8:A4754. [Google Scholar]
- 18.Kelley DS, Daudu PA, Taylor PC, et al. Effects of low-copper diets on human immune response. Am J Clin Nutr. 1995;62:412–6. doi: 10.1093/ajcn/62.2.412. [DOI] [PubMed] [Google Scholar]
- 19.ESHA Research. Salem, Oregon: Food processor plus nutrition and fitness program. Version 7.0. [Google Scholar]
- 20.Kramer TR, Burri BJ. Modulated mitogenic proliferative responsiveness of lymphocytes in whole-blood cultures after a low-carotene diet and mixed-carotenoid supplementation in women. Am J Clin Nutr. 1997;65:871–5. doi: 10.1093/ajcn/65.3.871. [DOI] [PubMed] [Google Scholar]
- 21.Statistical Analysis System Institute. Cary, NC: SAS/STAT user's guide. Version 6.11. [Google Scholar]
- 22.Nolan CM, Beaty HN, Bagdade JD. Further characterization of the impaired bactericidal function of granulocytes in patients with poorly controlled diabetes. Diabetes. 1978;27:889–94. doi: 10.2337/diab.27.9.889. [DOI] [PubMed] [Google Scholar]
- 23.Rabinowitz SG. Infection in the diabetic patient. In: Rifkin H, Raskin P, editors. Diabetes mellitus V. Bowie, Maryland: Robert J. Brady Co., Am Diabetes Assoc; 1981. pp. 213–7. [Google Scholar]
- 24.Casey JI. Host defense abnormalities in diabetic patients. In: Rifkin H, Raskin P, editors. Diabetes mellitus V. Bowie, Maryland: Robert J. Brady Co., Am Diabetes Assoc; 1981. pp. 219–23. [Google Scholar]
- 25.Masuzawa T, Miyaura C, Onoe Y, et al. Estrogen deficiency stimulates B lymphopoiesis in mouse bone marrow. J Clin Invest. 1994;94:1090–7. doi: 10.1172/JCI117424. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Olsen NJ, Kovacs WJ. Gonadal steroids and immunity. Endocrine Rev. 1996;17:369–84. doi: 10.1210/edrv-17-4-369. [DOI] [PubMed] [Google Scholar]
- 27.Helgason S, Schoultz BV. Estrogen replacement therapy and the mixed lymphocyte reaction. Am J Obstet Gynecol. 1981;141:393–7. doi: 10.1016/0002-9378(81)90600-1. [DOI] [PubMed] [Google Scholar]
- 28.Malarkey WB, Burleson M, Cacioppo JT, et al. Differential effects of estrogen and medroxyprogesterone on basal and stress-induced growth hormone release, IGF-1 levels, and cellular immunity in postmenopausal women. Endocrine. 1997;7:227–33. doi: 10.1007/BF02778145. [DOI] [PubMed] [Google Scholar]
- 29.Vollenhoven RF, McGuire JL. Estrogen, progesterone and testosterone: can they be used to treat autoimmune diseases? Cleve Clin J Med. 1994;61:276–84. doi: 10.3949/ccjm.61.4.276. [DOI] [PubMed] [Google Scholar]
- 30.Mahan LK, Escott-Stump S. Krause's food nutrition, diet therapy. 9. Pennsylvania: W.B. Saunders Co; 1996. Weight management and eating disorders; pp. 451–88. [Google Scholar]
- 31.National Research Council. Recommended dietary allowance. 10. Washington, DC: National Academy of Sciences; 1989. [Google Scholar]
- 32.Schoeller DA. Limitations in the assessment of dietary energy intake by self-report. Metabolism. 1995;44:18–22. doi: 10.1016/0026-0495(95)90204-x. [DOI] [PubMed] [Google Scholar]
- 33.Fricker J, Baelde D, Igoin-Apfelbaum L, et al. Underreporting of food intake in obese ‘small eaters’. Appetite. 1992;19:273–83. doi: 10.1016/0195-6663(92)90167-5. [DOI] [PubMed] [Google Scholar]
- 34.Lee RD, Nieman DC. Nutritional assessment. 2. Missouri: Mosby; 1996. Standards for nutrient intake; pp. 15–90. [Google Scholar]
- 35.Olivares M, Uauy R. Limits of metabolic tolerance to copper and biological basis for present recommendations and regulations. Am J Clin Nutr. 1996;63:846S–52S. doi: 10.1093/ajcn/63.5.846. [DOI] [PubMed] [Google Scholar]
- 36.Pennington J, Young B. Total Diet Study nutritional elements, 1982–89. J Am Diet Assoc. 1991;91:179–83. [PubMed] [Google Scholar]
- 37.Failla M, Hopkins RG. Copper and immunocompetence. In: Fischer PWF, Labbe MRL, Cockell KA, Gibson RS, editors. Trace elements in man and animals – 9: Proceedings of the ninth international symposium on trace elements in man and animals. Ottawa, Canada: NRC Research Press; 1997. pp. 425–8. [Google Scholar]
- 38.Bloemena E, Roos MTL, Van Heijst JLAM, et al. Whole-blood lymphocyte cultures. J Immunol Meth. 1989;122:161–7. doi: 10.1016/0022-1759(89)90260-3. [DOI] [PubMed] [Google Scholar]
- 39.Bocchieri MH, Talle MA, Maltese LM, et al. Whole blood culture for measuring mitogen induced T cell proliferation provides superior correlations with disease state and T cell phenotype in asymptomatic HIV-infected subjects. J Immunol Meth. 1995;181:233–43. doi: 10.1016/0022-1759(95)00007-w. [DOI] [PubMed] [Google Scholar]
- 40.Petrovsky N, McNair P, Harrison LC. Circadian rhythmicity of interferon-gamma production in antigen-stimulated whole blood. Chronobiologia. 1994;21:293–300. [PubMed] [Google Scholar]
- 41.Rajaram R, Nair BU, Ramasami T. Chromium (III) induced abnormalities in human lymphocyte cell proliferation: evidence for apoptosis. Biochem Biophys Res Comm. 1995;210:434–40. doi: 10.1006/bbrc.1995.1679. [DOI] [PubMed] [Google Scholar]
- 42.Borella P, Manni S, Giardino A. Cadmium, nickel, chromium and lead accumulate in human lymphocytes and interferes with PHA-induced proliferation. J Trace Elem Electrolytes Health Dis. 1990;4:87–95. [PubMed] [Google Scholar]
- 43.Bala S, Failla ML, Lunney JK. Alterations in splenic lymphoid cell subsets and activation antigens in copper-deficient rats. J Nutr. 1991;121:745–53. doi: 10.1093/jn/121.5.745. [DOI] [PubMed] [Google Scholar]
- 44.Lukasewycz OA, Prohaska JR. Lymphocytes from copper-deficient mice exhibit decreased mitogen reactivity. Nutr Res. 1983;3:335–41. [Google Scholar]
- 45.Prohaska JR, Lukasewycz OA. Copper deficiency during perinatal development: effects of the immune response of mice. J Nutr. 1989;119:922–31. doi: 10.1093/jn/119.6.922. [DOI] [PubMed] [Google Scholar]
- 46.Sullivan JL, Ochs HD. Copper deficiency and the immune system. Lancet. 1978;23:686. doi: 10.1016/s0140-6736(78)92806-4. [DOI] [PubMed] [Google Scholar]