Apr 1, 1984 - dismutase copper status, deficiency, ferroxidase, ceruloplasmin,. The American. Journal of Clinical. Nutrition 40: OCTOBER. 1984, pp 743-746.
Original
Research
Communications-rapid
Effect of zinc supplementation in adult man1-4
on copper status
Peter
R L ‘Abb#{233}, MSc
WF Fischer,
PhD,
Alexandre
Giroux,
and
Mary
KEY WORDS Zinc, superoxide dismutase
supplements,
Introduction The
antagonistic effect of high levels of zinc on copper absorption and status has been demonstrated in a variety of species, including man (1-5). Many of these studies have used large amounts of zinc at levels that could be considered to be pharmacological doses rather than realistic dietary quantities. Such levels have also been shown to lower serum ferroxidase (ceruloplasmin) (EC 1.16.3.1) activity in rats (6, 7), chicks (8), sheep (2), and man (4), as well as liver and heart Cu,Zn-superoxide dismutase (EC 1.15.1. l)and cytochrome C oxidase(EC 1.9.3.1) in rats (6). We have recently demonstrated that reductions in the copper metalloenzyme activities of rats fed either copper-deficient or high-zinc diets are greater and precede changes in plasma and tissue, levelsb of the element (6, 7). Thus, a measure of the changes in these enzyme activities is a better indicator of an alteration in copper status than is a measure of changes in tissue or plasma levels. The activity of erythrocyte dietary
The American Journal of Clinical Nutrition 40: OCTOBER © 1984 American Society for Clinical Nutrition
copper
status,
deficiency,
ferroxidase,
ceruloplasmin,
Cu,Zn-superoxide dismutase is dependent on copper but not zinc status, and thus is a viable index of tissue copper status (8). Zinc supplements, in doses of up to 50 mg or more per tablet, are readily available in pharmacies and health food stores. The effect of ingesting these moderately high amounts of zinc on the copper status of humans has not been investigated. The objective of the present study was to determine whether the ingestion of 50 mg of zinc per day by a group of adult males over a 6-wk period had any effect on their copper status. ‘From the Nutrition Research Division, Bureau of Nutritional Sciences, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario, K1A OL2, Canada. 2Publication 182 of the Bureau of Nutritional Sciences. Presented in part at the 68th Annual Meeting of the Federation of American Societies for Experimental Biology, St Louis, MO, April 1-6, 1984. 4Address reprint requests to: Dr Peter Fischer, Nutrition Research Division, Health Protection Branch, Tunney’s Pasture, Ottawa, Ontario, K1A 0L2, Canada. Received February 29, 1984. Accepted for publication May 15, 1984. 1984,
pp 743-746.
Printed
in USA
743
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ABSTRACT The effects of zinc supplementation on the copper status of healthy adult men, as assessed by the activities of the copper-metalloenzymes, plasma ferroxidase (ceruloplasmin), and erythrocyte Cu,Zn-superoxide dismutase, were determined. The subjects were given either two daily doses of 25 mg zinc or placebo for 6 wk. No significant differences in the plasma copper levels or the ferroxidase activities between the supplemented and control groups could be detected at 2, 4, or 6 wk. Plasma zinc increased and erythrocyte Cu,Zn-superoxide dismutase decreased in the supplemented group, the difference between the groups becoming significant at 6 wk (p < 0.05). This suggested that the zinc supplements decreased the copper status of the experimental group. Am iC/in Nuir 1984;40:743-746.
744
FISCHER
The activities of plasma ferroxidase and erythrocyte Cu,Zn-superoxide dismutase, as well as plasma copper concentration were used as indices of copper status. Methods Subjects
Analysis The erythrocytes were separated from the plasma by centrifugation at 1000 x g for 15 mm at 4C. The plasma was removed and frozen until analyzed for zinc, copper, and ferroxidase activity. The erythrocytes were washed three times with equal volumes of 0.9% NaCI and recentrifuged. After the final wash, 1.0 ml of packed cells were lysed with 3.0 ml of water and placed in an ice bath for 15 mm. Chloroform (0.6 ml) and ethanol (1.0) were added and the mixture was vortexed. This was centrifuged for 20 mm at 5000 x g at 4C. The supernatant was diluted 50 times with assay buffer and used immediately for Cu,Zn-superoxide dismutase determinations. The activity was measured spectrophotometrically by its ability to inhibit the superoxidemediated reduction of ferricytochrome C by xanthine oxidase plus xanthine (9). The concentration of superoxide dismutase was determined from a standard curve prepared from purified bovine blood superoxide dismutase (Sigma Chemical Co. St Louis, MO). One unit of activity is defined as that amount of enzyme which caused a 50% inhibition of the reaction (10). Plasma ferroxidase activity was determined using o-
AL
dianisidine dihydrochloride as substrate according to the method of Schosinsky et al(ll). One unit of activity is defined as that which catalyzes the oxidation of I amol of o-dianisidine per mm at pH 5.0 and 30C. Plasma zinc and copper concentrations were determined by flame atomic absorption spectroscopy following 1:10 dilution with 6% butanol in 0.05 N HO (12). Statistics All results are reported as the mean ± SEM and significant differences between means were determined by “Student’s” t test (13).
Results There were no significant differences in either the plasma copper levels or the ferroxidase activity between the two groups at 2, 4, or 6 wk. Plasma zinc (Fig 1), however, was significantly elevated in the supplement group by 2 wk (p < 0.01), and remained so at 4 (p < 0.05) and 6 wk (p < 005) when compared to the control group. Erythrocyte superoxide dismutase activity (Fig 2) decreased after 4 wk in the supplement group and was significantly lower (p
