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Department of Nutritional Sciences, University of California, Berkeley, CA 94720 and * Western Human Nutrition Research Center, University of California, Davis, CA 95616
3To whom correspondence should be addressed. E-mail: dkelley{at}whnrc.usda.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and tumor necrosis factor-
or neutrophil superoxide production. The results suggest that changes in lymphocyte proliferation and IL-2R expression may be early markers of mild zinc deficiency.
KEY WORDS: • zinc deficiency • immunity • proliferation • cytokines • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–4
). Patients with acrodermatitis enteropathica (AE),4
an inherited disorder of zinc malabsorption, frequently experience severe infections with fungi, viruses and bacteria (1
). Lymphocyte numbers and proliferative responses to the mitogen phytohemagglutinin (PHA) have been impaired in patients with AE and corrected with zinc supplementation (1
). Secretion of interferon- (IFN-) and tumor necrosis factor- (TNF-) by mitogen-stimulated peripheral blood mononuclear cells (PBMNC) was significantly decreased in healthy men after experimental zinc depletion (5
). Reduced interleukin-2 production has resulted from zinc depletion (5
,6
) and has been seen in mildly deficient individuals (4
,7
). Zinc deficiency has also been accompanied by alterations in neutrophil chemotaxis and superoxide generation (4
,8
), declines in natural killer cell activity (1
), impaired B lymphocyte antibody responses (4
) and depressed delayed-type hypersensitivity skin responses (9
,10
); these conditions have been corrected with zinc supplementation.
Mild-to-moderate zinc deficiency is frequently reported in developing countries and also is associated with depressed immunity (4
,11
,12
). Zinc deficiency is often assumed when zinc supplementation improves immune response. The prevalence of mild deficiency in industrialized countries is unknown due to the lack of sensitive clinical biomarkers of zinc status (13
). In the United States, inadequate zinc intakes are seen in children from low income families and frail elderly persons, and secondary zinc deficiency accompanies medical conditions such as renal disease, sickle cell anemia and acquired immunodeficiency syndrome (14
). Despite the clear association between zinc status and immune response, few recent clinical studies investigating zinc depletion have included measurements of immune parameters (14
). Previous studies have not fully characterized the effects of mild dietary zinc depletion on the immune response of healthy humans, and the primary effects of mild human zinc deficiency are not known.
In the present study, we investigated the effects of a marginal zinc intake of 4.6 mg zinc/d, which is 42% of the 2001 recommended dietary allowance (RDA) (15
), on several indices of immune response in healthy men. The purpose of this study was to characterize the effect of a low zinc intake on clinical measures of immune response and to identify the initial defects, if any, that would result from a zinc-restricted diet.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The research protocol was approved by the University of California Committee for Protection of Human Subjects, and informed consent was obtained from all subjects. Eight healthy nonsmoking men (27–47 y old) completed the study. The subjects had been screened to ensure that their plasma Zn and lymphocyte concentrations were normal (plasma Zn 
12 µmol/L, lymphocytes 1.5 billion/L) and that they had no history of chronic illness, autoimmune disease, alcohol or drug abuse, or mineral megadosing.
The subjects stayed in a metabolic unit for all but the first 3 wk of the 20-wk study. They were given a controlled 3-d rotating diet that contained 4.6 mg Zn/d. Zn intakes were adjusted by giving Zn gluconate supplements during the 5-wk baseline (BL) and 5-wk repletion periods (RP), and placebos during the 10-wk Zn-restriction period (ZR). The subjects were not told when Zn intakes were changed. During BL and RP, subjects’ Zn intakes totaled 13.7 mg/d, an amount close to the U.S. median intake of 13 mg (15
). During ZR, the subjects consumed 4.6 mg of highly available Zn. The phytate:Zn molar ratio of the diet was low, averaging 5.5:1 during ZR. As a comparison, diets typical of those in the U.S. have phytate:Zn ratios within the range of 5–15 (16
) and Zn absorption is significantly impaired when the phytate:Zn ratio is > 10:1 (17
).
The experimental diet consisted of conventional foods supplemented with an extra-energy formula of oil, sugar and dextromaltose to meet individual energy needs, and egg albumen powder to meet the protein RDA (0.8 g/kg) for each subject. Magnesium and calcium were supplemented at 125 and 200 mg/d, respectively, to meet the 1989 RDA for these minerals; other nutrients in the experimental diet were at or above the RDA. Iron supplements of 3 mg/d were provided to compensate for blood losses due to sampling. The men consumed deionized water ad libitum and amounts consumed were recorded daily. Additional details regarding the study diet have been published (18
).
Laboratory procedures.
Blood samples were collected weekly to monitor plasma Zn concentrations, and twice (1 wk apart) at the end of each metabolic period (BL, ZR, and RP) to measure leukocyte counts, PBMNC proliferation and cytokine secretion. Samples for neutrophil analysis were taken at an additional time point 3 wk after the start of ZR. Plasma samples were collected into zinc-free plastic Monovette syringes (Sarstedt, Newton, NC) or Vacutainers (Becton Dickinson, Rutherford, NJ). Solutions used for the cell separation procedure were tested and found to have zero, or negligible amounts of zinc. The results presented for leukocyte counts, PBMNC proliferation, and IL-2R secretion are the means of the values obtained on the two study days scheduled at the ends of the metabolic periods. For all analyses, blood was drawn from the antecubital veins of subjects who had fasted overnight (12 h). Blood samples for leukocyte analyses were used immediately. Plasma for Zn analysis was separated from red blood cells by centrifugation, 10 min at 230 x g and frozen at -20°C until assayed. For quality control of laboratory procedures, blood samples from 4 healthy men and women who were not in the study were tested along with samples from the 8 experimental subjects.
Plasma Zn concentration and blood cell count.
Zn concentrations in blood plasma were measured using flame atomic absorption spectrometry with a Smith-Hieftje 22 spectrometer (Thermo Jarrell Ash, Franklin, MA). Samples were vortexed and diluted eightfold in 0.125 mol/L HNO3 (Trace Metal Grade; Fisher Scientific, Pittsburgh, PA) and a bovine liver standard (National Institute of Standards and Technology, Gaithersburg, MD) was analyzed with each run to ensure spectrometer accuracy. Complete blood counts with differential estimations of white blood cells, lymphocytes, monocytes and granulocytes were performed using a System 9000 DIFF automated cell counter (Biochem Immunosystems, Allentown, PA). Readings were done in duplicate and averaged for each sample.
Isolation and culture of PBMNC.
PBMNC were isolated from whole blood using Histopaque-1077 (Sigma, St. Louis, MO) density gradient as previously described (19
). Briefly, cells were cultured in RPMI-1640 (Gibco, Grand Island, NY) with 10% autologous serum, L-glutamine (2 mmol/L), penicillin (100,000 U/L), streptomycin (100 mg/L) and gentamycin (20 mg/L). To measure cell proliferation, 100 µL of culture medium containing 100,000 PBMNC was added to each well of a 96-well flat-bottomed plate. Then, another 100 µL of medium was added, either with or without mitogens. The mitogen PHA (Sigma) was added to achieve final culture concentrations of 1.2, 2.5, 5.0, 10.0 and 20.0 mg/L. Cells were incubated for 72 h, and 37 kBq [3H]thymidine, in 50 µL, was added to each well for the final 24 h. PBMNC were collected on filter strips and radioactivity determined using a Packard ß-gas counter. Thymidine incorporation into cellular DNA (Bq/1000 cells) was used as the index of PBMNC proliferation.
PBMNC were cultured with PHA to stimulate secretion of IL-2R and IFN- and incubated with lipopolysaccharide (LPS; Sigma) to induce TNF- secretion. Mitogen concentrations in the cultures were 2.5 and 10 mg/L PHA for generation of IL-2R, 10 mg/L PHA for IFN-, and 1 and 10 mg/L LPS for TNF-. PBMNC with LPS were incubated for 24 h, and cells with PHA for 72 h, before media from wells were collected. Supernatant samples were stored at -70°C until cytokine analysis. ELISA kits were used to analyze the cytokines (Quantikine assays, R&D Systems, Minneapolis, MN) and IL-2R (Immunotech, Westbrook, ME). Samples from the three metabolic periods were analyzed simultaneously on one 96-well plate.
Neutrophil superoxide generation.
Neutrophils were isolated from whole blood using Neutrophil Isolation Media (NIM, Cardinal Associates, Santa Fe, NM), and the neutrophil pellet was suspended in 1 mL D-PBS (without Ca and Mg, no phenol red; Sigma) for cell counting. Cells were diluted to a concentration of 5 x 109 cells/L using D-PBS (with Ca and Mg, no phenol red) that was supplemented with glucose (1.35 mg glucose/L D-PBS). Cell samples were kept on ice for 
45 min until assayed. Superoxide (O2-) generation was determined using a kinetic microplate assay that measured the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome C in phorbol myristate acetate (PMA)-stimulated cells. The reagents used in the assay were cytochrome c (from horse heart, Sigma) diluted to 9.25 g/L in D-PBS/glucose, SOD (Sigma) diluted to 1 g/L in D-PBS/glucose and PMA diluted to 2.5 mg/L in triply deionized water. Samples were assayed in quadruplicate in a 96-well flat-bottomed plate and were measured with and without SOD; the SOD+ wells served as the blank for the SOD- wells. For this assay, the following were added to each well: 25 µL of the cytochrome c solution, 15 µL of either the SOD solution or triply deionized water and enough D-PBS/glucose to obtain a reaction mixture of 250 µL. After the plate was incubated at 37°C for 5 min, 125,000 cells were added to each well and the plate was incubated for another 3 min at 37°C. PMA (20 µL) was added and the plate immediately placed into a Ceres 900 microplate reader (BioTek Instruments, Winooski, VT) preset to 37°C and set to shake at a low speed throughout the assay. The plate was read every 4 min for a 40-min period at an absorbance of 550 nm.
Statistical analysis.
Data were analyzed by repeated-measures ANOVA followed by Tukey’s studentized range test for pairwise comparisons; P < 0.05 was considered significant. All data are presented in the text and figure legends as means ± SD. Analyses were performed using Statistical Package for Social Sciences software (SPSS, Chicago, IL) located on a mainframe computer at the University of California, Berkeley.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Leukocytes.
Circulating leukocytes remained constant, numbering [5.4 ± 1.0] x 109/L throughout the study. Their percentage distribution as lymphocytes, monocytes and neutrophils did not change, and were 33.8 ± 6.4, 7.7 ± 1.5 and 58.6 ± 7.2%, respectively.
Neutrophil superoxide generation.
There were no differences in superoxide production by neutrophils taken from 6 men during the study (samples for 2 men were unavailable for this assay). The shape of the absorbance curve was also not altered by zinc restriction.
PBMNC proliferation.
Proliferation of PBMNC cultured with different concentrations of PHA (0,1.2, 2.5, 5.0, 10.0, 20.0 mg/L) are shown in Figure 1
. Zinc restriction reduced (P < 0.01) PBMNC proliferation at all mitogen concentrations tested except 10 mg/L compared with the corresponding baseline values and the diminished PBMNC proliferation was not restored by 5 wk of zinc repletion.
|
Dietary zinc restriction reduced IL-2R secretion by PBMNC stimulated at a suboptimal PHA concentration (2.5 mg/L), 31% (Fig. 2A
; P = 0.058) compared with corresponding values at the end of BL. IL-2R secretion continued to decline and was 40% lower than BL values at the end of RP (P < 0.05). However, when cells were treated with PHA concentrations required for maximal stimulation (10 mg/L; Fig. 2
B), restricted zinc had no effect on secretion of IL-2R. The amount of IL-2R secreted at 10 mg/L PHA was 10 times more than that at 2.5 mg/L. The IL-2R secretion of unstimulated cultures was below the detection limit of the assay system. The secretion of IFN- and TNF- was unchanged throughout the study (results not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), we also found significant declines in PHA-stimulated PBMNC proliferation and IL-2R secretion by the end of a 42-d copper-restriction period that were not reversed after 24 d of copper repletion. Lymphocytes have long life spans, and certain classes of lymphocytes can survive for years (21
,22
).
There have been reports that patients with AE have had impaired lymphocyte responses to PHA, and that these were reversed by zinc supplementation (23
). Similarly, zinc-deficient hospital patients maintained on total parenteral nutrition have displayed dramatic improvements in PHA-stimulated lymphocyte proliferation after zinc therapy (24
,25
). Secretion of IL-2R has not been measured previously in zinc-deficient patients or in zinc-restricted humans. In zinc-deficient young adult mice, IL-2R on T cells were unaffected by zinc deficiency, but the mice exhibited reduced mitogen-induced proliferation of splenocytes compared with zinc-adequate controls (26
). Further study must be done to resolve whether IL-2R secretion is affected by low dietary zinc intakes in zinc-deficient humans.
In contrast to the IL-2R results, cytokine secretion did not change during the study. Although Beck et al. (5
) found that mild zinc deficiency was associated with decreased production of IFN- and TNF-, their subjects consumed a diet containing 2–3.5 mg zinc/d that included 1 g phytic acid to reduce bioavailability, and the depletion phase lasted 20–24 wk. The zinc-restriction regimen in our study was less severe and did not induce apparent zinc deficiency in our subjects; therefore; our results do not contradict those of Beck et al.
Leukocyte numbers were not affected by zinc restriction. Although lymphopenia is well documented in severe zinc deficiency (1
), its incidence during mild-to-moderate zinc deficiency is not known. Ruz et al. (10
) measured total leukocyte and lymphocyte concentrations in a zinc depletion study in which 15 healthy men were fed a high phytate diet containing 0.6 and 4 mg Zn/d for 1 and 6 wk, respectively. Leukocyte numbers were unchanged throughout the study, even though there were significant decreases in fasting plasma zinc concentrations at the end of depletion. This suggests that leukocyte and lymphocyte counts are not sensitive indicators of zinc status. Our data also confirm that leukocytes are not responsive to short-term, low zinc intakes.
There have not been previous studies that tested neutrophil superoxide generation in zinc-deficient patients or in zinc-restricted humans. However, adult rats consuming a zinc-deficient diet containing 0.5 µg Zn/g for 6 wk exhibited an enhancement of superoxide production by PMA-stimulated neutrophils and a shorter latency to the onset of superoxide production compared with rats fed a control diet with 25 µg Zn/g (8
). Our results showed that the oxidative burst function of neutrophils was unchanged by short-term dietary zinc restriction. Therefore, we could not confirm an association between zinc depletion and altered neutrophil superoxide production in human subjects.
In conclusion, we saw evidence of impaired PBMNC function during mild zinc-restriction in healthy adult men that was not restored after 5 wk of zinc repletion. The subjects did not exhibit increased rates of infection or illness, and other tests of immune function that we conducted indicated that immunity was largely maintained. The reductions in PBMNC proliferation and IL-2R secretion suggest that a 4.6 mg zinc intake is close to the threshold of zinc nutriture required to maintain an optimal immune response. However, a marginal zinc intake may be adequate to sustain overall immunity in healthy men for a short-term period.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by USDA/ARS Western Human Nutrition Research Center Intramural funding.
4 Abbreviations used: AE, acrodermatitis enteropathica; BL, baseline period; IL-2R, interleukin-2 receptor; IFN-, interferon-; LPS, lipopolysaccharide; PBMNC, peripheral blood mononuclear cells; PHA, phytohemagglutinin; PMA, phorbol myristate acetate; RDA, Recommended Dietary Allowances; RP, repletion period; SOD, superoxide dismutase; TNF-, tumor necrosis factor-, ZR, zinc-restriction period.
Manuscript received 2 January 2002. Initial review completed 8 February 2002. Revision accepted 25 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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