jurnal gizi

Journal of Gerontology: BIOLOGICAL SCIENCESCopyright 2007 by The Gerontological Society of America

2007, Vol. 62A, No. 6, 598608

Effect of Zinc Supplementation on the Immune Status of Healthy Older Individuals Aged 5570 Years:

The ZENITH Study

Clare F. Hodkinson,1 Mary Kelly,1 H. Denis Alexander,1,2 Ian Bradbury,1 Paula J. Robson,1 Maxine P. Bonham,1 Jacqueline M. OConnor,1 Charles Coudray,3

J. J. Strain,1 and Julie M. W. Wallace1

1Northern Ireland Centre for Food and Health (NICHE), University of Ulster, Coleraine, Northern Ireland. 2Department of Haematology, Belfast City Hospital, Northern Ireland.

3Centre de Recherche en Nutrition Humaine dAuvergne, Unite Maladies Metaboliques et Micro-nutriments INRA, Centre de Recherche de Clermont-Ferrand, Saint Gene`s Champanelle, France.

Aging is associated with alterations in the immune system, effects which may be exacerbated by inadequate zinc (Zn) status. We examined the relationship between Zn status and markers of immunity and the effect of supplementation with 15 mg or 30 mg Zn/d for 6 months on immune status in healthy individuals. Zn status was assessed by dietary intake and biochemical indices. Immune status was assessed by multiple flow cytometric methods. At baseline, Zn concentration was positively associated with lymphocyte subpopulation counts and T-lymphocyte activation. Zn supplementation of 30 mg/d significantly lowered B-lymphocyte count, albeit at month 3 only. Lower doses of Zn (15 mg Zn/d) significantly increased the ratio of CD4 to CD8 T lymphocytes at month 6. Overall, these findings suggest that total Zn intake (diet plus supplementation) of up to 40 mg Zn/d do not have significant long-term effects on immune status in apparently healthy persons aged 5570 years.

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LDER individuals are at risk of developing age-associated immune deficiencies, a process known as

immunosenescence. Theresultingimmunedysfunctioncontrib-utes to increased risk of susceptibility to infectious diseases, autoimmune and inflammatory disorders, and cancer. Although major restructuring is evident throughout the immune system (15), the most striking age-related changes have been observed in the adaptive immune system (69).

It has been suggested that the achievement of optimal nutritional status may help to alleviate the stress placed on the aging immune system, allowing for successful aging (10). One trace element that has been identified as essential for the maintenance and optimal functioning of the immune system is zinc (Zn). Zn deficiency has been shown to result in depressed innate and adaptive immunityspecifically, reduced antibody production, lymphocyte proliferation in response to mitogen stimulation, and decreased CD4/ CD8 ratios. These conditions can be reversed by Zn supplementation [see reviews (1114)].

To date, however, the majority of studies on Zn and immune function have focused on infectious disease in childhood (1519) or in elderly populations (2026). These studies have demonstrated that higher Zn intakes are associated with improved resistance to infection, symptom-atic relief, and reduced duration of illness; as well as improvements in antibody titer, delayed hypersensitivity reaction, and specific lymphocyte subpopulation numbers. Other studies investigating immune status have been based

on selected samples of aging volunteers or on clinically based samples, e.g., Senieur project of EURAGE (27). However, there is a distinct lack of studies that have examined the relationship between Zn status and immune status in healthy older people at risk for suboptimal Zn status such as late-middle-aged persons. It is estimated that dietary Zn intakes in 50% of 51- to 70-year-olds in Western populations fall below the recommended dietary allowance, indicating that this population may be at risk of suboptimal Zn status (28). It is unclear, however, at what point suboptimal Zn status may become deleterious to immune status and, consequently, health.

Although the deleterious effects of age have been more consistently reported with respect to adaptive immunity (6 9), we have previously reported that age-related alterations occur in markers of both innate and adaptive immunity in the current study population and, importantly, that such changes are largely sex specific (29). We hypothesized, given the findings of previous research (1526), that specific markers of adaptive immune status such as B lymphocytes, nave and memory T lymphocyte subsets, natural killer (NK) cells, the CD4/CD8 ratio, and intracellular cytokine production would respond to Zn supplementation. Other functional and phenotypic markers of immunity, for which there is less evidence of an effect of Zn, were included as secondary outcomes. There is no single marker that can provide a definitive picture of an individuals immune competence. Therefore, in the current study, an extensive

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ZN SUPPLEMENTATION AND IMMUNE STATUS

599

panel of phenotypic markers of immunity were included to provide comprehensive information on immune status (as defined by numbers of circulating lymphocyte subsets), and to facilitate the interpretation of an array of functional assays; important given that functional assays are associated with clinical endpoints and present a mechanistic un-derstanding of the immune response (30).

Consequently, the aim of the current study was to assess the associations between putative indices of Zn status and markers of immunity and to examine the effect of Zn supplementation on a comprehensive range of phenotypic and functional immune markers in free-living, apparently healthy late-middle-aged men and women, aged 5570 years.

METHODS

Study Population

A total of 147 individuals, aged 5570 years (77 women, 70 men) were recruited from across Northern Ireland through media coverage, leaflets, and the cooperation of national and local organizations with members spanning the age group of interest. Of the 147 persons recruited, 101 apparently healthy individuals were invited to participate in the study based on defined exclusion criteria, including: body mass index (BMI) , 20 or . 33 kg/m2; abnormal hematology, liver function, or kidney function; unusual dietary habits (e.g., vegetarian and vegan); current acute or chronic disease; poor neuropsychological performance; use of immune modulating medications, habitual use of vitamin and/or mineral supplements in the last 6 months; alcohol consumption of . 30 g/d (for men) or . 20 g/d (for women); and smoking . 10 cigarettes, cigars, or pipes per day. All the women recruited were postmenopausal (.12 months since their last menses) and were not receiving hormone replacement therapy. The University of Ulster Research Ethical Committee granted approval for the study. All volunteers gave informed signed consent in accordance with the declaration of Helsinki.

Study Design

The study design was a double-blind, randomized, placebo-controlled intervention. Volunteers were recruited over a 3-month period from March and were randomly assigned to receive either 15 mg/d or 30 mg/d of elemental Zn (as Zn gluconate) or an identical placebo capsule for 6 months. Supplements were supplied by E-Pharma (Ganna, France).

Collection of Peripheral Blood and Hematological Analysis

Following an overnight (.12 hours) fast, participants were asked to arrive at the research center at 8:30 AM on the study day. Body weight and height were measured at baseline, and BMI was calculated.

Blood and urine samples were collected from volunteers at baseline, at month 3, and at month 6 of the intervention. Venous anticoagulated blood (55 mL) was collected in K3EDTA, lithiumheparin, sodiumheparin anticoagulated Vacutainer tubes and serum separator tubes. K3EDTA whole blood was used for determination of immune status on a FACSCalibur

flow cytometer (BD Biosciences, Oxford, U.K.) within 4 hours of sample collection. K3EDTA whole blood was also used to assess full blood profile (conducted at Causeway Laboratory, Causeway NHSS Trust, Coleraine, Northern Ireland). Plasma, erythrocytes, and serum were separated and stored at 808C until analysis at the end of the study.

Trace Element Intake, Markers of Trace Element Status, and Measures of Inflammation

Dietary Zn, copper (Cu), and iron (Fe) intakes were assessed using a semiquantitative food diary. Participants were asked to record every item of food and drink consumed over four consecutive days. Amounts consumed were recorded in terms of household measures and units of food (e.g., slices of bread, cans of soda), and published standard food weights for the U.K. were used to convert these quantities to gram weights (31). Nutrient intakes were analyzed using the Weighed Intake analysis Software Package (WISP) 2.0 for Windows (Tinuviel Software, Warrington, U.K.), based on U.K. food composition data.

Serum, erythrocyte, and urinary Zn concentrations were determined by flame atomic absorption spectrometry on a Perkin Elmer 560 analyzer at Grenoble University Hospital, France. Urinary creatinine concentration was assessed by colorimetry, as previously described (32). Alkaline phosphatase (ALP) was analyzed using a commer-cially available kit (Roche Diagnostics, Lewes, U.K.) on a Hitachi 912 autoanalyzer (Roche Diagnostics). Hemoglo-bin (Hb), ferritin, and transferrin saturation were assessed as measures of Fe status. Hb was determined at Causeway Laboratory, Causeway NHSS Trust, and ferritin and trans-ferrin saturation were analyzed by immunonephelometry at Grenoble University Hospital. Erythrocyte Zn-Cu superox-ide dismutase activity (RBC SOD) and serum ceruloplasmin (CP) were assessed as putative indicators of Cu status. RBC SOD was analyzed using a commercially available kit (Randox, Crumlin, U.K.), and CP was determined by turbidimetry (DakoCytomation, Ely, U.K.) on a Hitachi 912 autoanalyzer (Roche Diagnostics). Serum C-reactive protein (CRP), complement protein-3 (C3), and complement protein-4 (C4), were determined using commercially avail-able kits (Roche Diagnostics) on a Hitachi 912 autoanalyzer.

Markers of Immunity

The panel of fluorochrome-conjugated monoclonal anti-bodies used in the current study for immunophenotyping, apoptosis, phagocytosis, and intracellular cytokine assays as performed by flow cytometry has been previously described by Hodkinson and colleagues (29). Immunophenotyping was performed as previously described by Hodkinson and colleagues (33).

The determination of early lymphocyte apoptosis by two-color flow cytometry was conducted using an Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences). Briefly, peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation from sodiumheparin anticoagulated whole blood using ACCUSPIN System Histopaque 1077 tubes (Sigma-Aldrich, Dorset, U.K.). PBMC were washed twice in fresh sterile filtered phosphate-buffered saline (PBS) and then resuspended in binding

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600HODKINSON ET AL.

Table 1. Baseline Characteristics of Apparently Healthy Men and Postmenopausal Women Aged 5570 Years Receiving Zinc (Zn) Supplementation or Placebo

Characteristics

Placebo Group (N 31)

15 mg Zn Group (N 28)

30 mg Zn Group (N 34)

Age (y)

62.6

6 0.75

62.4

6 0.91

62.4

6 0.80

BMI (kg/m2)

26.7

6 0.62

26.7

6 0.68

27.1

6 0.55

Energy (kJ)*

8487

6 360

7735

6 336

7844

6 330

Dietary Zn intake (mg/d)*

9.38

6 0.60

9.52

6 0.68

8.99

6 0.57

Serum Zn concentration (lmol/L)y

13.2

6 0.28

13.0

6 0.30

12.9

6 0.19

Erythrocyte Zn concentration (lmol/L)y

231

6 7.74

216

6 10.26

220

6 8.29

Urinary Zn/creatinine ratio (lmol/mmol creatinine)z

0.71

6 0.06

0.61

6 0.04

0.62

6 0.04

ALP concentration (U/L)

78.2

6 2.52

77.6

6 2.62

75.0

6 3.34

Notes: All vales are mean 6 standard error of the mean. There were no significant differences in the baseline characteristics among the three groups. *Energy and dietary Zn intakes were assessed by a 4-day food diary and analysed by Weighed Intake analysis Software Package (WISP) 2.0 for Windows.

ySerum and erythrocyte Zn concentrations were determined by flame atomic absorption spectrometry. The normal laboratory ranges for serum and erythrocyte Zn concentration are 11.318.8 lmol/L and 120250 lmol/L, respectively.

zUrinary Zn concentration was determined by flame atomic absorption spectrometry and urinary creatinine by colorimetry.

Total alkaline phosphatase (ALP) was measured by a colorimetric assay. The normal adult laboratory range for ALP is 3090 U/L. BMI body mass index; ALP alkaline phosphatase.

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buffer (BD Biosciences). PBMC were stained using Annexin V-FITC and propidium iodide (PI). Lymphocytes were gated using a forward-scatter (FSC) versus side-scatter (SSC) dotplot. Lymphocytes staining positive for Annexin-V FITC and negative for PI were determined using fluorescence channel 1 height (FL1-H) versus fluorescence channel 2 height (FL2-H) dotplots. The percentage of lymphocytes undergoing early apoptosis was obtained from fluorescence-activated cell sorter (FACS) analysis, and absolute counts were calculated using the lymphocyte white blood cell differential (3 109/L).

The quantification of phagocytic capacity and activity of granulocytes and monocytes were determined using a PhagoTest kit (Orpegen Pharma, Heidelberg, Germany). Sodiumheparin whole blood (100 lL) was incubated with opsonized Escherichia coliFITC. Monocytes and granulo-cytes were gated using FSC versus SSC dotplot. SSC versus E. coliFITC dotplots were used to measure phagocytic capacity and activity for both cell types. The percentage of E. coliFITCpositive cells provided a measure of phago-cytic capacity. Mean fluorescence intensity (MFI) was used to quantify the mean number of E. coli ingested per cell, as a determinant of phagocytic activity.

Intracellular cytokine production by activated monocytes was based on a previously described method by McNerlan and colleagues (34). Briefly, 1 mL of sodiumheparin whole blood was incubated with lipopolysaccharide (LPS) at 1 lg/ mL and Brefeldin A (BFA) at 10 lg/mL (Sigma-Aldrich). After incubation, 100 lL of activated blood was incubated with either 10 lL of immunoglobulin G (IgG) 2a FITC (isotype control) or CD14 FITC (BD Pharmingen, Oxford, U.K.). Erythrocytes were lysed, the remaining cells were washed and centrifuged, and the cell pellet was resuspended with 100 lL of permeabilizing medium B (Caltag, Invitrogen, Paisley, U.K.). IgG1 phycoerythrin (PE; isotype control), interleukin-1b (IL-1b) PE, or IL-6 PE (10 lL) was added for a further incubation. Cells were washed and fixed with 500 lL of 13 Cell Fix solution (BD Biosciences). Samples were analyzed immediately.

Monocytes were gated using FSC versus SSC dotplots, and percentages of cytokine-positive CD14 cells were obtained from FL-1 H versus FL-2 H dotplots. Intracellular

cytokine production, as determined by the antibody binding capacity (ABC), was quantified using PE QuantiBRITE beads (BD Biosciences) for standardization of PE MFI.

Statistical Analysis

Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 11.0. Markers of immune status did not display normal distribution and, therefore, were normalized as appropriate prior to analysis. Partial correlations were used to analyze associations between serum and erythrocyte Zn concentration and immune status, controlling for age (p .05). Repeated-measures analysis of variance (ANOVA) was used to determine the effect of Zn supplementation on immune status, in Zn and control groups (p .05), with sex, age, and the appropriate baseline variable as covariates. In this procedure, responses at month 3 and month 6 were used as within-subject variables, and treatment was used as the between-subject factor. The main effect of treatment was compared by using Bonferronis procedure for confidence interval (CI) adjustment.

RESULTS

Participant Characteristics

A total of 93 apparently healthy individuals completed the intervention; 31 in the placebo group, 28 in the 15 mg Zn/d group, and 34 in the 30 mg Zn/d group. The mean (6 standard error of the mean [SEM]) age was 62.4 (6 0.47) years. Table 1 shows the mean (6 SEM) for baseline characteristics for each treatment group. No significant differences were seen among groups at baseline in any of the variables. Serum Zn, erythrocyte Zn, urinary Zn/creatinine ratio and ALP concentrations were all within the normal adult reference ranges. Mean (6 SEM) Hb, ferritin, trans-ferrin saturation, RBC SOD, and CP were 14.5 (6 0.13) g/dL, 93.0 (6 7.49) lg/L, 30.2 (6 1.14)%, 1011 (6 21.7) U/g Hb, and 0.21 (6 0.004) U/L at baseline, respectively. No significant differences were observed among groups for putative measures of Fe and Cu status at baseline (data not

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Table 2. Significant Associations Between Serum and Erythrocyte Zinc (Zn) Concentration and Markers of Immunity in

Healthy Men and Postmenopausal Women Aged 5570 Years at Baseline

Serum Zn (lmol/L)

Erythrocyte Zn (lmol/L)

Immune Parameter

Mean

SEM

r1

r1

CRP (mg/dL)

0.20

0.04

.261*

.008

Granulocyte phagocytic capacity (%)

47.4

2.08

.074

.221y

CD3 T lymphocytes (%)

67.9

0.84

.093

.228y

CD3 T lymphocytes (3 109/L)

1.19

0.04

.174

.234y

CD3/CD16/CD56 NKT cells (%)

6.50

0.95

.080

.226y

CD3/CD16/CD56 NKT cells (3 109/L)

0.12

0.02

.123

.249y

CD3/CD25 intermediate-activated T lymphocytes (%)

26.1

0.84

.025

.083

CD3/CD25 intermediate-activated T lymphocytes (3 109/L)

0.30

0.11

.184

.211y

CD3/HLA-DR late-activated lymphocytes (%)

10.1

0.80

.093

.223y

CD3/HLA-DR late-activated lymphocytes (3 109/L)

0.13

0.02

.058

.263y

CD3/CD45RA nave T lymphocytes (%)

49.1

1.48

.055

.098

CD3/CD45RA nave T lymphocytes (3 109/L)

0.60

0.03

.084

.237y

T lymphocyte IL-2 receptor density (MFI)

68.2

1.53

.058

.263y

Notes: Analyzed by partial correlation between Zn concentration (serum and erythrocyte) and markers of immunity, controlling for age. Where necessary, markers of immunity were normalized prior to analysis.

*Significant association between serum Zn concentration and marker of immunity, p .05. ySignificant association between erythrocyte Zn concentration and marker of immunity, p .05.

SEM standard error of the mean; CRP C-reactive protein; NKT natural killer-like T; MFI mean fluorescence intensity.

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shown). Immune status as assessed by immunophenotyping was within normal reference ranges and comparable to that reported by McNerlan and colleagues (35), who examined a similar population from Northern Ireland, and by Bisset and colleagues (36), who assessed immune status in healthy 19- to 70-year-olds in Switzerland.

Associations Between Serum and Erythrocyte Zn Concentration and Markers of Immune Status

At baseline, serum Zn concentration was inversely associated with age in all participants (r 0.226, p

.015), whereas erythrocyte Zn concentration was inversely associated with age in women only (r 0.252, p 0.042). Partial correlations, controlling for age, were used to examine the associations between erythrocyte and serum Zn concentrations and markers of immunity at baseline (Table 2). Results show significant positive associations between erythrocyte Zn concentration and the following markers of immunity: CD3 T lymphocytes (% and absolute count), CD3/CD(1656) NK-like T (NKT) cells (% and absolute count), CD3/CD25 intermediately activated T lymphocytes (absolute count), CD3/HLA-DR late-activated T lymphocytes (% and absolute count), and CD3/CD45RA nave T lymphocytes (absolute count). A significant inverse association was observed between erythrocyte Zn concentration and granulocyte phagocytic capacity (Table 2). Serum Zn concentration was inversely associated with CRP concentration and positively associated with IL-2 receptor density on T lymphocytes (Table 2).

Effect of Zn Supplementation on Measures of Zn, Fe, and Cu Status

Postsupplementation mean (6 SEM) serum Zn concen-tration for placebo, 15 mg Zn/d, and 30 mg Zn/d groups were 12.4 (6 0.28) lmol/L, 13.0 (6 0.47) lmol/L, and 14.3 (6 0.77) lmol/L, respectively, and erythrocyte Zn concen-

tration for placebo, 15 mg Zn/d, and 30 mg Zn/d groups were 235 (6 8.69) lmol/L, 233 (6 10.3) lmol/L, and 228 (6 9.35) lmol/L, respectively. There was no significant Time 3 Treatment interaction, as analyzed by repeated-measures ANOVA, for serum Zn concentration. However, there was a significant main effect of treatment (p .008), whereby participants in the 30 mg Zn/d group had significantly higher serum Zn concentrations than the 15 mg Zn/d and placebo groups had. Analysis showed no significant Time 3Treatment interactions or treatment effect for either erythrocyte Zn or ALP concentration (data not shown). Postsupplementation mean (6 SEM) urinary Zn/ creatinine ratio for placebo, 15 mg Zn/d, and 30 mg Zn/d groups were 0.80 (6 0.80) lmol/mol creatinine, 0.62 (6 0.54) lmol/mol creatinine, and 0.90 (6 0.91) lmol/mol creatinine, respectively. Urinary Zn concentration demon-strated no significant Time 3 Treatment interaction, but did show a significant effect of treatment (p .027), where individuals receiving 30 mg Zn/d had a higher urinary Zn/ creatinine ratio than did those receiving 15 mg Zn/d.

There was no significant Time 3 Treatment interaction of Zn supplementation on Hb, ferritin concentration, transferrin saturation, or CP. RBC SOD showed a significant (p .046) within-subject Time 3 Treatment interaction and a signifi-cant between-subject (p .043) treatment effect, where individuals receiving 30 mg Zn/d had higher RBC SOD than did those receiving 15 mg Zn/d at month 3 only.

Effect of Zn Supplementation on Markers of Inflammation

Mean (6 SEM) serum CRP, C3, and C4 for all participants were 0.20 (0.04) mg/dL, 1.28 (0.02) g/L, and 0.26 (0.01) g/L, respectively. C3 and C4 concentrations were within the normal adult reference ranges. Although 10 individuals had subclinical inflammation, as indicated by elevated serum CRP concentration (. 0.5 mg/dL), no significant interaction or supplementation effects were

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602HODKINSON ET AL.

observed for C3, C4 complement proteins, or CRP con-centration (data not shown).

Effect of Zn Supplementation on Leukocyte Subpopulations

The percent expression and absolute counts of leukocyte and lymphocyte subpopulations in peripheral whole blood were within normal adult ranges, and the Time 3 Treatment interactions for Zn supplementation can be seen in Table 3 (% expression) and Table 4 (absolute counts).

For CD3/CD45RA nave T lymphocytes (% expression), within-subject effects showed a significant Time 3 Treat-ment interaction (p .036) as seen in Table 3, but between-subject treatment effects were nonsignificant (p .919). As shown in Table 4, monocyte counts (3 109/L), showed a significant Time 3 Treatment interaction (p .030), but between-subject treatment effects were nonsignificant (p .160).

For percent expression of B lymphocytes, within-subject effects showed a nonsignificant Time 3 Treatment in-teraction (p .051), as shown in Table 3. However, a significant main effect of treatment (p .028) was seen, whereby the percent expression of B lymphocytes was significantly lower (p .045) in the 30 mg Zn/d group than in the placebo group. For the B-lymphocyte absolute count (3 109/L), within-subject effects showed a significant Time 3 Treatment interaction (p .028; see Table 4) and a significant treatment effect (p .016). The absolute count of B lymphocytes was significantly lower (p .001) in the 30 mg Zn/d group than in the 15 mg Zn/d group, at month 3 only. There was no significant Time 3Treatment interaction on the percent expression (Table 3), or absolute count of lymphocytes in early apoptosis (Table 4).

For the (CD3/CD4: CD3/CD8) T helper to T-cytotoxic lymphocyte (CTL) ratio responses, within-subject effects showed a significant Time 3 Treatment interaction (p .026), where the T helper/CTL ratio in the 15 mg Zn/d group at month 6 was significantly higher (p .043) than in the placebo group (see Table 4).

Effect of Zn Supplementation on Phagocytosis and Intracellular Cytokine Production in Activated Monocytes

Repeated-measures ANOVA showed no significant Time 3 Treatment interaction or main effect of treatment on the phagocytic capacity (%) or activity (MFI) of either granulocytes or monocytes (Table 5). There was no significant Time 3 Treatment interaction and no effect of treatment on the percent expression of either IL-1b or IL-6 and ABC by activated monocytes (Table 6).

Response to Supplementation in Individuals According to Baseline Serum and Erythrocyte Zn Concentration

Of 93 participants, six had serum Zn concentrations below the normal reference range of 11 lmol/L, with the lowest recorded value at 10.6 lmol/L. All six of these participants had adequate Zn intakes and did not have self-reported indications of overt clinical Zn deficiency. A median split on

the basis of serum Zn (12.9 lmol/L) and erythrocyte Zn (225 lmol/L) revealed no significant difference in response to Zn supplementation regardless of initial Zn status using repeated-measures ANOVA (data not shown).

DISCUSSION

The present study was undertaken to evaluate the associations between putative indices of Zn status and markers of immune status and to determine the effect of Zn supplementation on selected markers of immunity in apparently healthy individuals aged 5570 years. Mean estimated dietary intake at baseline was 9.28 mg Zn/d, just below the U.K. reference nutrient intake of 9.5 mg Zn/d (37), and comparable with intakes previously reported (38). Thus, the upper level of Zn intake (at supplementation of 30 mg Zn/d) was approximately 40 mg/d for participants in the current study, which exceeds the European tolerable upper level (UL) of 25 mg Zn/d, and is equivalent to the UL in the United States (39,40).

At baseline, erythrocyte Zn concentration was inversely associated with granulocyte phagocytic capacity and positively associated with T-lymphocyte subsets, markers of T-lymphocyte activation, and NKT cell numbers. These findings suggest that a higher cellular Zn concentration maintains T-lymphocyte populations, and that such immune cells may be more readily primed for response to exogenous stimuli. Human circadian rhythms demonstrate high morning cortisol concentration, high Zn concentrations, and low phagocytic activity, with respective decreases and increases during the course of the day (41,42). This observation is further supported by our finding of a significant inverse association between morning erythro-cyte Zn concentration and granulocyte phagocytic capacity. During periods of acute and/or chronic inflammation, Zn is redistributed from the labile serum pool to other body compartments, e.g., liver and erythrocytes (4346). The redistribution of Zn during inflammation is supported by our observation of an inverse association between serum Zn concentration and the inflammatory marker CRP. In addition, IL-2 receptor density on T lymphocytes was positively associated with serum Zn. As IL-2 receptor density is associated with T-lymphocyte activation (47), this may explain why T-lymphocyte activation is dependent on Zn concentration. Overall, the current study emphasizes that, in the absence of a sensitive clinical indicator of Zn status (48), the measurement of Zn concentration in multiple body compartments is recommended to explore adequately the relationship between Zn and immune status. Future studies examining the influence of Zn status on immune function should also consider using neutrophil and/ or lymphocyte Zn concentration and associated measures of Zn metallothionein in immune cells (49,50).

Postsupplementation, the percent expression of nave T lymphocytes, the absolute count of monocytes and B lymphocytes, and T helper/CTL ratio showed significant Time 3 Treatment interactions with Zn. However, only B

lymphocytes

and T helper/CTL ratios demonstrated

a significant

main effect of treatment, suggesting that

the Time 3

Treatment interactions observed for other


Table 3. Responses of Circulating Lymphocyte Subpopulations (% Expression) to Zinc (Zn) Supplementation for 6 Months in

Healthy Men and Postmenopausal Women Aged 5570 Years

Baseline

Month 3

Month 6

p Value Time 3

Placebo

15 mg Zn

30 mg Zn

Placebo

15 mg Zn

30 mg Zn

Placebo

15 mg Zn

30 mg Zn

Immune Parameters (%)

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Treatment

CD3/CD19

13.0 6 0.96

13.2 6 0.91

12.1 6 0.74

13.9 6 0.94

14.2 6 0.82

11.5 6 0.69

13.9 6 1.05

13.7 6 0.99

12.6 6 0.78

.051

N

30

28

34

31

28

34

29

28

34

CD3

66.6

6 1.37

67.3

6 1.63

69.4

6 1.36

66.8

6 1.29

67.6

6 1.66

71.2

6 1.17

66.1

6 1.41

66.7

6 1.79

70.0

6 1.18

.683

N

30

28

34

31

28

34

29

28

34

CD3/CD16/CD56

16.5 6 1.22

15.2 6 1.24

13.3 6 1.09

15.7 6 1.17

14.3 6 1.42

12.3 6 0.97

15.9 6 1.34

13.6 6 1.21

11.9 6 0.90

.646

N

31

28

34

31

28

34

29

28

34

CD3/CD16/CD56

4.57 6 1.00

6.03 6 1.30

8.62 6 2.16

4.95 6 1.03

6.20 6 1.29

8.72 6 2.08

4.50 6 0.93

5.56 6 1.05

8.80 6 3.00

.283

N

31

28

34

31

28

34

29

28

34

CD3/CD25

26.9 6 1.51

27.1 6 1.62

24.6 6 1.24

31.1 6 1.42

29.7 6 1.62

29.2 6 1.42

30.0 6 1.31

30.8 6 1.69

29.7 6 1.28

.456

N

31

28

33

30

25

34

28

28

34

CD3/HLA-DR

9.10 6 0.88

8.76 6 0.89

12.2 6 1.88

11.0 6 1.21

10.6 6 1.21

13.0 6 1.63

9.88 6 1.11

10.6 6 1.33

13.4 6 1.93

.595

N

31

28

34

31

27

34

27

28

34

CD3/CD4

66.8 6 2.46

64.5 6 2.57

59.7 6 2.32

65.5 6 3.11

63.3 6 2.56

58.0 6 2.67

65.6 6 2.72

68.1 6 2.06

60.6 6 2.30

.319

N

31

28

34

31

28

34

28

28

34

CD3/CD8

26.8 6 2.06

30.2 6 2.30

33.4 6 2.42

25.6 6 1.71

29.4 6 1.67

30.6 6 2.49

29.5 6 2.66

26.5 6 1.94

33.3 6 2.55

.090

N

31

28

34

31

28

34

28

28

34

CD3/CD45RA

47.1 6 2.23

50.4 6 3.25

49.8 6 3.25

46.8 6 2.78

46.6 6 3.60

48.2 6 3.44

48.6 6 2.79

52.7 6 3.12

49.6 6 2.84

.036

N

30

25

33

28

26

33

27

28

34

CD3/CD45RO

48.8 6 2.41

50.6 6 2.47

53.4 6 2.32

53.2 6 1.84

53.4 6 2.14

53.5 6 2.17

50.5 6 2.33

52.0 6 2.28

53.7 6 2.33

.669

N

31

26

34

31

28

34

28

28

34

CD3/CD4/CD45RA

40.8 6 2.49

42.6 6 3.37

39.7 6 2.85

43.7 6 2.56

43.5 6 4.46

41.0 6 3.36

48.2 6 3.16

47.5 6 3.37

44.9 6 3.83

.856

N

30

25

33

28

26

33

25

28

33

CD3/CD4/CD45RO

51.6 6 2.94

56.4 6 2.05

59.4 6 2.16

53.0 6 2.32

58.3 6 2.77

61.2 6 2.82

54.3 6 2.58

55.4 6 2.72

59.3 6 2.54

.056

N

31

26

34

31

28

34

28

28

34

CD3/CD8/CD45RA

52.2 6 3.39

56.1 6 4.03

58.1 6 3.86

55.1 6 3.09

59.4 6 3.92

59.1 6 3.37

60.1 6 2.87

63.9 6 3.26

62.6 6 3.32

.877

N

30

25

33

26

24

30

27

28

34

CD3/CD8/CD45RO

45.8 6 2.58

43.3 6 2.97

48.3 6 3.43

52.9 6 3.42

48.5 6 3.47

52.3 6 3.77

46.8 6 2.76

45.0 6 3.09

49.4 6 3.20

.590

N

31

26

33

30

28

34

27

28

34

Annexin-V/PI lymphocytes*

34.6 6 2.14

35.1 6 3.90

29.9 6 3.12

35.5 6 2.32

40.1 6 4.06

28.2 6 3.46

.497

N

12

11

13

12

11

12

Notes: Analysis performed by repeated-measures analysis of variance (p , .05) with Bonferronis procedure for confidence interval (CI) adjustment. In this procedure, Time 3Treatment interaction was used as within-subject variable, and treatment was used as a between-subjects factor. For CD3/CD45RA (p .036), the within-subject effects for Time 3 Treatment interaction was significant, but there was no main effect of treatment. For B-lymphocyte responses, the within-subjects effects showed a nonsignificant (p .051) Time 3Treatment interaction, but a significant main effect of treatment (p .028) treatment was observed, where B lymphocytes

(%) were lower in the 30 mg Zn group compared to the control group.

*Early lymphocytes apoptosis was measured on a subset of participants (n 36) at month 0 and month 6. SEM standard error of the mean; PI propidium iodide; data not collected for this variable.


603

Downloaded from org/.oxfordjournals.http://biomedgerontology by guest on November 11, 2015

Table 4. Responses of Circulating Leukocyte Subpopulations (3 109/L) to Zinc (Zn) Supplementation for 6 Months in Healthy Men and Postmenopausal Women Aged 5570 Years

Baseline

Month 3

Month 6

p Value Time 3

Placebo

15 mg Zn

30 mg Zn

Placebo

15 mg Zn

30 mg Zn

Placebo

15 mg Zn

30 mg Zn

Immune Parameters (3 109/L)

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean

6 SEM

Mean 6 SEM

Mean

6 SEM

Mean 6 SEM

Treatment

Neutrophils

2.63

6 0.13

2.70

6 0.14

3.24

6 0.17

2.81

6 0.12

2.70

6 0.15

2.80

6 0.17

2.77

6 0.15

2.70

6 0.14

3.10 6 0.15

.097

N

31

28

34

31

28

34

29

28

34

Lymphocytes

1.82 6 0.09

1.51

6 0.08

1.86

6 0.09

1.69 6 0.08

1.61

6 0.07

1.69

6 0.07

1.68

6 0.08

1.55

6 0.08

1.73 6 0.07

.489

N

31

28

34

31

28

34

29

28

34

Monocytes

0.44

6 0.02

0.40 6 0.02

0.46

6 0.02

0.52

6 0.03

0.43

6 0.02

0.47

6 0.03

0.46 6 0.02

0.41

6 0.03

0.50 6 0.03

.030

N

31

28

34

31

28

34

29

28

34

Eosinophils

0.18

6 0.02

0.20

6 0.03

0.20

6 0.02

0.19

6 0.02

0.21

6 0.02

0.19

6 0.02

0.19

6 0.02

0.21

6 0.02

0.21 6 0.02

.694

N

31

28

34

31

28

34

29

28

34

CD3/CD19

0.23 6 0.02

0.19 6 0.01

0.22 6 0.02

0.23ab 6 0.02

0.23b 6 0.02

0.20a 6 0.02

0.23 6 0.02

0.21

6 0.02

0.22 6 0.02

.028

N

30

28

34

31

28

34

29

28

34

CD3

1.30 6 0.07

1.03 6 0.07

1.30 6 0.07

1.21 6 0.06

1.10 6 0.07

1.21

6 0.06

1.22 6 0.06

1.05

6 0.07

1.22 6 0.06

.417

N

30

28

34

31

28

34

29

28

34

CD3/CD16/CD56

0.30 6 0.03

0.23 6 0.02

0.24 6 0.02

0.27 6 0.02

0.23 6 0.02

0.21

6 0.02

0.27 6 0.02

0.21

6 0.02

0.20 6 0.02

.474

N

31

28

34

31

28

34

29

28

34

CD3/CD16/CD56

0.09 6 0.02

0.10 6 0.02

0.17 6 0.06

0.09 6 0.02

0.11 6 0.02

0.17

6 0.05

0.08 6 0.02

0.10

6 0.02

0.16 6 0.05

.098

N

31

28

34

31

28

34

29

28

34

T-cell IL-2R density1

67.5

6 2.72

69.5

6 2.00

67.7

6 3.00

69.6 6 2.59

67.2

6 4.57

73.0

6 1.74

70.5

6 1.89

76.5

6 1.96

73.0 6 1.74

.093

N

31

28

34

31

28

34

29

28

34

CD3/CD25

0.32 6 0.02

0.26 6 0.01

0.31 6 0.02

0.33 6 0.02

0.32 6 0.02

0.34

6 0.02

0.33 6 0.02

0.32

6 0.02

0.35 6 0.02

.468

N

30

28

33

30

25

34

28

28

34

CD3/HLA-DR

0.12 6 0.02

0.10 6 0.02

0.18 6 0.02

0.13 6 0.02

0.12 6 0.02

0.17

6 0.02

0.11 6 0.02

0.13

6 0.02

0.18 6 0.02

.177

N

30

28

34

31

27

34

27

28

34

CD3/CD4

0.81 6 0.05

0.63 6 0.03

0.76 6 0.04

0.74 6 0.05

0.68 6 0.04

0.68

6 0.04

0.73 6 0.04

0.69

6 0.04

0.73 6 0.04

.443

N

30

28

34

31

28

34

28

28

34

CD3/CD8

0.33 6 0.03

0.34 6 0.04

0.45 6 0.05

0.29 6 0.03

0.34 6 0.04

0.40

6 0.05

0.34 6 0.04

0.30

6 0.04

0.42 6 0.04

.068

N

30

28

34

31

28

34

28

28

34

CD3/CD45RA

0.60 6 0.05

0.53 6 0.06

0.65 6 0.05

0.55 6 0.05

0.53 6 0.05

0.61

6 0.06

0.55 6 0.04

0.57

6 0.06

0.61 6 0.05

.792

N

29

25

33

28

26

33

27

28

34

CD3/CD45RO

0.58 6 0.04

0.52 6 0.04

0.69 6 0.05

0.59 6 0.03

0.53 6 0.03

0.65

6 0.04

0.56 6 0.04

0.59

6 0.04

0.65 6 0.04

.499

N

30

26

34

31

28

34

28

28

34

CD3/CD4/CD45RA

0.52 6 0.05

0.43 6 0.04

0.51 6 0.04

0.51 6 0.05

0.50 6 0.06

0.50

6 0.05

0.55 6 0.05

0.51

6 0.05

0.55 6 0.06

.403

N

29

25

33

28

26

33

25

28

33

CD3/CD4/CD45RO

0.61 6 0.05

0.59 6 0.06

0.78 6 0.05

0.58 6 0.03

0.63 6 0.04

0.76

6 0.06

0.60 6 0.04

0.58

6 0.04

0.73 6 0.05

.117

N

30

26

34

31

28

34

28

28

34

CD3/CD8/CD45RA

0.66 6 0.07

0.60 6 0.07

0.79 6 0.08

0.65 6 0.06

0.67 6 0.07

0.76

6 0.07

0.67 6 0.04

0.70

6 0.07

0.78 6 0.07

.511

N

29

25

33

26

24

30

27

28

34

CD3/CD8/CD45RO

0.54 6 0.04

0.43 6 0.03

0.62 6 0.06

0.57 6 0.04

0.51 6 0.04

0.62

6 0.05

0.52 6 0.04

0.46

6 0.04

0.59 6 0.04

.660

N

30

26

33

30

28

34

27

28

34

CD3/CD4: CD3/CD8

3.28 6 0.37

2.67 6 0.30

2.32 6 0.25

3.02 6 0.30

2.46 6 0.24

2.47

6 0.28

3.06b 6 0.40

3.15a 6 0.33

2.50ab 6 0.30

.026

N

31

28

34

31

28

34

28

28

34

CD4/CD45RA: CD4/CD45RO

0.97 6 0.12

0.94 6 0.13

0.75 6 0.08

0.91 6 0.10

0.88 6 0.11

0.81

6 0.11

1.01 6 0.12

0.99

6 0.12

0.88 6 0.11

.824

N

30

23

33

28

26

33

25

28

33

CD8/CD45RA: CD8/CD45RO

1.34 6 0.15

1.56 6 0.19

1.67 6 0.26

1.29 6 0.15

1.56 6 0.19

1.52

6 0.17

1.48 6 0.16

1.84

6 1.55

1.60 6 0.17

.691

N

30

23

33

26

24

30

26

28

34

Annexin-V/PI lymphocytes*

1.11 6 0.58

0.57 6 0.09

0.59 6 0.07

0.58 6 0.05

0.67

6 0.08

0.53 6 0.08

.411

N

12

11

13

12

11

12

Notes: Analysis performed by repeated-measures analysis of variance (p , .05) with Bonferronis procedure for confidence interval adjustment. In this procedure, Time 3 Treatment interaction was used as within-subject variable, and treatment was used as a between-subjects factor. For a given month, means with superscript letters are significantly different, p , .05. For monocytes (p .030), within-subject effects for Time 3 Treatment interactions were significant. For B-lymphocyte responses (3 109/L), the within-subjects effects showed a significant (p .028) Time 3 Treatment interaction, and the between-subject effects showed a significant (p .001) treatment effect at month 3 only. For CD3/CD4: CD3/CD8 ratio responses, the within-subjects effects showed a significant (p .026) Time 3 Treatment interaction, and the between-subject effects showed a significant (p .043) treatment effect at month 6 only.

*Early lymphocytes apoptosis was measured on a subset of participants (n 36) at month 0 and month 6 only.

SEM standard error of the mean; IL-2R interleukin-2 receptor; PI propidium iodide; data not collected for this variable.

604

HODKINSON ET AL.


Table 5. Response of Phagocytic Capacity and Activity to Zinc (Zn) Supplementation for 6 Months in Healthy Men and Postmenopausal Women Aged 5570 Years

Baseline

Month 3

Month 6

p Value Time 3

Placebo

15 mg Zn

30 mg Zn

Placebo

15 mg Zn

30 mg Zn

Placebo

15 mg Zn

30 mg Zn

Immune Parameters

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Treatment

Granulocyte phagocytic capacity (%)

42.2

6 2.99

50.4

6 4.04

49.8

6 3.67

70.8

6 3.28

73.4

6 3.91

71.5

6 3.17

77.9

6 3.18

81.4

6 2.78

81.7

6 3.02

.723

N

31

27

34

30

27

33

29

25

33

Granulocyte phagocytic activity (MFI)

291

6 19.5

374

6 33.0

353

6 29.4

507

6 58.6

559

6 53.9

529

6 48.0

718

6 57.0

754

6 66.9

801

6 76.0

.709

N

31

27

34

30

27

33

29

25

33

Monocyte phagocytic capacity (%)

27.3

6 2.26

36.8

6 4.12

33.5

6 3.07

60.0

6 2.23

62.8

6 4.03

59.5

6 2.95

68.7

6 3.01

71.3

6 2.96

71.6

6 3.08

.527

N

31

27

34

30

27

33

29

25

33

Monocyte phagocytic activity (MFI)

203

6 17.3

259

6 23.2

242

6 19.8

387

6 32.9

340

6 24.6

345

6 25.6

381

6 27.4

395

6 21.6

401

6 29.7

.575

N

31

27

34

30

27

33

29

25

33

Notes: Analysis performed by repeated-measures analysis of variance (p , .05) with Bonferronis procedure for confidence interval adjustment. In this procedure, Time 3 Treatment interaction was used as within-subject variable, and treatment was used as a between-subjects factor. No significant Time 3 Treatment interaction was observed for the above parameters, nor was there any main effect of treatment.

SEM standard error of the mean; MFI mean fluorescence intensity.

Table 6. Responses of Intracellular Cytokine Production by Activated Monocytes to Zinc (Zn) Supplementation for 6 Months in

Healthy Men and Postmenopausal Women Aged 5570 Years

Baseline

Month 3

Month 6

p Value Time 3

Placebo

15 mg Zn

30 mg Zn

Placebo

15 mg Zn

30 mg Zn

Placebo

15 mg Zn

30 mg Zn

Immune Parameters

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Mean 6 SEM

Treatment

CD14/IL-1b (%)

45.6

6 3.85

47.3

6 4.86

48.3

6 4.16

51.4

6 3.24

53.3

6 3.31

48.3

6 3.59

66.8

6 2.92

60.7

6 3.63

63.9

6 2.95

.217

N

31

28

31

31

28

34

29

28

34

CD14/IL-1b (ABC)

1161

6 44.2

1265

6 62.5

1183

6 40.9

1182

6 58.0

1249

6 93.7

1208

6 73.5

2205

6 164

2420

6 183

2256

6 132

.114

N

31

28

31

31

28

34

29

28

34

CD14/IL-6 (%)

49.9

6 3.84

51.1

6 4.79

55.4

6 3.85

50.3

6 3.10

60.7

6 2.59

55.3

6 3.43

50.3

6 3.01

50.2

6 2.89

46.7

6 3.45

.835

N

31

28

31

31

28

34

29

28

34

CD14/IL-6 (ABC)

3344

6 451

4243

6 763

3086

6 254

2636

6 209

2903

6 184

2510

6 188

2218

6 186

2296

6 128

2259

6 225

.818

N

31

28

31

31

28

34

29

28

34

Notes: Analysis performed by repeated-measures analysis of variance (p , .05) with Bonferronis procedure for confidence interval adjustment. In this procedure, Time 3 Treatment interaction was used as within-subject variable, and treatment was used as a between-subjects factor. No significant Time 3 Treatment interaction or main treatment effect was observed for percent cytokine expression or cytokine antibody binding capacity (ABC).

SEM standard error of the mean; IL interleukin.


605


606HODKINSON ET AL.

parameters were largely driven by seasonal effects on immune function (5155).

If confirmed, the finding that B-lymphocyte counts were significantly lower in the 30 mg Zn/d group compared to the 15 mg Zn/d and placebo groups at month 3 only potentially indicates a negative influence of high doses of Zn sup-plementation, which may be of particular concern in older individuals vulnerable to immune deficiencies. The effi-ciency of humoral immunity is reduced with age (5659), which results in decreased effectiveness of vaccination (60,61), increasing the susceptibility to, and mortality from, infection. A positive relationship between Zn nutrition and antibody titer postvaccination in the elderly population has been reported in some (62), but not all studies (63,64). The potential negative effect of higher doses of Zn supplemen-tation (30 mg Zn/d) in non-Zn-deficient individuals requires confirmation. Furthermore, it will be important to clarify further the effect of Zn supplementation on humoral immunity and response to vaccination in aged individuals.

The T helper/CTL ratio has been reported to decline with age, and to be a predictor of survival in old age (65,66). Postsupplementation at month 6, individuals receiving 15 mg Zn/d had significantly higher T helper/CTL ratios compared to the placebo group, suggesting that longer term Zn supplementation at a moderate dose (15 mg Zn/d) may result in maintenance of these lymphocyte subpopulations with age. Measurement of a large number of parameters increases the likelihood of chance findings; therefore, further investigation is required to confirm the effects of Zn supplementation in healthy individuals observed in the current study. No significant effect of Zn supplementation on the functional markers of immunity, phagocytosis and intracellular cytokine production by activated monocytes, were observed in this study, possibly owing to the apparent lack of Zn deficiency in the study population.

Previous studies have shown that marginal Zn status in elderly individuals (70 years) is associated with decreased immunity (25,67) and that Zn supplementation restores immune responses in this age group (20,21,24,68,69). These findings are in contrast to our results, where only B lym-phocytes and the T helper/CTL ratio appeared responsive to Zn supplementation. However, our finding of an overall lack of effect of supplementation on immune status is supported by other previous studies, which reported little effect of Zn supplementation in young adult men (38) and elderly individuals (aged 64100 years) (63).

However, one major limitation that plagues all studies of Zn supplementation is the lack of a specific and sensitive marker of Zn status. Indeed in the current study, only one of the putative markers of Zn status, serum Zn, increased postsupplementation, and this was only evident in the 30 mg Zn/d treatment group. However, participants in the group receiving 30 mg Zn/d demonstrated significantly higher urinary Zn/creatinine ratios compared to the group receiving 15 mg Zn/d. This finding (if confirmed) would suggest that, in apparently healthy individuals, supplemental doses of 30 mg Zn/d might exceed requirements. The tight regulation of Zn homeostasis within the body is well known, and this may partly explain why individuals with apparently adequate dietary intakes of Zn did not demonstrate

enhancement of immune status postsupplementation. Al-though adequate Zn status at baseline may be a key factor, we cannot rule out the possibility that other aspects of immunity not assessed in those individuals studied may have responded to Zn supplementation.

The results of the current study contribute to the limited scientific literature examining the effect of Zn supplemen-tation at the UL and no observed adverse effect level (NOAEL) on immune status (39,40). To our knowledge, this is the first trial to examine such effects in this age group. Supplementation with 30 mg Zn/d has previously been reported to have no negative effect on putative measures of Cu status (38), a finding largely supported by our results.

Conclusion

Zinc supplementation appears to have a differential response on humoral and cell-mediated immunity in healthy older adults with near adequate dietary Zn intake. Moderate Zn supplementation at the level of 15 mg/d may help to maintain the T helper/CTL ratio and consequently enhance adaptive immunity; however, higher doses of supplementa-tion in the region of 30 mg/d may affect B-lymphocyte counts, exacerbating age-related changes. Further interven-tion studies should focus on the effects of Zn on other aspects of immunity, with relevance to an aging population, such as response to vaccination.

ACKNOWLEDGMENTS

This work was supported by the European Commission Quality of Life and Management of Living Resources Fifth Framework Program, Contract No: QLK1-CT-2001-00168, and by the Department of Education and Learning (DELNI), U.K.

We thank Dr. E. E. A. Simpson, School of Psychology, University of Ulster, for her role in participant recruitment and application of health and lifestyle and psychosocial questionnaires; the Causeway Laboratory, Causeway NHSS Trust, Coleraine, Northern Ireland for their analysis of full blood profiles; Dr. J. L. Lafond, Grenoble University Hospital, France, for his analysis of serum ferritin and transferrin saturation; and Mary Toohey, The Northern Ireland Clinical Research Support Centre (CRSC), Education and Research Centre, Royal Group of Hospitals Trust, Belfast for her advice and assistance with statistical analysis. We gratefully acknowledge the participation the participants in the study. C. F. H. was responsible for study execution, data analysis, and writing of the manuscript. M. K. assisted with study execution. C. C. was the coordinator of the ZENITH study and was responsible for analysis of biological zinc indices. I. B. provided statistical advice. H. D. A. provided advice on flow cytometry methodology and assisted in the preparation of the manuscript. P. J. R., J. M. O. M. P. B., J. J. S., and J. M. W. W. were responsible for study design, supervised study execution and data analysis, and assisted with preparation of the manuscript.

CORRESPONDENCE

Address correspondence to Julie M. W. Wallace, PhD, Northern Ireland Centre for Food and Health (NICHE), University of Ulster, Coleraine, Northern Ireland BT52 1SA. E-mail: [email protected]

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Received March 24, 2006

Accepted January 30, 2007

Decision Editor: Huber R. Warner, PhD


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