Journal of the American College of Nutrition

ISSN: 0731-5724 (Print) 1541-1087 (Online) Journal homepage: http://www.tandfonline.com/loi/uacn20

Effects of one year of supplementation with zinc and other micronutrients on cellular immunity in the elderly. J D Bogden, J M Oleske, M A Lavenhar, E M Munves, F W Kemp, K S Bruening, K J Holding, T N Denny, M A Guarino & B K Holland To cite this article: J D Bogden, J M Oleske, M A Lavenhar, E M Munves, F W Kemp, K S Bruening, K J Holding, T N Denny, M A Guarino & B K Holland (1990) Effects of one year of supplementation with zinc and other micronutrients on cellular immunity in the elderly., Journal of the American College of Nutrition, 9:3, 214-225, DOI: 10.1080/07315724.1990.10720372 To link to this article: http://dx.doi.org/10.1080/07315724.1990.10720372

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Effects of One Year of Supplementation with Zinc and Other Micronutrients on Cellular Immunity in the Elderly John D. Bogden, PhD, James M. Oleske, MD, Marvin A. Lavenhar, PhD, Elizabeth M. Munves, PhD, RD, Francis W. Kemp, BS, Kay S. Bruening, MA, RD, Kimberly J. Holding, BS, Thomas N. Denny, BS, Michael A. Guarino, MPH, and Bart K. Holland, PhD Departments of Preventive Medicine and Community Health (J.D.B., MAL., F.W.K., K.S.B., KJ.H., and B.K.H.), Pediatrics (JM.O. and T.N.D.), and Medicine (E.M.M.), University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, and the Bergen County Department of Health Services (MA.G.), Paramus, New Jersey

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Key words: zinc, vitamins, trace elements, delayed dermal hypersensitivity, mitogens, natural killer cell activity The objective of this study was to determine the effects of a year of Zn supplementation on Zn concentrations in circulating cells and on cellular immune functions in the elderly. Subjects, aged 60-89, were given a placebo, 15 mg Zn, or 100 mg Zn daily for 12 months. All subjects alsoreceiveda multivitamin/mineral supplement that contained no additional Zn. Blood samples were drawn and immune functions assessed prior to and at 3, 6,12, and 16 months after beginning Zn supplementation. Subject diets were also assessed at each visit. Dietary folate, pyridoxine, a-tocopherol, copper, zinc, and magnesium were consistently belowrecommendedintakes. Although plasma Zn increased sig­ nificantly in the 100 mg Zn treatment group, concentrations of Zn in erythrocytes, mononuclear cells, polymorphonuclear leukocytes, and platelets were not significantly increased by zinc supplementation. Natural killer cell activity was transiently enhanced by the 100 mg/day dose of Zn. There was a progressive improvement in delayed dermal hypersensitivity (DDH) and in lymphocyte proliferativeresponsesto two mitogens; this may have been due to one or more components of the multivitamin/mineral supplement administered to all study subjects. The enhancement of DDH was significantly greater in the placebo group than in either zinc treatment group. Thus, zinc had a beneficial effect on one measure of cellular immune function while simultaneously having an adverse effect on another measure of cellular immunity.

INTRODUCTION Studies of experimental animals and of humans pro­ vide extensive evidence that severe zinc deficiency can result in profound adverse effects on cellular immunity that are prevented by zinc supplementation [1-4]. How­ ever, the effects of zinc supplementation on cellular im­ mune functions in people who do not have frank zinc deficiency have not been definitively delineated. It is well established that cellular immune functions decline with age [5,6]. In a prior study of 100 unsupple-

mented free-living elderly subjects [7], we found that more than 90% had dietary zinc intakes below the RDA of 15 mg/day. In addition, a substantial percentage of study subjects had evidence of depressed cellular im­ munity reflected by poor in vitro proliferative responses of lymphocytes to stimulation by mitogens and by anergy to skin test antigens. Furthermore, responses to skin test antigens were significantly correlated with plasma zinc concentrations. In a second study [8], we assessed the effects of sup­ plementation of elderly subjects with either 15 or 100

Supported in part by grant No. 1 ROI AG04612 from the National Institute on Aging and a grant from the New Jersey State Commission on Cancer Research. Presented in part at the UCLA Colloquium "Metal Ion Homeostasis: Molecular Biology and Chemistry," Frisco, Colorado, April 13, 1988, and at the FASEB meeting, Las Vegas, Nevada, May 3,1988. Address reprint requests to Dr. John D. Bogden. Department of Preventive Medicine and Community Health, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103-2757.

Journal of the American College of Nutrition, Vol. 9, No. 3, 214-225 (1990) © 1990 John Wiley & Sons, Inc.

CCC 0731-5724/90/030214-12$04.00

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Effects of Supplementation with Zinc mg of zinc per day for 3 months. Delayed dermal hypersensitivity (DDH) and lymphocyte proliferative respon­ ses (LPR) to mitogens were not influenced by zinc sup­ plementation in most subjects, but there was a subgroup of elderly subjects who did experience enhanced DDH or LPR after zinc administration. The inability of zinc administration to enhance the cellular immune functions in most subjects may have been related to the fact that such supplements did not increase zinc concentrations in circulating cells, including immunologically active cells, despite increases in plasma zinc at the highest dose studied. Periods of supplementation greater than 3 months may be required to adequately define effects of zinc ad­ ministration on cellular immune functions in the elderly. The objective of the present study was to determine the effects of a full year of zinc supplementation on plasma and cellular zinc concentrations, DDH, LPR, and natural killer cell (NK) activity in a sample of healthy elderly subjects. The results reported here demonstrate that periods of supplementation greater than 3 months produce effects on cellular immunity that differ from those that may occur with a shorter duration of sup­ plementation.

METHODS Apparently healthy free-living elderly (aged 60-89) subjects were recruited at three senior citizen centers in Bergen County, New Jersey. These centers contain satel­ lite medical facilities of the Bergen County Department of Health Services and have medical offices. The study protocol was approved by the University of Medicine and Dentistry of New Jersey-New Jersey Medical School Institutional Review Board. Informed consent was obtained from subjects prior to participation in the study; the results reported in this paper are data from the 63 subjects enrolling and completing 16 months of participation. Excluded from enrollment in the study were subjects with a history of cancer or those taking corticosteroid or estrogen drugs. In addition, subjects having a recent infectious disease or other condition [9] that could affect plasma zinc concentrations were not enrolled until a minimum of 3 weeks after the disease had subsided. Also excluded were subjects who had been taking zinc supplements. The study had a double-blind partial crossover design. The volunteers were randomly assigned to one of the three treatment groups using a table of random numbers. Depending upon treatment group, each person was given a 3-6-month supply of placebo, 15 mg, or 100-mg Zn capsules and was instructed to consume one capsule

each day with their evening meal. After 1 year, all sub­ jects were given the placebo for the final 4 months of participation. The capsules were identical in appearance, used lactose as a filler, and contained zinc in the form of zinc acetate. These capsules were prepared by Twin Cities Pharmacy (South Plainfield, NJ) or at the Rutgers University School of Pharmacy (Piscataway, NJ) and were analyzed for their Zn content prior to use. Zn con­ tents were within 7% of target values for all capsules analyzed. In addition to the Zn capsules, each volunteer was given a supply of vitamin/mineral supplements which did not contain Zn. Subjects were instructed to consume the supplement daily with breakfast to minimize the ef­ fects that the supplement might have upon Zn absorption from the Zn capsule which was to be consumed at the evening meal. Included in the supplement capsules were the following vitamins and minerals: vitamin A, 5500 IU; vitamin C, 120 mg; vitamin B„ 3 mg; vitamin B2, 3.4 mg; niacin, 30 mg; vitamin B6, 3 mg; vitamin B12, 9 μg; vitamin D, 400 IU; vitamin E, 30 IU; pantothenic acid, 10 mg; folic acid, 0.4 mg; biotin, 15 μg; iodine, 150 μg; iron, 27 mg, magnesium, 100 mg; copper, 2 mg; manganese, 5 mg; chromium, 15 μg·, selenium, 10 μg; molybdenum, 15 μg, potassium, 7.5 mg; chloride, 7.5 mg; calcium, 40 mg; phosphorus, 31 mg. These capsules were manufactured by E. R. Squibb and Sons (Princeton, NJ). These multivitamin/mineral supplements were provided to discourage participants from taking thenown vitamins and to prevent other micronutrient deficiencies that could limit the ability of zinc sup­ plementation to enhance cellular immune functions. Sub­ jects were not permitted to consume any other nutritional supplements for the 16-month duration of the study. Subjects had an initial visit and were seen again 3, 6, 12, and 16 months (±10 days) later. A medical and nutri­ tional history was obtained at each subject visit. The medical history included questions about education, cur­ rent and/or former occupations, smoking, alcohol use, recent major personal problems or achievements, prior diseases, and current illnesses and medications. The nutritional history included questions about eating and exercise habits, prior use of vitamin and mineral supple­ ments, and food purchasing and preparation practices. Also included were a food frequency checklist and a 24-hr recall of the previous day's meals. To obtain the medical/nutritional history, the same master's degreelevel registered dietitian (KSB) interviewed all subjects; about 1 hour was required to administer the question­ naire. Food models were used to help subjects estimate portion sizes. Subject heights and weights also were measured, and pill counts were conducted at the 3, 6,12, and 16 month visits.

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Effects of Supplementation with Zinc DDH to seven recall antigens was assessed using the Merieux Multitest CMI skin test antigen applicator (Merieux Institute Inc., Miami, FL) [10,11]. The seven antigens administered simultaneously by this applicator were tetanus, diphtheria, Streptococcus, tuberculin, Can­ dida, proteus, and trichophyton, as well as a control (glycerin) injection. Reactions were assessed 48 + 2 hours after injection by measuring mean induration diameter (mm). Induration of 2 mm or greater was con­ sidered a positive reaction. Data are reported as the num­ ber of positive responses to the seven antigens ad­ ministered and the sum of induration for positive respon­ ses. All skin tests were administered and read by the same individual, who did not know the subject's treat­ ment group assignment. Batches of 7-ml royal blue-stoppered zinc-free vacutainers (Becton Dickinson, Rutherford, NJ), both heparinized and without an anticoagulant, were checked for zinc contamination before use. Blood samples were drawn between 0930 and 1100 hours from nonfasting subjects. Fifty milliliters of whole blood were withdrawn by venipuncture; 35 ml were collected into heparinized vacutainers and 15 ml into vacutainers without an an­ ticoagulant. Dipotassium EDTA (JT Baker, Phillipsburg, NJ) was added to some vacutainers to prevent denaturation of plasma. Blood samples were transported to the laboratory within 2 hours of collection. Laboratory procedures were conducted using techni­ ques designed to minimize the possibility of contamina­ tion of blood samples with zinc and cytotoxic substan­ ces. Serum and plasma were separated from the whole blood samples by centrifugation at 4°C. Samples exhibit­ ing visible hemolysis were not analyzed. Erythrocytes (RBC), mononuclear cells (MNC), polymorphonuclear leukocytes (MNC), and platelets were isolated and washed as previously described [7]. Plasma copper and zinc and RBC, PMN, and platelet zinc concentrations were determined by flame atomic absorption spectrophotometry and MNC zinc concentra­ tions were determined by flameless atomic absorption techniques [12]. In some cases, the volume of blood col­ lected was insufficient to conduct analyses for cellular zinc. Serum concentrations of albumin, alkaline phosphatase, and cholesterol were determined by automated procedures at the University Hospital Pathology Laboratory using a Technicon Model SMAC Π (Technicon, Tarrytown, NY). Red and white cell counts were done with a Coulter Model ZM cell counter after dilu­ tion using a Coulter Dual Diluter Π (Coulter Electronics, Luton, UK). Whole blood hemoglobin concentrations were determined with a Coulter Model HGBR2 Hemoglobinometer (Coulter Electronics, Hialeah, FL).

216

Serum albumin and cholesterol were determined to help assess subject nutritional status, and alkaline phosphatase was done because it is a zinc metalloenzyme. Hemoglobin concentrations and cell counts were done to quantify cellular zinc concentrations. In vitro proliferative responses to mitogens and an­ tigens were assessed by standard techniques [7,12-15]. Mitogens used were phytohemagglutinin (PHA) (Difco Labs, Detroit, MI), concanavalin A (ConA) (Pharmacia, Piscataway, NJ), and pokeweed mitogen (PWM) (Grand Island Biological, Grand Island, NY). MNC pellets were suspended in RPMI-1640 (KC Biological, Lenexa, KS) containing 100 mM sodium pyruvate (Gibco, Grand Is­ land, NY), 200 mM glutamine (Hazelton/KC, Lenexa, KS), 100 U/ml penicillin-streptomycin (Hazelton/KC, Lenexa, KS), and 20% autologous serum. Cells were exposed to mitogens in round-bottomed microtiter plates (Corning Glass Works, Corning, NY). For PHA, PWM, and ConA plates, cells were pulsed with tritiated thymidine 72 hours after culture and harvested 6 hours later. PHA was evaluated at three dilutions (1:50, 1:200, and 1:400), ConA at two dilutions (1:80 and 1:160) and PWM at two dilutions (1:10 and 1:20). The dilutions used resulted in final concentrations in culture of 4.0, 1.0, and 0.5 μg/ml for PHA, 49.4 and 24.7 μg/ml for ConA, and 20 and 10 μg/ml for PWM. All assays were performed in quadruplicate together with normally reac­ tive controls, and the results, excluding obvious outliers, were averaged. Values excluded as outliers were at least two times the mean of the other three replicates. Thymidine (3H) radioactivity was measured in a Beckman LS6800 scintillation counter (Beckman, Irvine, CA). If the normal control response for PHA 200 was < 10,000 counts per minute, the data for samples from that run were not used. A stimulation index (SI) was calculated by dividing raw counts by background. Nor­ mally reactive controls used were laboratory personnel with a prior history of normal responses to this panel of mitogens. Natural killer cell (NK) activity was assessed by a "Cr release assay [16]. Data reduction and analysis were performed using IBM PC/AT and PC/XT microcomputers (IBM Corp., Armonk, NY). Software used included dBase ΙΠ (Ashton-Tate, Culver City, CA), Statistical Analysis System (SAS) (SAS Institute Inc., Cary, NC), and Nutritionist ΙΠ (N-Squared Computing, Silverton, OR). The latter pro­ gram was used to calculate the content of the diet for zinc, magnesium, copper, energy, iron, calcium, protein, vitamin A, oc-tocopherol, folate, vitamin C, pyridoxine, and cyanocobalamin. The numbers of subjects completing each phase of the study from an original group of 158 were: 3 months, 110; 6 months, 98; 12 months, 83; and 16 months, 63.

VOL. 9, NO. 3

Effects of Supplementation with Zinc Table 1. Plasma Trace Metals After 1 Year of Supplementation Group

Initial

6 months

3 months

12 months

16 months

Zinc Placebo

13.3 ±0.4

IS mg zinc 100 mg zinc

(23)

13.8 ±0.5

(23)

13.6 ±0.4

(24)

12.6 ± 0.4

(24)

13.5 ±0.5

(20)

13.3 ±0.4

(20)

13.3 ±0.4

(17)

12.4 ±0.2

(20)

16.6 ±0.9

(18)B

17.2 ± 1.0

(19)B

16.8 ±0.6

(17)B

12.8 ±0.5

(18)A

(22)

13.1 ±0.4

12.9 ±0.5

(20)

13.1 ±0.5

(19)A

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Copper Placebo

19.1 ±0.9

(24)

17.1 ±0.7

(23)

18.1 ±0.7

(23)

18.5 ±0.7

(24)

17.9 + 0.8

(24)

15 mg zinc

18.1 ±0.8

(20)

17.8 ±0.8

(20)

18.3 ±0.7

(20)

18.8 ±0.8

(Π)

18.3 ±0.8

(20)

100 mg zinc

17.1 ±0.7

(19)

17.4 ±0.8

(18)

17.1 ±0.7

(19)

18.3 ±0.8

(17)

18.3 ±0.7

(18)

Data are mean ± SE (n). Units are μπιοΙ/L. Mean values in the same row with different superscripts are significantly (p < 0.05) different. 12-16 months, no zinc supplementation.

Statistical analyses were performed separately on all subjects and on the subgroup completing the full 16 months of participation. Since the results were similar, only data for the 63 subjects completing the entire study are reported here. Subjects who did not complete the study withdrew because of illness or inability to comply with the study protocol. A preliminary descriptive data analysis was com­ pleted to summarize the characteristics of the study sub­ jects at intake with respect to their plasma and cellular zinc concentration distributions and other measures of zinc nutriture, their performance on tests of immune function, and their age and sex distributions. These sum­ mary data were used to assess the comparability of patients randomly assigned to the three treatment groups. After treatment was initiated, follow-up mean posttreatment zinc levels and immune response scores were com­ pared with corresponding mean pretreatment scores for each of the study subgroups. Analysis of variance (ANOVA) for repeated measures was used to assess ef­ fects of treatment over time in the three treatment groups. When significant F values were obtained, pairwise comparisons were made by Duncan's multiple range test. To assess the significance of treatment intervention on specific or composite indices of immune response, a multiple regression approach to ANOVA was applied hierarchically as follows: (1)

The criterion or dependent variable (Y) was defined as the posttreatment immune func­ tion or zinc nutriture variable.

(2)

(3)

Two dummy (0,1) treatment variables designating membership in the three treat­ ment group were entered in the first step. The additional independent variables age, sex, and the pretreatment value of the de­ pendent variable being evaluated, as well as their interactions with treatment status, were entered into the model in decreasing order of significance using a stepwise SAS procedure. Independent variables are reported as significant determinants of de­ pendent variables if p was < 0.05 for the effect of the independent variable in the model.

Finally, associations between selected variables were as­ sessed by calculations of Pearson correlation coeffi­ cients.

RESULTS Study Sample The number of subjects completing 16 months of zinc/vitamin supplementation was 63. Of these, 24 were in the placebo group, 20 in the 15 mg Zn per day treat­ ment group, and 19 in the 100 mg Zn per day treatment group. Study subject ages ranged from 60 to 89 years. Mean subject age did not differ significantly among the three groups; mean (±SE) ages were 71.2 ± 1.3, 71.0 ± 1.3, and 72.1 ± 1 . 4 years for the placebo, 15-mg, and

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION

217

Effects of Supplementation

with

Zinc

Table 2. Cellular Zinc Concentrations After 1 Year of Supplementation Group

3 months

Initial

6 months

12 months

16 months

Erythrocyte Placebo 15 mg zinc 100 mg zinc

0.016 ± 0.001 0.016 ± 0.001 0.01510.000

(22) (19) (18)

0.017 ±0.001 0.016 ±0.001

(22) (20)

0.016 ±0.001 0.016 ±0.001

(22) (20)

0.016 ±0.000 0.017 ± 0.001

0.016 ±0.001

(Π)

0.017 ± 0.002

(19)

0.016 ±0.001

(17) (15)

(23)

0.016 ±0.001 0.017 ±0.001

(22) (19)

0.016 ±0.001

(18)

Polymorphonuclear leukocyte Placebo

0.068 ± 0.007

(19) A 3 C

0.082 ±0.008

(21)A

0.070 ± 0.009

(21)A'B

0.056 ± 0.004

(20)B-C

0.051 ±0.003

(23)c

15 mg zinc 100 mg zinc

0.057 ±0.005 0.065 ±0.009

(17)A (14)

0.078 ±0.007 0.058 ±0.006

(17)AB (13)

0.085 ±0.013 0.073 ±0.006

(15)B (17)

0.056 ± 0.005

0.058 ±0.005 0.050 ±0.007

(18)A

0.055 ±0.011

(14)A (10)

(11) (10) (12)

0.073 ±0.006

(21)

0.071 ±0.009

(22)

0.065 ±0.006

(17) (18)

0.049 ±0.006

(17) (18)

(15)

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Mononuclear cell Placebo

0.058 ±0.008

(12)

0.069 ±0.011

(16)

0.076 ±0.016

15 mg zinc 100 mg zinc

0.045 ±0.003

(7) (11)

0.084 ±0.010

(14)

0.078 ±0.011

0.068 ±0.009

(11)

0.071 ±0.015

0.081 ±0.008

0.076 ±0.007

0.062 ±0.008

Platelet Placebo 15 mg zinc 100 mg zinc

0.0079 ±0.0013 (16) 0.0070 ±0.0013 (15) 0.0088 ±0.0015 (14)

0.0074 ±0.0014 (21) 0.0070 ±0.0008 (19) 0.0056 ± 0.0006 (16)

0.0074 ± 0.0011 (22) 0.0070 ±0.0013 (15) 0.0080 ±0.0013 (17)

0.0049 ±0.0004 (21) 0.0079 ±0.0015 (15) 0.0061 ± 0.0008 (14)

0.0088 ± 0.0016 (21) 0.0052 ± 0.0006 (19) 0.0074 ±0.0015 (15)

Data are mean ± SE (n). Units are μηιοΐ zinc/billion cells. Mean values in the same row with different superscripts are significantly (p < 0.05) different. 12-16 months, no zinc supplementation.

100-mg treatment groups, respectively. The percentages of females in each treatment group were also similar: 58.3, 70.0, and 63.2% in the placebo, 15-mg, and 100mg treatment groups, respectively. For males completing the study, the mean body weight was 78.4 + 2.6 kg and the mean height was 171 ± 2 cm. For females, mean body weight was 65.4 ± 2.5 kg and mean height 155 ± 1 cm. Pill counts indicated that study subjects consumed 82.7 ± 1.4% of the zinc or placebo capsules and 92.8 ± 1.3% of the vitamin capsules provided. Pill counts did not differ significantly among the three treatment groups and did not vary significantly during the course of the study.

Plasma and Cellular Concentrations Plasma zinc concentrations were similar in each of the three treatment groups at the start of the study. They were significantly increased at 3, 6, and 12 months only in the 100-mg Zn treatment group (Table 1). They remained elevated for the duration of zinc administration and returned to baseline values after zinc administration was discontinued (12—16 months). The plasma zinc con­ centration in the 100-mg Zn group was significantly cor­ related (r = 0.53 and 0.51, p < 0.05) with the pill counts for zinc capsules done at the 3 and 6 month visits.

218

Table 2 contains the zinc concentrations in circulating cells. Mononuclear cell, platelet, and erythrocyte ^g/billion cells or μξ/g hemoglobin) zinc concentrations were not significantly changed in any treatment group. Polymorphonuclear leukocyte zinc was transiently in­ creased in the 15-mg Zn treatment group but not in the 100-mg Zn group. Plasma copper (Table 1) was not significantly in­ fluenced by zinc supplementation. Serum albumin, alkaline phosphatase, and cholesterol were also not sig­ nificantly influenced by zinc administration. Mean (±SE) serum cholesterol concentrations were 6.31 ±0.16, 6.10 ± 0.13, 6.23 + 0.16, 6.15 ± 0.13, and 6.26 + 0.16 mmol/L at the initial, 3, 6, 12, and 16 month visits, respectively. Corresponding serum albumin concentrations were 43.5 ± 0.3, 43.2 ± 0.4, 43.3 ± 0.3, 43.1 ± 0.3, and 42.9 ± 0.3 g/L. None of the study subjects had a serum albumin concentration below our lower limit of normal (30.0 g/L) at any time. Cellular Immune Functions DDH, as assessed by positive responses and indura­ tion (Table 3), increased continuously during the course of the study. Final values were more than twice the ini­ tial values. The increase in DDH was significantly

VOL. 9, NO. 3

Effects of Supplementation with Zinc Table 3. Delayed Dermal Hypersensitivity After 1 Year of Supplementation Group

16 months

12 months

6 months

3 months

Initial

Number of Dositive responses Placebo

A

1.58 ± 0.30 (24)

2.17 ± 0.32 (23)Α·Β

2.71 ±0.37 (21 )B-C

2.95 ± 0.46(20)CD

3.53 +0.41 (19)D

15 mg zinc

1.40 ± 0.40 (20)A

1.85 ± 0.40(20)AB

1.75 ± 0.53 (16)Α·Β

2.53 ± 0.49 (19)B

3.55 ± 0.52 (20)c

B

2.81 ± 0.49 (16)B

c

3.33 ± 0.27 (55)D

100 mg zinc All groups combined

A

1.53 ± 0.35 (19)

A

1.51 ± 0.20 (63)

A

1.65 ± 0.38 (17)

B

1.92 ±0.21 (60)

A

1.84 ± 0.32 (19)

2.53 +0.42 (19)

B

2.14 ± 0.24 (56)

2.67 ± 0.26 (58)

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Sum of induration 10.57 ± 1.78 (20)A

16.92 ± 3.54 (19)B

6.22 + 2.36 (16)A

10.24 ± 2.26 (19)B

12.47 ± 2.01 (20)B

A

B

Placebo

7.52 ± 2.24 (24)A

7.65+ 1.24 (23)A

12.26 ±2.57 (21)A-B

15 mg zinc

4.62 ± 1.46 (20)A

6.10 ± 1.62 (20)A A

A

100 mg zinc

5.47±1.36(19)

All groups combined

5.98 ± 1.05 (63)A

5.94 ± 1.29 (17)

7.16 ±1.58 (19)

10.76 ± 1.72 (19)

11.03 ±2.39(16)B

6.65 ± 0.80 (60)A

8.80 ±1.32 (56)B

10.53 ± 1.09 (58)B

13.59± 1.59 (55)c

Data are mean ± SE (n) of the total number of positive responses or sum of induration of positive responses (mm) to panel of seven recall antigens. Mean values in the same row with different superscripts are significantly (p < 0.05) different. 12-16 months, no zinc supplementation.

DELAYED DERMAL HYPERSENSITIVITY NUMBER OF POSITIVE RESPONSES

DELAYED DERMAL HYPERSENSITIVITY INDURATION

[ZZI

zz:

Placebo

O

Placebo

15 mg Zinc

15 mq Zn

100 mg Zn

100 mg Zn

3 6 12 16 MONTHS OF PARTICIPATION

O

3 6 12 16 MONTHS OF PARTICIPATION

Fig. 1. Mean (±SE) number of positive skin test responses to a panel of seven skin test antigens. n = 17-24 for all values. The increase in the number of positive responses with time is significantly (p < 0.01) greater in the placebo group than in the 15mg and 100-mg Zn per day treatment groups.

Fig. 2. Mean (±SE) of total induration of positive responses to a panel of seven skin test antigens. n = 17-24 for all values. The increase in the total in­ duration of positive responses with time is significant­ ly (p < 0.01) greater in the placebo group than in the 15-mg and 100-mg Zn per day treatment groups.

(p < 0.01) greater in the placebo group than in the zinc treatment groups (Figs. 1 and 2). The suppression of the increase in DDH by zinc persisted even after zinc sup­ plementation was discontinued at 12 months, both in the treatment group receiving 15 mg zinc per day (Fig. 2) and especially in the group given 100 mg zinc per day (Figs. 1 and 2). Skin test responses to each of the seven antigens administered were increased during the study

(Table 4). Of 22 initially anergic subjects, 18 reacted to at least one skin antigen by the 16 month visit. Results for the various dilutions used for PHA, ConA, and PWM were similar; therefore, only data for one dilu­ tion for each mitogen are reported in Table 5. LPR to PHA and PMW, but not ConA. increased during the course of the study, particularly in the placebo group. NK cell activity was increased transiently (at 3 but not 6

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION

219

Effects of Supplementation with Zinc Table 4. Delayed Dermal Hypersensitivity After 1 Year of Supplementation: Responses to Individual Skin Test Antigens Initial value n = 63

3 months, n = 60

6 months, n = 58

21 ±5 A 16±5 A 14±4 A 35±6 A 35±6 A 17±5 A 13±4A

22 + 5A 25±6 A 7±3 A 47±6 A 55±6 B 27±6 A 13±4A

30±6 A 27±6 A 18±5 A 50±7 B 48±7 A 23±6 A 16±5 A

Antigens

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Tetanus Diphtheria Streptococcus Tuberculin (PPD) Candida Trichophyton Proteus

12 months, n = 58

16 months, n = 55

41±7B

49±7 B 58±7 C 27±6 B 73±6 C 60±7 B 35±6 B 31 ±6 B

B

33±6 28±7 B 55±7 B 59±7 B 26±6 A 24±6 A

Data are percent of positive responses to individual skin test antigens (mean ± SE). All treatment groups combined. Mean values in the same row with different superscripts are significantly (p < 0.05) different.

Table 5. In Vitro Lymphocyte Proliferative Responses to Mitogens Group

3 months

Initial

6 months

12 months

16 months

Phytohemagglutinin A (1/200 dilution) Placebo 15 mg zinc 100 mg zinc All groups combined

A

22 ± 3 (22) 21 ± 5 (Π) Α

19 ± 5 (Π) 21 ± 3 (56)A

41 ± 12 (22)Α·Β A

23+4 (20) 44+11 (14) 35 ± 6 (56)ABC

33 ± 15 (19)Α·Β 26±15(12) A 36 ±24 (14) 32 ± 10 (45)Α·Β

74±29(10) B 71 ± 17 (12)B 35 ± 13 (10) 61 ± 12 (32)c

41 ±20 67 ±20 64 ±18 57 ±11

(19)A* (18)B

25 ± 9 (10) 18±4 (12) 22 ±11 (10) 21 ± 5 (32)

16 ±5 16±4 17±4 16±2

(19)

(18) (55)B'C

Concanavalin A (1 /80 dilution) Placebo 15 mg zinc 100 mg zinc All groups combined

15 ± 3 10 ± 3 11 ± 3 12±2

(22) (17) (17) (56)

24 + 6 (22) 13 ±2 (20) 21+4 (14) 19 ± 3 (56)

16 ± 5 19 ± It 13 ± 5 16 ± 4

(19) (12) (14) (45)

(18) (18) (55)

Pokeweed Mitogen (1/20 dilution) Placebo 15 mg zinc 100 mg zinc All groups combined

7±1 5±1 6±2 6±1

A

(22) (16)

(17) (55)A

10 ±2 7+1 12 ± 2 10 + 1

(22)Α·Β (20) (14) (56)AB'C

8 ±3 8 ±2 11 ± 4 9±2

(19)Α·Β (12) (14) (45)AB

18 + 7 13±3 13±7 15 ± 3

(10)B (12) (10) (32)c

11 ± 4 13 ± 4 14 ± 3 13 ± 2

(19)AB (18) (18) (55)B'C

Data are mean ± SE (n) stimulation indices. Mean values in the same row with different superscripts are significantly (p < 0.05) different. 12-16 months, no zinc supplementation. Concentrations in culture are: PHA, 1.0 μg/ml; ConA, 49.4 μg/ml; PWM, 10 μg/ml.

and 12 months) only in the 100-mg Zn treatment group. Since the results were similar for each of the four tar­ get/effector cell ratios used, only data for the 50:1 ratio are reported in Table 6.

220

Multiple regression analysis indicated that there were no significant effects of subject age or sex on DDH or LPR, though values for DDH at 3, 6, 12, and 16 months were significantly associated with the initial (month 0) values of DDH.

VOL. 9, NO. 3

Effects of Supplementation with Zinc Table 6. Natural Killer Cell Activity, % 51Cr Release Group

Initial

3 months

6 months

12 months

Placebo

25.5 ± 5.3

(13)

29.2 ± 7.7

(12)

23.9 ± 9.0

(9)

40.0 ± 8.0

(Π) (14)

22.0 ± 6.8

15 mg zinc

25.8 ± 6.0

(14)

13.8 ±6.8

(11)

30.5 ± 6.8

100 mg zinc

26.9 ± 5.8

(10)A

52.3 + 9.7 (1DB

34.5 ± 10.4 (7)A

28.3 ± 6.0

(11) (12)A

16 months 32.6 ± 4.7

(16)

38.6+10.1 (10) 26.0 ± 10.9

(7)A

Data are mean ± SE (n). Target/effector cell ratio = 50:1. Mean values in the same row with different superscripts are significantly (p < 0.05) different. 12-16 months, no zinc supplementation.

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Diets There were no consistent significant différences among the three treatment groups for the five repetitions of the 13 dietary components assessed by either 24-hr recall or the food frequency checklist method. Table 7 contains nutrient intake from food as determined by the food frequency checklist approach. There were modest fluctuations in intakes from food of energy and selected nutrients consumed by study subjects in each treatment group during the course of the investigation. In par­ ticular, dietary iron in males and dietary energy, iron, protein, oc-tocopherol, and folate in females varied sig­ nificantly with time. Intake from food of energy, folate, pyridoxine, cctocopherol, copper, zinc, and magnesium were consis­ tently below recommended intakes for both males and females. For males, percentages below Vi of the RDA were 46% for folate, 33% for pyridoxine, 80% for cctocopherol, 51% for copper, 67% for zinc, and 33% for magnesium. Corresponding values for females were 75% for folate, 41% for pyridoxine, 65% for oc-tocopherol, 60% for copper, 81% for zinc, and 29% for magnesium.

DISCUSSION A substantial increase in DDH responses, which was both clinically and statistically significant, occurred during the 16 months of the study in each of the three treatment groups and was clearly not due to zinc sup­ plementation. The reasons for this increase are not known but could include the booster effect that may result from repeated application of skin test antigens [16,17], the multivitamin/mineral supplement ad­ ministered to all study subjects, changes in subject diets, or other unknown factors. We believe that the most like­ ly explanation is the multivitamin/mineral supplement given daily to all study participants. This supplement contains a number of single nutrients that are known to be required for optimal cellular immune functions [18].

These include magnesium, 100 mg (29-33% of RDA), iron, 27 mg (270% of RDA), selenium, 10 μg (5-20% of recommended intake), pyridoxine, 3 mg (136-150% of RDA), folate, 0.4 mg (100% of RDA), cyanocobalamin, 9 μg (300% of RDA), vitamin A, 5500IU (110-138% of RDA), and cc-tocopherol, 30 IU (200-250% of RDA). Furthermore, the elderly subjects in the study had mean dietary intakes for several vitamins that were consistent­ ly less than % of the RDA [19], specifically for folate, oc-tocopherol, and pyridoxine. In addition, copper in­ takes were consistently below the recommended range. The usefulness of the RDAs is limited by incomplete food tables for some nutrients, inadequate data for the elderly, and by other factors [20,21]. Nevertheless, future studies of single nutrients and cellular immune functions in the elderly might focus on the above four micronutrients. Future studies might also include investigation of magnesium, since its intake from food by many study subjects was low. There is prior evidence for inadequate magnesium intake and absorption in the elderly, and this element is required for immunocompetence [18,22-24]. The 100 mg of magnesium provided in the supplement administered could convert the marginal magnesium in­ takes of many of our subjects to more adequate levels. It is unlikely that changes in subject diets during the 16-month course of the study resulted in enhanced DDH responses, since the data show only modest fluctuations in the dietary content of the 13 nutrients assessed. It is also unlikely that the booster effect from repeated testing can explain the enhanced DDH responses found. Although evidence for boosting has been widely reported in the literature, studies using the skin antigen test kits employed in this study show either minimal or no evidence of boosting [25-28]. In addition, maximum boosting tends to occur with the first repetition of skin testing [29,30] (here the 3 month visit), and in our study DDH continuously improved during the 16-month course of the study. For tuberculin, if boosting does not occur with a second skin test given 1 week or more after the initial injection, then it is not likely to occur with

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION

221

Effects of Supplementation

with Zinc

Table 7. Selected Dietary Nutrients

Nutrient

Initial value

6 months

12 months

16 months

Age 51 + RDAor recom. intake

1607 ± 126

1694 ± 116

1747 ± 153

1802 ± 150

2050-2400

14.3 ± 1.3BC

15.4 ± 1.2C

14.6 ± 1.2B'C

3 months Males

Energy (kcal) Iron (mg)

13.3 ± 1.5

12.1 ± 1.2A

Zinc (mg)

8.6 ± 0.8

7.8 ± 0.5

9.3 ±0.8

8.6 ± 0.5

8.8 ± 0.7

15

Copper (mg)

1.4 ±0.1

1.4 ±0.1

1.4 ±0.1

1.5 ±0.1

1.5 ±0.1

2-3

Calcium (mg)

700 ± 6 6

693 ± 63 68+4

699 ± 6 7

719 ± 69

784 ± 61

800

68 ± 5

74 ± 5

73 ± 4

73 ± 5

56

Vitamin A (IU)

9510 ± 1339

9143 ± 1122

10027 ± 1645

8208 ± 1037

10233 ± 1400

5000

Tocopherol (mg) Folate ^ g )

4.3 ± 0.6 289 ± 3 4

5.0 ± 0.6 279 ± 25

5.0 ± 0.5 280 ± 3 0

4.9 ± 0.5 310 ±32

5.3 ± 1.4 306 ± 28

10 400

Vitamin C (mg)

121 ± 14

145 ± 20

132 ± 13

60

1.8 ±0.2

119 ± 11 1.7 ±0.2

127 ± 13

Pyridoxine (mg)

1.9 ±0.2

2.0 ± 0.2

2.0 ± 0.2

2.2

Protein (g)

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1635 ± 109 A3

10

Cyanocobalamin ^ g )

5.2 ± 1.3

5.2 ±0.9

9.1 ± 3.0

5.0 ± 1.1

6.9 ± 1.4

Magnesium (mg)

261 ± 22

272 ± 20

277 ± 2 1

305 ± 25

287 ± 20

350

3

1611 ±78 B

1600-1800

13.1 ±0.8 B

10

Females Energy (kcal)

1472 ± 97

AB

1382 ±60* A

1515 ± 7 1 A B

1525 ± 84 AB

A

11.4±0.6

A

Iron (mg)

10.0 ± 0.8A

10.1 ±0.6

Zinc (mg)

7.7 ± 0.5

7.5 ± 0.4

7.9 ±0.4

7.4 ± 0.5

8.4 ± 0.5

15

Copper (mg) Calcium (mg)

1.2 ±0.1 791 ± 76

1.2 ± 0.1 759 ± 6 0

1.3 ±0.1 775 ± 5 9

1.4 ±0.1 783 ± 50

1.4 ±0.1 847 ± 69

2-3 800

60±3A

60±3A

63 ± 3 A

62 ± 3 A

69±4B

44

8563 ±918

9270 ± 1050

10169 ± 1377

7245 ± 673

8731 ±972

4000

5.1 ±0.4 A

4.3 ±0.2 A

7.2 ± 1.5B

5.0 ± 0.3A

Protein (g) Vitamin A (IU) Tocopherol (mg)

A

5.4 ±0.4A-B

8

188 ± 13

251 ± 2 1 B

117 ± 9

112 ± 11

117 ± 1 0

60

1.6 ±0.1

1.5 ±0.1

1.7 ±0.1

2.0

4.0 ±0.5

4.3 ± 0.9

3.6 ± 0.4

4.9 ±0.6

3

255 ± 16

252 ± 14

258 ± 12

269 ± 14

200 ± 18

216 ± 1 6

Vitamin C (mg)

98 ± 1 2

103 ± 11

Pyridoxine (mg)

1.4 ±0.1

1.6 ±0.1

Cyanocobalamin ^ g )

3.6 ± 0.5

Magnesium (mg)

239 ± 13

Folate ^ g )

11.5 ±0.6

AB

221 ± 16

AB

A

400

300

Data are mean ± SE. AH treatment groups combined, n = 22 or 23 for all tabular values for males and 39 or 40 for females. Mean values in the same row with different superscripts are significantly (p < 0.05) different Determined by food frequency checklist method.

subsequent injections [30,31]. Nevertheless, a definitive assessment of whether the multivitamin/mineral supple­ ment used in this investigation can enhance DDH must await additional study with appropriate control groups. It will be important to conduct such studies because anergy in the elderly is associated with increased mortality, in­ cluding greater mortality subsequent to surgery and as a result of infectious diseases [32-34]. Even if future studies find that boosting is in part responsible for the enhancement of DDH observed, our data suggest that administration of 15 mg and especially 100 mg of Zn per day may diminish or retard the increase in DDH.

222

Subject age and sex did not significantly influence the progressive enhancement of DDH observed. Thus, the benefits potentially available from micronutrient sup­ plementation may occur in both sexes and for the entire age range (60-89) studied. Chandra [35] has reported that elderly subjects with clinical, anthropométrie, or laboratory evidence of nutri­ tional deficiencies experienced improved cell-mediated immunity after supplementation for 8 weeks with a for­ mulation providing 500 kcal/day as protein, fat, and car­ bohydrates, as well as minerals, vitamins, and trace ele­ ments. Enhancement of DDH in our study subjects

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Effects of Supplementation with Zinc developed over a longer period of time. This may be due to differences between Chandra's formulation and the multivitamin/mineral supplement that we used and/or to differences in initial nutritional and immunological status of subjects in the two studies. Nevertheless, both Chandra's study and the present investigation suggest that nutritional supplementation of the elderly may result in improved cellular immune functions. The lower than recommended intakes of energy, cop­ per, zinc, folate, pyridoxine, magnesium, and atocopherol observed in the present study have been pre­ viously described in the elderly [7,8,23,36-38]. The results for NK activity are in agreement with prior experimental and in vitro studies that suggest that high doses of zinc may enhance natural killer cell ac­ tivity. They are consistent with the results of Bunk et al [39], who found that doses of zinc higher than needed for optimal growth enhance natural killer cell activity in the mouse. Ventura et al [40] have found in vitro Zn enhancement of human natural killer cell activity of cells from elderly but not young subjects, suggesting that there may be differences in the response of the young and elderly to supplemental Zn. Thus, even though some elderly individuals may have relatively preserved NK cell function in spite of reduced T-cell function, this does not preclude the possibility that NK activity in some healthy elderly subjects can improve with Zn sup­ plementation at a dose of 100 mg Zn per day. However, the improved NK activity did not persist beyond 3 months, suggesting eventual adaptation to the effects of zinc on NK activity. Except for NK activity, cellular immune functions were not significantly improved by administration of zinc at doses of 15 and 100 mg/day. In fact, zinc at both doses studied appears to have slowed down the progres­ sive improvement in DDH (Fig. 1 and 2) that we ob­ served. The effects persisted after zinc supplementation was discontinued at 12 months, suggesting that tissue zinc stores may continue to suppress the increase in DDH for several months after zinc supplementation is stopped. Perhaps zinc supplementation can interfere with the absorption or metabolism of other nutrients, such as copper and folate, that are required for optimal cellular immune functions. Zinc inhibition of copper absorption is well known [41,42], and recent studies suggest an an­ tagonistic relationship between zinc and folate [43,44]. In the present study, lymphocyte proliferative respon­ ses to mitogens were significantly enhanced for PHA but not ConA. Since PHA and ConA may stimulate different T-cell subpopulations [45], these data suggest that dif­ ferent T-cell subsets may have different nutritional re­ quirements. Responses to PWM, a T-cell-dependent Bcell mitogen, were also significantly increased. These

results are similar to those of Talbott et al [46], who found that supplementation of elderly subjects for 1-2 months with 50 mg pyridoxine hydrochloride per day increased PHA and PWM, but not ConA, responses. The pill counts conducted suggest good subject com­ pliance with the study protocol. Compliance is further documented by the observation of expected increases in plasma zinc in subjects receiving the 100-mg/day dose; this increase was significantly correlated with pill counts for the subjects taking this dose. Zinc supplementation, even at a dose of 100 mg zinc per day for 1 year, did not increase RBC, MNC, or platelet zinc concentrations, even though plasma zinc was significantly increased by a daily dose of 100 mg Zn. There were significant fluctuations in PMN zinc concentrations in the placebo and 15-mg Zn groups, but not in the group receiving 100 mg Zn per day. The pat­ tern of these changes suggest that they were not caused by zinc supplementation. The above results suggest that monitoring of zinc concentrations in circulating cells during zinc supplementation of the elderly is of little value. Other investigators have found depressed NK cell ac­ tivity in zinc-deficient sickle cell disease patients [47] and diminished interleukin-2 production in elderly sub­ jects with evidence of mild zinc deficiency [48], but high doses of zinc have also been reported to inhibit polymorphonuclear leukocyte chemotaxis [49]. Administration of the 100-mg/day dose of zinc in the present study resulted in a significant increase in NK cell activity at 3 months, but this dose and the 15-mg/day dose retarded the progressive increase in DDH that was observed in all treatment groups. Thus, zinc supplementation may exert beneficial effects on some measures of cellular immune function while simultaneously having adverse effects on other measures of cellular immunity. The results of this study suggest that additional inves­ tigation could lead to development of a safe micronutrient formulation that could exert favorable effects on cellular immunity in the elderly. Such studies could potentially lead to substantial health benefits for the elderly, since impaired cellular immunity in this age group is associated with greater morbidity and mortality.

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JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION

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Received May 1989; revision accepted November 1989.

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