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The Effect of Zinc Depletion on the Concentration of Circulating Hormones and Transport Proteins
B. SUTHERLAND, T N.M. LOWE , * B.J. BURRL AND J.C. KJNG Department of Nutritional Sciences, U.C. Berkeley, Berkeley, CA 94720, and the 'USDA Western Human Nutrition Research Center, San Francisco, CA 94129, USA Keywords zinc deficiency, hormones, protein, men
The pathologies that develop with prolonged zinc (Zn) deficiency may be explained in part by changes in protein metabolism, for instance growth retardation, dermatitis and abnormal immune function. Several hormones perform key roles in the regulation of protein metabolism: insulin, cortisol, testosterone and the thyroid hormones. Reduced plasma levels of testosterone, thyroid hormones and plasma transport proteins, as well as altered cortisol and insulin response to stimuli, have been reported to occur with Zn deficiency (Wada and King 1986, Baer et al. 1985, Guigliano and Millward 1987). The purpose of this study was to determine if acute Zn depletion alters plasma concentrations of the thyroid hormones, insulin, cortisol, testosterone, and the transport proteins (albumin, transthyretin, transferrin and retinol bindin g protein).

Method
Eight men participated in the 85 d Zn depletion/repletion study which was divided into three metabolic periods: a 16 d baseline (MP1), a 40 d depletion (MP2) and a 29 d repletion (MP3). Zinc intake was 12 mg/d during baseline and repletion. and 1 mg/d during depletion. The subjects were fed a synthetic formula; energy and protein intakes were held constant after adjustments during the baseline period. The subjects were confined to a metabolic unit. Their daily exercise consisted of two 3-mile, moderately-paced walks. Fasting plasma Zn was measured weekly to monitor the change in Zn status. The plasma proteins measured were albumin, transthyretin, transferrin and retinol binding protein (RBP); the hormones were insulin, cortisol, testosterone and the thyroid hormones. Fasting plasma proteins and hormones were measured on day 6 of MP1, days 6 and 27 of MP2 for two subjects who depleted rapidly, days 6 and 35 of MP2 for the other six subjects, and on day 21 of MP3 for all subjects. Plasma Zn was measured by atomic absorption spectrophotometry (AAS). Plasma albumin, transthyretin and transferrin were measured on an automated centrifugal analyzer; albumin by a bromocresol green binding assay, transthyretin and transferrin by immunoprecipitation. RBP was measured by high pressure liquid chromatography (Burn and Kutnink 1989). All hormones were measured with commercially available radioimmunoassay kits. Statistical analysis of data was performed using a one factor repeated measures ANOVA and Tukey's follow up test.

Results
The ages of the 8 subjects ranged from 20-35 years; they had a height of 1.78 0.06 m (mean S.D.), weight of 77 10 kg and BMII of 24.3 2.5 kg/M 2 . They were all healthy with no recent change in weight. Their usual diet contained meat at least twice a week and provided more than 8 mg Zn/d. They did not take mineral supplements for at least six weeks prior to the study and they all reported no use of cigarettes or drugs. By the end of depletion mean plasma Zn concentration was 5 l.tmolfL; with Zn repletion the level returned to baseline value (12 l.tmol/L). RBP concentration was significantly lower (p < 0.05) by the end Correct citation: Sutherland, B., Lowe, N.M., Burn, B.J., and King, J.C. 1997. The effect of zinc depletion on the concentration of circulating hormones and transport proteins. In Trace Elements in Man and Animals - 9. Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals. Edited by P.W.F. Fischer, M.R. L'Abbe. K.A. Cockell, and R.S. Gibson. NRC Research Press, Ottawa, Canada. pp. 609-611.

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Table 1. Response of plasma hormones and proteins to Zn depletion/repletion.' Metabolic Period Proteins and hormones Baseline Early Depletion Late Depletion Albumin (g/L) 421 411 441 Transthyretin (gIL) 0.210.13 0.190.24 0.210.19 2.10.09ab 2.00.07a 2.10.08" Transferrin (g/L) Retinol BindingProtein (gIL) 0.060.01" 0.050.002ab 0.050.002a Total T4 (nmol/L) 814b 804" 733a Free T4 (nmol/L) 141a 151" 14la Total T3 (nmol/L) 2.20.2 2.00.1 2.10.2 Free T3 (pmol/L) 4.10.3 4.30.3 4.60.3 rT3 (nmollL) 0.370.05" 0.290.03a 0.290.05a TSH (mUlL) 1.30.2 1.30.2 1.50.3 Insulin (pmollL) 6611 677 779 C-peptide (sg/L) 1.70.2 1.90.2 2.10.2 Cortisol (nmol/L) 40844" 30938a 34250a Total Testosterone (ngldL) 571.848.6 568.850.3 594.255.5 Free Testosterone (pglmL) ,25.41.7b 20.41.Oa 20.71.3a
'Values are mean SEM. Values in a row not sharing similar superscripts are significantly different at p < 0.05.

Repletion -411 0.230.13 2.20.08b 0.050.002a 794" 141' 2.10.1 4.80.2 0.29+-0.02a 1.50.2 656 2.00.2 31536' 577.158.3 21.01.2

of depletion and remained lower at day 21 of repletion (Table 1). An additional RBP measurement was done on repletion day 30 at which point the level had returned to baseline. Plasma albumin and transthyretin concentrations were unchanged with Zn depletion, transferrin increased significantly (p < 0.05) from early to late depletion but did not decline in repletion. The following significant changes (p <0.05) in plasma hormone concentrations were observed (Table 1): total and free thyroxine (T4) and reverse triiodothyronine (rT3) decreased by day 6 of depletion; total T4 returned to baseline by the end of depletion while free T4 and rT3 remained low. Free testosterone and cortisol levels also declined by day 6 of depletion and remained low throughout the rest of the study. Toward the end of depletion the following clinical manifestations of Zn deficiency were observed: skin rashes on the perioral and groin regions, lethargy and moodiness. These signs were reversed within a few days of Zn repletion.

Conclusions
Acute Zn depletion was associated iith a decrease in plasma levels of cortisol, total and free T4, revers T3 and free testosterone. This decrease was seen within 6 d of depletion suggesting an early adaptive response to the acute decline in Zn intake. Several of the changes observed suggest that acute Zn depletion altered protein synthesis: the rapid decline in total and free T4, reverse T3 and free testosterone, and the fall in plasma RBP by the end of depletion. The decrease in thyroid hormones was not associated with a change in resting metabolic rate.

Literature Cited
Baer, MT., King, iC., Tamura, T., Margeri S., Bradfield, R.B., Weston, W.L., & Daugherty, N.A. (1985) Nitrogen utilization, enzyme activity, glucose intolerance and leukocyte chemotaxis in human experimental zinc depletion. Amer. J. Clin. Nutr. 41:1200-1235. Bum, B.J. & Kutnink, M.A. (1989) Liquid-chromatography assay for free and transthyretin-bound retinol binding protein in serum from normal humans. Clin. Chem. 35:582-586. Guigliano, R. & Millward, D.J. (1987) The effects of severe zinc deficiency on protein turnover in muscle and thymus. Br. J. Nutt. 57:139-155. Wada, L. & King, J.C. (1986) Effects of low zinc intakes on basal metabolic rate, thyroid hormones and protein utilization in adult men. J. Nutr. 116:1045-1053.

Trace Elements in Man and Animals - 9 Discussion

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Qi. Steve Cunnane, University of Toronto, ON, Canada: Are the data consistent with Janet's report of the 5 mg study, where RQs declined? A. We didn't see any changes in basal metabolic rate in this study. However, the 5 mg study didn't show the same changes in T3 levels that we saw. No changes were noted in that study, but they didn't separate total versus free T3. The differences between these two studies may represent differences in response to acute depletion and chronic. Q2. Hans-Peter Roth, Technical University of Munich, Germany: How do you explain the observation that insulin levels decreased, then increased late in zinc deficiency? A. This was seen in our previous study also, though it was actually reflected in that study as impaired glucose tolerance, as we didn't measure insulin levels directly in that study. Q3. Les Kievay, USDA-ARS, Grand Forks, ND, USA: Your data were expressed in terms of percent change? Were any of your measurements outside of the clinically normal ranges for these parameters? A. No, all of our results were within normal ranges for humans, at all times. Q4. Klaus Eder, Technical University of Munich, Germany: What were the sources of fats in the diet? New studies have indicated that dietary fats can influence serum thyroid hormones. A. We used safflower oil, at 30% of the diet. I'm not familiar with that literature, but we'll certainly look into it. Thank you.