Zinc metabolism and immune function in healthy elderly people

Clind

Nurrition (1994) 13.280-285

@ Longman

Group Ltd 1994

Zinc metabolism elderly people

and immune

G. C. STURNIOLO+, R. D’INCA”, M. C. MONTINO”, F. ROMAGNONIO and R. NACCARATO*

function

in healthy

M. SANZARI*, D. MARAN’, G. VALERIO”,

*Divisione di Gastroenterologia, lstituto di Medicina Interna, Universit2 di Padova; +Diparfimento di Medicina lntema e Terapia Cfinica, Universitg di Messina; *Laboratorio Analisi Ospedafe Geriatric0 di Padova; ‘Divisione Acuti Ospedale Geriatric0 di Padova. (Correspondence to: GCS, Dipartimento di Medicina lnterna e Terapia Clinica, Universith degli Studi, 98100 Messina, Italy)

ABSTRACT-Changes in immune function have been described in the elderly, and zinc is known to influence immune parameters. Zinc supplementation has, however, produced contradictory results. We evaluated zinc metabolism and immunity in 15 healthy noninstitutionalised elderly people on a self-selected diet and compared the results with those obtained from 10 young healthy subjects. The elderly subjects had reduced serum zinc levels and their zinc absorption values were lower than in the young subjects. Circulating immune parameters were within the normal range, while delayed immune response was altered in 8 of the 15 elderly subjects. Zinc supplementation (25 mg/day) did not result in a significant improvement in dermal hypersensitivity responses although some effect was seen in 5 cases. It is concluded that zinc supplementation is unnecessary in elderly healthy subjects even if they present zinc metabolism alterations, but may be indicated in the presence of disease.

(9-12). The purpose of this study was to evaluate zinc status and immune function in a group of healthy elderly non-institutionalized subjects on a self-selected diet. We then investigated the influence of oral zinc supplementation on immunological parameters.

Introduction In the elderly, zinc deficiency occasionally becomes clinically evident, but marginal deficiency is believed to be much more common (1). Low zinc intake or reduced gastrointestinal tract absorption have been suggested as factors associated with marginal zinc deficiency in the elderly, but the condition may also be part of the physiological process of aging (2,3). According to the recommended dietary allowances zinc intake should be approximately 10-15 mg a day (4). However, zinc is not equally distributed in all foods and some elderly people may not receive the recommended quantity because they normally eat vegetable rather than animal proteins, which contain more biologically available zinc (5). Besides social and economic factors affecting the quality and quantity of nutrient intake, the increased need for drugs and the reduced gastric acidity associated with age, are other potential causes of reduced zinc absorption (6). The elderly frequently complain of slow wound healing, decreased taste acuity and impaired immunity (I, 7, 8), and some of these manifestations may be related to zinc deficiency - zinc treatment has been found to ameliorate some of these conditions

Methods Subjects and protocol

15 healthy elderly subjects (6 male and 9 female, mean age 82 years, range 74-86), living at home and eating a self-selected diet, entered the study. They were consecutive asymptomatic subjects presenting at our Geriatric Hospital for routine health checks. They had no history of diabetes, cancer, major surgical interventions or chronic inflammatory diseases. Medical history and examination and routine biochemical tests were performed in each subject. None of the subjects was taking NSAIDs, steroids, antibiotics, insulin or iron supplements. Cardiovascular medications, laxatives and sedatives were allowed. 15 out of 60 screened subjects were studied over a period of 6 months. At the beginning of the study nutritional status was evaluated on nomograms for determining body 280

CLINICAL NUTRITION

mass index, triceps and subscapular skinfold thickness (13) and biochemical determination of albumin, prealbumin, retinol binding protein and fibronectin. Obesity was defined as 20% exceeding the ideal body weight (13). Immune function was ascertained on the basis of total white blood cells, circulating T lymphocytes and their subpopulations of helper, suppressor, HLADR and natural killer cells. Delayed dermal hypersensitivity to 7 common antigens was also evaluated. Zinc metabolism was assessed on the basis of serum and red blood cell (RBC) zinc levels and Zinc Tolerance Test (ZnIT) results. Subjects were then asked to take a 3-week supplementation course with zinc sulphate. They were instructed to take 1 tablet of zinc sulphate (55 mg) at lunch and 1 tablet at the evening meal. Blood was collected within 1 week from the end of the supplementation period for zinc determination and re-evaluation of the immunological parameters. In 10 subjects (5 male, 5 female, mean age 24 years, range 19-40) who were taking no medication, zinc metabolism was evaluated for comparison. Sample collection and analysis

Zinc absorption was evaluated by means of ZnTT (14). Patients were fasted for at least 12 h prior to and throughout the test. The areas under the plasma zinc concentration time curves (AUC) after oral administration of 55 mg zinc sulphate, which is equal to 12.5 mg (760 mmol) of elemental zinc were calculated. Each subject, irrespective to his body weight, was administered the same amount of zinc. Zinc was measured in heparinized blood samples immediately before administration of the metal and after 60, 120, 180,240 and 360 minutes. Blood samples, taken using zinc-free plastics, were centrifuged and kept at -20°C until assayed. Plasma and serum zinc were assayed using atomic absorption spectrophotometry (15). RBC zinc was determined following methods described elsewhere (16). Cellular immune function was evaluated measuring total circulating T lymphocytes and their subpopulations and cutaneous hypersensitivity reactions to purified protein derivatives of 7 antigens: Candida, tuberculin, Trycophyton, Streptococcus, diphteria, Proteus mirabilis and tetanus. The control injection was glycerine (Multitest CMI, skin test antigen applicator, Merieux Institute, Lyon, France) (17). All the tests were performed by the same investigator who read the results at 48 f 2 h by measuring mean induration diameters (in millimetres). Data are reported as the number of positive responses and the

281

sum of induration diameters. Induration of > 4 mm was considered a positive skin test. A score of less than 2 mm was considered anergy, between 2 and 4 mm hypoergy and more than 4 mm normoergy. Results are expressed as mean + SD. A statistical analysis of the results was made using Student’s t test for paired and unpaired data. All data were analyzed for statistical significance at the 5% significance level (p < 0.05) . Results Zinc metabolism

The mean serum zinc levels of the elderly were significantly lower than that of young controls (13.42 + 1.84 ymol/dl vs 18.66 + 3.84; p
parameters

The mean weight of the young subjects was 61 + 11 kg, that of the elderly subjects was 67 f 18 kg. 25% of them were obese, none was underweight. The nutritional parameters, triceps and subscapular skin-fold thickness, were within the normal range following reference tables (Table 1) (18). Nutritional proteins such as albumin and libronectin were within the normal range in all the patients (Table 2) while RBP and prealbumin levels were in the low range values in 2 and 3 patients respectively. No apparent reason was found for these abnormalities. All the subjects had normal biochemical routine tests and normal values for total circulating T lymphocytes and their subpopulations. Skin tests were positive for a mean of 2.2 + 0.7 antigens. The score revealed 6 anergic and 3 hypoergic subjects. The mean score of the remaining 7 subjects was 14.5 f 9.0 mm.

282 ZINC METABOLISM AND IMMUNITY IN THE ELDERLY

PLASMA ZINC E4INCR.I

60 -

0

60

120

Fig. 1. Zinc Tolerance Test: Mean plasma concentration *p < 0.01.

skin-fold thickness of elderly women and men (reference values adjusted for age in brackets*) Triceps Subscapula skin fold thickness (mm) 17.2 + 8.2 (19.5 + 9.3) 14.4 f 8.5 (15.9 + 7.2)

21.6 f 9.8 (23.3 f 7.5) 11.9f5.7(11.8f5.5)

Results presented as mean f SD. *From the National Center for Health Statistics, Department Health and Human Services (I 8).

Table 2

Plasma levels of nutritional

240

360

MIN

curve for zinc (o/oof basal values f SE) in elderly (0) and young (0) subjects:

Table 1 Triceps and subscapular

Female Male

180

of

was found between immunological parameters in the blood before and after treatment (Table 3). In one subject who was anergic at the beginning of the study a marked improvement was observed in the delayed cutaneous response. In another, the number of positive reactions increased two-fold (from 2 to 4 antigens) and the intensity of responses four-fold (mean induration diameter for each antigen, 9 mm). One subject had a borderline response to Candidu antigen and developed a 5 mm reaction after zinc supplementation. Two more subjects reacted to a higher number of antigens (from 3 to 5) after zinc

proteins in 15 elderly

subjects

Albumin g/100 ml Pre-albumin mg/llXl ml Retinol binding protein mg/lC!XIml Fibronectin mgllO0 ml

Median

Range

Normal range*

4.1 16 3.8 38

3.1-5.3 8-29 1.1-5.2 3@-61

(3.5-5) (1240) (36) (23-58)

* as in use in our laboratory

Response to zinc supplementation The 3-week course of zinc sulphate supplementation

did result in changes in serum (13.4 f 1.8 l.tmol/dl vs 16.1 * 4.8) but not in RBC zinc levels (1.3 f 0.2 ng/106 vs 1.2 F 0.1). No significant difference

Table 3

White blood cells and lymphocyte values in 15 elderly subjects before and after zinc supplementation (normal values in brackets*)

White blood cells (10s/mm3) Total lymphocytes ( 103/mm3) CD19 (147 f 85) HLA-DR (221 f 110) CD3(1129f404) CD4 (727 rt: 255) CD8 (509 f 227) CD4/CD8(1-2.6%) CD16(257f 123)

Results expressed as mean f SD * as in use in our laboratory

Before ZnSO,

After ZnSO,

6158~2400 169Ok670 159+777 229+ 167 987f475 674f284 330f240 2.67 + 1.3 364+_208

7030+2021 1653+700 122f54 207f94 9915544 666f420 378f284 1.96k 0.7 320f264

CLINICAL NUTRITION

0

i I

‘

BEFORE

AFTER

ZnSO4

Fig. 2. Mean cutaneous induration diameters following Multitest CMI before and after zinc supplementation.

supplementation. One of them had a flat absorption curve despite normal plasma zinc levels before starting zinc supplementation. None of the remaining subjects showed any modifications in skin induration areas (Fig. 2)

Discussion

Although zinc metabolism was altered in our elderly subjects, none presented clinical signs of zinc deficiency. Zinc status is difficult to assess in humans since determination of the metal in the plasma involves measurement of circulating, easily exchangeable zinc which makes-up less than 10% of the blood zinc pool, while the tissue concentration is strictly bound to proteins. Available indexes have poor sensitivity in acute conditions and in the diagnosis of marginal deficiency states (19). Several parameters are therefore used in association. We assessed zinc status by measuring plasma, erythrocyte zinc concentrations and zinc absorption. Low plasma zinc concentrations have been found in several studies on elderly patients, and our results confirm these findings (3, 8, 20). Low plasma zinc levels may be caused by low albumin levels since

283

approximately two-thirds of plasma zinc is bound to albumin. A correlation has been found between albumin and plasma zinc levels in patients with cirrhosis (21) and inflammatory bowel disease (22). In our experimental conditions there was no correlation even though albumin levels were slightly altered in 5 of our patients (levels were, however, always above 3 g/100 ml). We also found unexplained low levels of nutritional proteins in some patients. Active inflammation was excluded on the basis of normal white blood cell counts and a2 globulin levels. Zinc concentration in RBCs cannot be considered a reliable index of zinc status since RBCs have a slow turnover and the zinc contained in this tissue is not easily exchangeable (23). Our subjects were in good health and it is therefore not surprising that RBC zinc levels were within the normal range. We did not measure zinc urinary output because elderly women are frequently incontinent and this compromises the reliability of 24 h urine collection test results. We found a significant reduction in the AUC after the administration of a quasi-physiological dose of zinc (55 mg of zinc sulphate). 4 subjects had a flat curve, although all possible causes of malabsorption were ruled out. Low gastric acidity (24) or low zinc intake (5) might have been a possibility. Since intake estimates are difficult to obtain and often inaccurate (25), we did not attempt to quantify the daily zinc intake of our patients. However, no major nutritional deficiencies were detected in their medical histories, nutritional status and laboratory data. Zinc is essential to the immune function of animals and humans (26-29). Few studies have focused on the influence of zinc on the immune system of elderly people (1, 8, 1 I, 12). Immune responsiveness decreases with increasing age and T lymphocytes appear to be more affected by this than other components with decreased total numbers of circulating T lymphocytes, decreased response of lymphocytes to stimulation and deficient delayed dermal hypersensitivity (30). A diminished IL-2 production has recently been reported in elderly people with mild zinc deficiency (3 1). We evaluated the immunological status of our subjects by counting T cell subpopulations and measuring delayed cutaneous hypersensitivity, which is one of the most sensitive indicators of cell-mediated immunity, and found no alterations in the number of circulating immune cells but a significant proportion (40%) of subjects showed no skin test reactivity to a panel of 7 common antigens. Bodgen et al found a similar percentage of anergic subjects in 100 subjects over 60 years of age (32). Duchateau et al (11) found that zinc supplementation increased the total number of lymphocytes and reversed impaired delayed hyper-

284

ZINC METABOLISM AND IMMUNITY IN THE ELDERLY

sensitivity reactions in 15 institutionalized subjects over 80 years of age. A correlation between serum zinc and dermal responses was found by Orlandoni (33) in 48 elderly subjects. We attempted a supplementation trial with 12.5 mg of elemental zinc twice daily. This dose is higher than the physiological daily recommended intake but much lower than that reported to be toxic with a possible negative influence on chemotaxis, phagocytosis (34) and copper absorption (35). The 3-week supplementation period with zinc was considered a good compromise between compliance and ability to correct marginal deficiency. Plasma zinc levels increased and skin reactivity ameliorated in one-third of the patients. Skin tests are used routinely for testing cellular immune function towards common environmental and viral antigens. One of the clinical applications is in the monitoring of the evolution of AIDS patients. A positive response should be expected given the every day contact with these antigens, while sensitization to the minimal amount of skin applied antigen is unlikely to occur. More sophisticated tests are probably necessary in order to obtain a better selection of patients likely to benefit from zinc supplementation. Parameters other than skin delayed hypersensitivity like production of cytokines or PHA induced proliferation of T cells might be useful. Our findings indicate either: 1) that our subjects may have had a reduced gastrointestinal absorption, but nevertheless maintained sufficient zinc levels; 2) that the 3-week supplementation period have been insufficient to see any changes in the parameters tested; or 3) that any effect of supplementation could not be identified because most of the parameters studied were within or just below the normal range when the study was started. A clear benefit for zinc supplementation was demonstrated only in 1 anergic patient with a flat zinc absorption curve. It is therefore difficult to prove that the parameters studied are interdependent. The different study designs and nonstandardised subject selection may account for the contradictory results reported by different authors. Institutionalised subjects may have different diets and elderly people in their seventies may differ from those in their eighties. The immunostimulatory effect of zinc can be direct rather than indirect (via correction of zinc deficiency). High doses can therefore affect immune parameters, also in people without zinc deficiency. In conclusion, our findings do not confirm that zinc supplementation should be given to healthy people, even if there is evidence of reduced zinc absorption. However, marginal zinc deficiency can cause increased susceptibility to some infections. It may therefore be

advisable to consider supplementation when patients’ needs increase. Acknowledgements The Authors thank Miss Simonetta Milani for her technical and secretarial assistance. The work was supported in part by a grant from the Minister0 Pubblica Istruzione 60% 1991 and in part by a grant from the Consiglio Nazionale delle Ricerche (CNR) (Contract number 89.02708.04).

References 1. Sandstead H H, Hemiksen L K, Greger J L, Prasad A S, Good

2.

3.

7

8.

9. 10.

11.

12. 13.

14. 15.

R A. Zinc nutriture in the elderly in relation to taste acuity. immune response, and wound healing. Am J Clin Nutr 1982; 36: 1046-1059. McClain C J, Stuart M A. Zinc metabolism in the elderly. In: Morely J E, Glick Z, Rubenstein L 2, eds. Geriatric Nutrition. New York: Raven Press Ltd, 1990: 161-169. Swanson C A, Mansourian R, Dirren H, Rapin C H. Zinc status of healthy elderly adults: response to supplementation. Am J Clin Nutr 1988; 48: 343-349. Food and Nutrition Board. Recommended Dietary Allowances (9th). National Academy of Science: Washington, 1980. Greger J L. Dietary intake and nutritional status in regard to zinc of institutionalized aged. J Gerontol 1977; 32: 549-553. Champagne E T. Low gastric hydrochloric acid secretion and mineral bioavailability. Adv Exp Med Biol 1989; 249: 173-l 84. Bales C W, Steinman L C, Freeland-Grabes J H, Stone J M, Young R K. The effect of age on plasma zinc uptake and taste acuity. Am J Clin Nutr 1986; 44: 664669. Bodgen J D, Oleske J M, Munves E M et al. Zinc and immunocompetence in the elderly: baseline data on zinc nutriture and immunity in unsupplemented subjects. Am J Clin Nutr 1987; 46: 101-109. Hallbook T, Lanner E. Serum-zinc and healing of venous leg ulcers. Lancet 1972; 2: 780-782. Stieclemann M, Harrell 1. Relation of immunocompetence to selected nutrients in elderly women. Nutr Rep Int 1980; 21: 931-940. Ducheateau J, Delepesse G, Vrijens R, Collet H. Beneficial effects of oral zinc supplementation on the immune response of old people. Am J Med 1981; 70: 1001-1004. Wagner P A, Jemigan J A, Bailey L B et al. Zinc nutriture and cell-mediated immunity in the aged. Int J Vit Nutr Res 1983; 53: 94-101. Bray G A. In: Schneider H A, Anderson C E, Coursin D B, eds. Nutritional Support and Medical Practice, 2nd ed. Philadelphia: Harper and Row. 1983: 46&490. Sulliv&J F, Jet&n M M, Burch R E. A zinc tolerance test. J Lab Clin Med 1979; 93: 485492. Prasad A S. Determination of zinc in biologic fluids by atomic absorption spectrophotometry in normal and cirrhotic subjects. J Lab Med 1965; 66: 508-516.

16. Milne D B, Ralston N V C, Wallwork J C. Zinc content of ceIIular components of blood: methods for ceI1 separation analysis evaluated. Clin Chem 1985; 31 (1): 65-69. 17 Kniker W T, Anderson C T, Roumiantzeff M. The

and

MLJLTITESTsystem: a standardized approach to evaluation

CLINICALNUTRITION

18.

19. 20.

21.

22.

23.

24.

25.

26.

of delayed hypersensitivity and cell mediated immunity. Ann All 1979; 43: 73-79. Schneider H A, Anderson C E, Coursin D B, eds. Nutritional Support and Medical Practice: Appendix 2nd ed. Philadelphia: Harper and Row, 1983: 649-656. Solomons N W. Gn the assessment of zinc and copper nutriture in man. Am J Clin Nutr 1979; 32: 856-871. Wagner P A, Krista. M L, Bailey L B et al. Zinc status of elderly black Americans from urban low-income households. Am J Clin Nutr 1980; 33: 1771-1777. Stumiolo G C, Martin A, Mastropaolo G et al. Zinc absorption in cirrhosis and chronic inflammatory bowel disease. In: Histidine III. Laboratory and Clinical Aspects. Therapeutic use of Histidine and Zinc. Third International Workshop: Bermuda, 1982: 119-129. McClain C, Soutor C, Zieve L. Zinc deficiency: a complication of Crohn’s disease. Gastroenterology 1980; 78: 272-279. Prasad A S. Clinical spectrum and diagnostic aspects of human zinc deficiency. In: Essential and Toxic Trace Elements in Human Health and Disease. Alan R. Liss Inc.. 1988: 3-53. Stumiolo G C, Montino M C, Rossetto L et al. Inhibition of gastric acid secretion reduces zinc absorption in man. J Am Co11Nutr 1991; 4: 372-375. Abdulla M, Jagerstad M, Norden A et al. Dietary intake of electrolytes and trace elements in the elderly. Nutr Metabol 1977; 21 (1): 41-44. Beisel W R. Single nutrients and immunity. Am J Clin Nutr 1982; 35: 417-468.

285

27. Oleske J M, Westphal M L, Starr S S et al. Zinc therapy of depressed cellular immunity in acrodermatitis enteropathica. Am J Dis Child 1979; 133: 15-18. 28. Golden M H N, Golden B E, Harland P S E G, Jackson A A. Zinc and immuno-competence in protein-energy malnutrition. Lancet 1978; 1: 1226-1227. 29. Arakawa T, Tamura T, Igarashi Y et al. Zinc deficiency in two infants during parenteral alimentation for diarrhea. Am J Clin Nutr 1976; 19: 197-204. 30. Murasko D M, Nelson B J, Silver R et al. Immunologic response in an elderly population with a mean age of 85. Am J Med 1986; 81: 612-618. 3 1. Kaplan J, Hess J W, Prasad A S. Impairment of immune function in the elderly: association with mild zinc deficiency. In: Essential and Toxic Trace Elements in Human Health and Disease. Alan R. Liss Inc., 1988: 309-317. 32. Bodgen J D, Oleske J M, Lavenhar M A L et al. Zinc and immunocompetence in elderly people: effects of zinc supplementation for 3 months. Am J Clin Nutr 1988; 48: 655-663. 33. Orlandoni P, Gaggiotti G, Spazzalirmo L et al. Skin tests in elderly patients. Riv It Nutr Parent 1990; 8: 99-105. 34. Chandra R K. Excessive intake of zinc impairs immune responses, JAMA 1984; 252 (11): 1443-1446. 35. Prasad A S, Brewer G J, Schoomaker E B, Rabbani P. Hypocupremia induced by zinc therapy in adults. JAMA 1978; 240: 21662168. 37. Allen J I, Kay N E, McClain C J. Severe zinc deficiency in humans: association with a reversible T-lymphocyte dysfunction. Ann Int Med 1981; 95: 154-157.

Submission date: 20 October 1993; Accepted ajier revision: 9 June 1994