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Endpoint:
direct observations: clinical cases, poisoning incidents and other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Used in EU risk assessment for zinc metal. Study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Low dietary zinc alters indices of copper function and status in postmenopausal women
Author:
Milne DB, Davis CD & Nielsen FH
Year:
2001
Bibliographic source:
Nutrition. 17(9): 701-708

Materials and methods

Study type:
study with volunteers
Endpoint addressed:
repeated dose toxicity: oral
Principles of method if other than guideline:
A 190 d study was conducted to ascertain the effect of moderately excessive (811 µmol/d; 53 mg/d) and deficient (45.9 µmol/d 3 mg/d) intakes of zinc on copper metabolism and use in humans fed low (15.7 µmol/d; 1 mg/d) and luxuriant amounts (47.2 µmol/d; 3 mg/d) of copper.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Zinc gluconate

Method

Type of population:
other: post-menopausal women
Subjects:
- Number of subjects exposed: 25
- Sex: Female
- Age: 50-76 yr (64.9±6.7 yr)
- Height: 159.6±7.6 cm
- Weight: 65.1±9.5 Kg
- Known diseases: No underlying disease
- Diet: Fed constant weighed basal diet of conventional foods, low in copper (9.6 mmol/8.4 MJ; 0.6 mg/2000 kcal) and zinc (45.9 mmol/8.4 MJ; 3 mg/2000 kcal) on a 3 d menu rotation.
- Other: Zinc was supplemented as zinc gluconate and copper was supplemented as cupric sulfate in beverages served with the meals.
Ethical approval:
confirmed and informed consent free of coercion received
Route of exposure:
oral
Reason of exposure:
intentional
Exposure assessment:
measured
Details on exposure:
After a 10 d equilibration period, in which they were fed a diet providing 31.5 µmol (2 mg) Cu and 91.8 µmol (9 mg) Zn/8.4 MJ (2000 kcal), the women were divided into two groups. the women were divided into two groups. One group was fed a basal diet supplemented with 15.7 µmol (1 mg) Cu/8.4 MJ (2000 kcal), and the other group was fed a diet containing 47.2 µmol (3 mg) Cu/8.4 MJ (2000 kcal). After equilibration, both groups were fed the basal diet providing 45.9 µmol (3 mg) Zn/8.4 MJ (2000 kcal) for 90 d; this was followed by another 10-d equilibration period before dietary zinc was increased to 811 µmol (53 mg)/8.4 MJ (2000 kcal) for 90 d.
Examinations:
- Urine and faeces analysis:
- Time or frequency of sampling: Last 18 d of each 90 d dietary period
- Method of analysis for copper and zinc balance: (a) Urinary copper and zinc were determined by analysis with inductively coupled argon plasma emission spectroscopy of a diluted aliquot. (b) Faeces copper and zinc were determined by analysis with inductively coupled argon plasma emission spectroscopy after wet digestion of aliquots of freeze-dried material with nitric and perchloric acids. (c) Dietary zinc and copper analysis

- Haematology:
- Time or frequency of sampling: Weekly, from antecubital veins
- Fasted: Yes, 12 h before drawing of samples
- Volume of sample: 235 mL/mo
- Elemental analysis: Plasma zinc and copper concentrations were determined by flame atomic absorption spectrometry after dilution with deionised water
- Parameters measured:
(1) Copper status indicators: Plasma copper concentrations, serum ceruloplasmin (CP) determined enzymatically and immunochemically (using human ceruloplasmin (40 U/mg of protein; Sigma, St. Louis, MO, USA) as a standard); Erythrocyte superoxide dismutase (ESOD) activity, Platelet cytochrome-c oxidase activity.
(2) Cholesterol findings and effect on amount of anti-oxidants: Hematologic indices (determined by Coulter S+IV hematology analyzer (Coulter Electronics, Inc., Hialeah, FL, USA)), Serum cholesterol, HDL-cholesterol, and low-density lipoprotein (LDL) cholesterol concentrations (determined using Cobas Fara II Centrifugal analyzer (Roche Diagnostics Systems, Montclair, NJ, USA), Glutathione and gluthathione peroxidase activity and plasma zinc concentrations.
(3) Effects on red blood cells
(4) Iron status: Serum iron, hematocrit, and percentage of transferrin saturation

- Other: Electrical activity of heart: Examined using Holter Electrocardiograms
- Parameters examined: Ventricular premature discharges
- Time or frequency of observation: Twice during initial equilibration, and then during 4, 7, 9, 10, 11, and 12 wks of each 90 d dietary period on all subjects



Medical treatment:
No

Results and discussion

Clinical signs:
No data
Results of examinations:
- Urine and faecal analysis:
(a) Copper balance: Only the women fed high dietary zinc (811 µmol or 53 mg) and luxuriant copper (47.2 µmol or 3 mg) had a positive copper balance (2.83 µmol/d; 0.18 mg/d). Copper balance was not positive in all other groups. Loss of dietary copper faeces was low, while urinary copper was not affected by dietary treatment. (See attached pdf, Table 1, Pg 703)
(b) Zinc balance: It reflected dietary zinc intake and was not significantly affected by copper intake. The percentage of dietary zinc intake excreted in the feces and the daily excretion in the urine were increased when dietary zinc was increased. Zinc balance was near zero but did not become non-positive during low zinc intake. (See attached pdf, Table 2, Pg 704)

- Haematology:
(1) Copper status indicators: Copper-status indicators were variably affected by dietary treatment.

(a) Plasma copper concentrations: Was not significantly affected by zinc intake. It was lower in women fed low-copper diet than in high copper diet fed women, regardless of dietary zinc. This decrease in plasma copper was significant (P < 0.02) when expressed as a percentage change (Table 4) from equilibration concentrations (Table 3) to concentrations during low and during high dietary copper intake. For details see attached pdf, Table 3 and 4, Pg 704, 705.

(b) Serum ceruloplasmin levels and specific activity: Was not significantly affected by dietary zinc treatment. The serum-immunoreactive ceruloplasmin was decreased (P < 0.05) and the specific activity of ceruloplasmin defined as the ratio between enzymatic and immunoreactive ceruloplasmin (ENZ/IMM Cp) was increased during low dietary zinc intake than during high dietary zinc intake. High zinc with luxuriant copper dietary treatment resulted in values closest to those obtained during the initial equilibration for immunoreactive ceruloplasmin and ENZ/IMM Cp. For details see attached pdf, Table 3 and 4, Pg 704, 705.

(c) Platelet cytochrome-c oxidase (U/109) and ESOD: Both were significantly affected by dietary zinc treatment.
ESOD activity was significantly lower (P < 0.02) at the end of the zinc-supplementation period than at the end of the low-zinc dietary period (Table 3).ESOD activity decreased significantly during the low-zinc dietary period in women fed low dietary copper (Table 4). Conversely, ESOD activity increased during the low dietary zinc period in women fed luxuriant copper. It decreased in both groups when dietary zinc was changed from low to high. For details see attached pdf, Table 3 and 4, Pg 704-705.

Platelet cytochrome-c oxidase activity, on a platelet-number basis, increased significantly when supplemental zinc was fed (P < 0.0007). Dietary copper had no significant effect on this measure. When expressed as milligrams of protein, neither zinc nor copper significantly affected cytochrome-c oxidase activity. This apparent discrepancy was the result of a reduction in platelet protein during the low-zinc dietary period followed by an increase in platelet protein when the zinc supplement was fed. For details see attached pdf, Table 3, Pg 704.

(2) Cholesterol findings and effect on amount of anti-oxidants:
(a) Cholesterol levels: Higher total cholesterol levels were observed in women fed with low-copper diet than in high-copper diet (P < 0.05; Table 5). During the low-zinc dietary period, total cholesterol increased over the initial equilibration value; and a greater increase in cholesterol was observed inwomen consuming the low-copper diet. Increasing the zinc intake to 811 mmol (53 mg)/d lowered serum cholesterol concentrations (Table 6). LDL cholesterol changes were similar to the total-cholesterol changes. HDL-cholesterol (Tables 5 and 6), very LDL-cholesterol, and triacylglycerol were not significantly affected by the dietary manipulations. For details see attached pdf, Table 5 and 6, Pg 706.

(b) Whole-blood glutathione and erythrocyte glutathione peroxidase activity: Were significantly affected by the dietary zinc but not by dietary copper.- Glutathione concentrations were increased from equilibration during the low-zinc dietary period and returned to essentially equilibration concentrations during the high-zinc dietary period (Table 5). For details see attached pdf, Table 5 and 6, Pg 706.

- Erythrocyte glutathione peroxidase activity also was increased by low dietary zinc and decreased by high dietary zinc. However, the decrease did not result in a return to initial equilibration activity; women fed high-copper diet and high dietary zinc were the closest, which returned to initial equilibration activity. For details see attached pdf, Table 5 and 6, Pg 706.

(c) Plasma zinc concentrations: Low dietary zinc did not decrease plasma zinc concentrations below those present during initial equilibration, however, high dietary zinc significantly increased the plasma zinc concentrations. For details see attached pdf, Table 5 and 6, Pg 706.

(3) Effects on red blood cells: The concentration of zinc in red blood cells was not affected by dietary zinc, nor was the concentration of copper affected by dietary copper (Table 7). However, high dietary zinc significantly decreased the concentration of copper in red blood cells, expressed as per gram of hemoglobin or per liter of packed cells. For details see attached pdf, Table 7, Pg 706.

(4) Iron status: Measured indicators of iron status were unaffected by the dietary treatments. Hemoglobin was lower at the end of the high-zinc dietary period than the low-zinc dietary period in women with low and high dietary intakes of copper (P < 0.001).

- Other: Electrical activity of heart: 3 women fed the diet marginal in copper (15.7 µmol/d; 1 mg/d) and low dietary zinc (45.9 µmol (3 mg)) exhibited an increase in ventricular premature discharges, resulting in obligatory supplementation with copper before the end of the study. After the switch to high dietary zinc, the return to control numbers occurred. None of the women receiving 47.2 µmol (3 mg) of Cu/d had significant changes in their electrocardiograms.
Effectivity of medical treatment:
Not applicable
Outcome of incidence:
Not applicable

Any other information on results incl. tables

Remarks on thestudy, reported by Davis et al. (2000) and Milne et al. (2001):

From personal communication with the authors it appears that for ESOD activity the initial equilibration values varied markedly between individuals, and that for women who were assigned to the low copper group ESOD activity was substantially higher than for those assigned to the high copper group. This implicates that for this indicator, the assignment of the subjects to the two groups was suboptimal, which might also be the case for other indicators.

The frequent blood sampling (an average of no more than 235 ml per month was drawn) might have compromised the physiology of the subjects (as was suggested for haemoglobin).

The subjects served as their own controls: values upon both treatments (i.e. low and high zinc administration) were compared with values upon first equilibration. However, as the second treatment is not independent of the first treatment, the study design is not optimal.

 

Applicant's summary and conclusion

Conclusions:
The inadequate intake of zinc (45.9 µmol/d; 3 mg/d) was more effective than a moderately high intake of zinc (811 µmol/d; 53 mg/d) in inducing changes associated with a decreased copper status in postmenopausal women.
Executive summary:

A 190 d study was conducted to ascertain the effect of moderately excessive and deficient intakes of zinc on copper metabolism and use in humans fed low and luxuriant amounts of copper.

Twent five postmenopausal women housed in a metabolic unit completed the study as designed. After a 10-d equilibration period in which they were fed a diet providing 31.5 µmol (2 mg) Cu and 91.8 µmol (9 mg) Zn/8.4 MJ (2000 kcal), the women were divided into two groups. One group was fed a diet containing 15.7 µmol (1 mg) Cu/8.4 MJ (2000 kcal), and the other group was fed a diet containing 47.2 µmol (3 mg) Cu/8.4 MJ (2000 kcal). After equilibration, both groups were fed the basal diet providing 45.9 µmol (3 mg) Zn/8.4 MJ (2000 kcal) for 90 d; this was followed by another 10-d equilibration period before dietary zinc was increased to 811 µmol (53 mg)/8.4 MJ (2000 kcal) for 90 d.

The women were in positive copper balance only when the diet provided 47.2 µmol (3 mg) Cu and 811 µmol (53 mg) Zn/d. Immunoreactive ceruloplasmin concentrations and platelet cytochrome-c oxidase activity on a platelet number basis were significantly lower and the ratio between enzymatic and immunoreactive ceruloplasmin was significantly higher during low dietary than during high dietary zinc intake. Serum cholesterol was higher in women fed 15.7 µmol (1 mg) Cu/d than in those fed 47.2 µmol (3 mg) Cu/d. Total and low-density lipoprotein cholesterol concentrations decreased with zinc supplementation. Whole-blood glutathione concentration and erythrocyte glutathione peroxidase activity were lower during high than during low dietary zinc intake.

Hence, the inadequate intake of zinc (45.9 µmol/d; 3 mg/d) was more effective than a moderately high intake of zinc (811 µmol/d; 53 mg/d) in inducing changes associated with a decreased copper status in postmenopausal women.