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1 From the Centre de Recherche en Nutrition Humaine d'Auvergne, Unité Maladies Métaboliques et Micronutriments, INRA, St Genès Champanelle, France (CF-C, NM, MR, JCT, AM, and CC); the Laboratoire de Nutrition Humaine, Clermont-Ferrand, France (MB-B); the Laboratoire de Biologie du Stress Oxydant, UFR de Pharmacie, La Tronche, France (MA); and the Department of Food and Nutritional Sciences, University College, Cork, Ireland (KDC)
2 The Zenith Study is supported by the European Commission "Quality of Life and Management of Living Resources" Fifth Framework Program, contract no. QLK1-CT-2001-00168. 3 Reprints not available. Address correspondence to C Feillet-Coudray, Unité Maladies Métaboliques et Micronutriments, INRA, 63122 Saint Genès Champanelle, France. E-mail: feillet{at}clermont.inra.fr.
ABSTRACT
Background: Zinc supplementation may be beneficial for health. Assessing exchangeable zinc pools may be a useful approach to evaluate zinc status.
Objective: We evaluated the effects of long-term supplementation with 2 moderate doses of zinc on the mass of exchangeable zinc pools.
Design: Three groups of healthy, late-middle-aged men (n = 16 per group) participated in a stable-isotope zinc kinetic study after 6 mo of daily supplementation with 0 (placebo), 15, or 30 mg Zn. At the end of the supplementation period, each subject received an intravenous injection of 0.89 mg 70Zn, and the plasma zinc disappearance curve was monitored for the next 10 d. Two approaches were used to determine the characteristics of the exchangeable zinc pools: 1) formal 3-compartmental modeling and 2) a simplified determination of the total mass of the rapidly exchangeable zinc pool (EZP).
Results: In the placebo group, the exchangeable zinc pool masses for the 3 considered pools were as follows: 2.15, 12.7, and 100.5 mg Zn. The rapidly exchangeable zinc pool mass in the placebo group was 143 mg Zn. Zinc supplementation significantly increased the exchangeable zinc pool masses regardless of the approach used to determine these pools. In addition, these data confirm that exchangeable zinc pool masses correlate positively with total zinc intake and negatively with subject age and do not correlate with plasma zinc concentrations.
Conclusion: Our data show that long-term supplementation with 2 moderate doses of zinc is an efficient way to increase exchangeable zinc pool masses in late-middle-aged men.
Key Words: Exchangeable zinc pools • kinetic modeling • stable isotope • zinc supplementation • late-middle-aged subjects • zinc status
INTRODUCTION
Zinc plays an important role in nutrition and health (1). It is involved in many enzymatic reactions, and several hormones are also known to be zinc-dependent (2). Thus, zinc plays an essential role in a wide range of fundamental cellular reactions, and zinc deficiency may be implicated in various metabolic disorders. In developed countries, marginal zinc intake may induce a high prevalence of marginal zinc deficiency, which in turn may contribute to various chronic and degenerative diseases associated with aging (1). Inadequate zinc intakes have been shown in elderly adults (3). Zinc supplementation may thus be beneficial for health, notably in late-middle-aged populations. This age bracket represents an important biological age for understanding the mechanisms of pre-aging before the appearance of aging-related diseases.
Sensitive and specific markers of zinc status are needed to achieve the most reliable estimates of zinc requirements. The approach most often used for assessing zinc status is the measurement of plasma zinc concentrations. However, many factors that are not directly related to zinc nutriture affect this measurement (4). Other static measures, such as urinary zinc excretion or zinc content in blood cells, can be performed, but they are not always reliable indicators of zinc status (4). Functional indicators may also be measured, such as plasma alkaline phosphatase activity or other zinc metalloenzyme activities in tissues; however, they have some limitations (4). The study of exchangeable zinc pools with the use of a stable isotope has been developed as a useful approach for evaluating zinc status (5). However, although numerous studies have explored the effect of zinc depletion alone or the effect of zinc depletion and repletion in humans (6-11), few data, if any, are available in the literature on the effect of moderate doses of zinc supplementation on these zinc kinetic parameters.
Thus, the aim of this study was to explore the effect of long-term supplementation with 2 moderate doses of zinc on exchangeable zinc pool characteristics in late-middle-aged subjects. We compared the effects of a placebo with those of two doses of supplemental zinc (15 and 30 mg/d), which correspond to 1.5 and 3 times the recommended dietary allowances of zinc for the present study population.
SUBJECTS AND METHODS
Subjects
Forty-eight healthy men aged 58–68 y participated in this study. The clinical examination was carried out by the medical staff at the Unit of Nutritional Exploration of the Human Nutrition Research Center in Clermont-Ferrand, France. All subjects were nonsmokers and judged to be healthy on the basis of a routine blood screening, a medical history, a physical examination, and a psychological profile. At the time of recruitment and throughout the duration of the study, none of the selected subjects took vitamin or mineral supplements or medications that may have affected mineral metabolism. Each subject's body weight (in kg) and standing height (in cm) were recorded while they were wearing light indoor clothing and no shoes. Body mass index (in kg/m2) was calculated to screen for obesity. Skinfold thickness was also assessed in seated and relaxed subjects with the use of a Harpenden caliper, according to the procedure described by Durnin and Womersley (12). Skinfold thicknesses were measured at the biceps, triceps, subscapular, and suprailiac regions. The mean of 3 measurements per site was used in subsequent calculations. Fat-free mass was estimated from skinfold thickness with the use of Durnin and Womersley equations (12). Dietary zinc intake was estimated with use of 4-d food-intake records (which included weekend days) and the GENI program (MICRO 6; Villiers les Nancy, France). The subjects were asked to maintain their habitual diet and exercise patterns for the duration of the study. The study was approved by the local Human Ethical Committee in Clermont-Ferrand under number AU 478 (CCPPRB Auvergne). All subjects were fully informed of the purposes of the study and gave their written informed consent.
Experimental design
The study was a randomized, double-blind, placebo-controlled intervention trial conducted in late-middle-aged men. Each group contained 16 subjects, who were randomly assigned to receive placebo, 15 mg Zn/d, or 30 mg Zn/d for 6 mo. The supplemental zinc was given as zinc gluconate, which was prepared and supplied by E-Pharma (Creapharm, Gannat, France). The placebo capsule contained 199 mg lactose and 1 mg magnesium stearate. The 7.5-mg Zn capsule contained 56.9 mg Zn gluconate, 142.1 mg lactose, and 1 mg magnesium stearate. The 15-mg Zn capsule contained 113.7 mg Zn gluconate, 85.3 mg lactose, and 1 mg magnesium stearate. Zinc capsules were distributed to the subjects at the beginning of the trial and at 3 mo. At 3 and 6 mo, the subjects were asked to return any remaining capsules, and the degree of apparent compliance was estimated from the number of delivered capsules and the number of returned capsules. Compliance, expressed as a proportion of the intended supplements consumed during zinc supplementation, did not differ among the groups; the mean compliance was >98% in all groups. Zinc kinetic studies, with use of the 70Zn stable isotope, were performed in all subjects after 6 mo of supplementation (see below).
Preparation of stable-isotope solution
Zinc has 5 naturally occurring stable isotopes. The least naturally abundant stable isotope, 70Zn (natural abundance 0.62%), was used in this study. This isotope was purchased in the oxide form, and it had an enrichment of 95.4% (Chemgas, Paris, France). Bottles of the 70Zn stable-isotope solution were prepared by the hospital pharmacist (Clermont-Ferrand University Hospital Center, France). Briefly, 153.7 mg ZnO (125 mg Zn corresponding to 119.3 mg 70Zn) were first moistened with 2 mL milliQ water (MilliPore, Saint-Quentin en Yvelines, France), and then 1 mL concentrated HCl (12 mol/L) was added gradually with stirring to completely transform the oxide form into the chloride form. The solution volume was then adjusted to 200 mL with water containing 0.9% NaCl. This solution was gradually neutralized with 6 mL of 1 mol NaOH/L, to reach a pH between 4 and 7. The volume of this solution was then adjusted to 2000 mL with water containing 0.9% NaCl. The pH and osmolarity (270–320 mOsmol) were then checked. The solution was finally filtered on a 0.22-µm filter and divided into 130 vials (each containing 15-mL aliquots) that were then autoclaved for 20 min at 121 °C. The pharmaceutical quality of the stable-isotope solution (nontoxicity, sterility, and pyrogenicity) was certified by the Clermont-Ferrand Hospital pharmacy. The zinc concentration and the 70Zn isotopic enrichment of the prepared solutions were determined by inductively coupled plasma mass spectrometry (ICP-MS DRC6100; Perkin-Elmer, Paris, France) in our laboratory before use.
Tracer administration at the end of zinc supplementation
Six months after zinc supplementation, the subjects were admitted to the Human Nutrition Unit of the Clermont-Ferrand Hospital after an overnight fast. An intravenous catheter was inserted into the left arm of each subject, and 1 mg (0.89 ± 0.02 mg) 70Zn in 15 mL of saline solution was perfused over 2 min. (The end of perfusion was considered to be 0 h.) Another intravenous catheter was also inserted into the left arm of each subject for blood sampling at –0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 10 h. A blood sample, which was taken after the subjects had fasted for 12 h, was also obtained on days 1, 2, 3, 5, 7, and 10. A urine sample was collected from fasting subjects in the morning of the day before the isotope was administrated.
Analysis
Blood samples (2 mL) were collected in zinc-free heparin-coated tubes, and a blood aliquot was used for the measurement of hematocrit. The blood sample was centrifuged (1000 x g, 15 min, 4 °C), and the plasma was separated. Erythrocytes that were collected 15 min before the start of the isotope study were washed with saline solution and hemolyzed. Urine samples were acidified with pure hydrochloric acid (final acid concentration: 1%). The samples were then stored at –20 °C until analyzed.
The 70Zn content in plasma samples was determined by ICP-MS (Perkin-Elmer). Before analysis, the plasma was diluted in 0.014 mol HNO3/L, so that total zinc concentrations in the analyzed samples were 100 µg/L. Natural zinc and indium (SPEX Claritas; SpexCertiPrep Inc, Metuchen, NJ) were used as external and internal standards, respectively. All isotope analyses were carried out at least twice. For the 70Zn/66Zn measurement, the within-day and between-day relative variations were 0.62% and 1.11%, respectively. The limit of detection for 70Zn/66Zn enrichment was 1.5%.
Total zinc concentrations were measured in plasma and urine samples (diluted in 1% HCl) with a flame atomic absorption spectrophotometer (AA800; Perkin-Elmer) at 214 nm. Urinary creatinine concentrations were measured by colorimetry. The standard method used at the hematologic laboratory of Clermont-Ferrand Hospital was used to measure hematocrit in blood samples.
Determination of exchangeable zinc pools
First approach: kinetic analysis
Zinc kinetics were determined with the use of a multicompartmental model as described by Pinna et al (7), which, in turn, is based on a model developed by Wastney et al (13). A schema of the proposed model is shown in Figure 1. Compartmental modeling of the data was performed with the aid of the SAAM II (stimulation, analysis, and modeling) software package (SAAM Institute Inc, Seattle, WA; 14). Plasma stable-isotope data were expressed as tracer/tracee, with use of the following formula as described by Lowe et al (15):
RESULTS
Subject characteristics and zinc nutriture at baseline
Age, body mass index, fat-free mass (FFM), and mean zinc intakes and plasma zinc concentrations were not significantly different between groups at baseline (Table 1). In the kinetic study, all subjects tolerated the intravenous 70Zn infusion well.
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TABLE 1. Characteristics of the subjects and zinc status at baseline and after 6 mo of zinc supplementation1
Effect of zinc supplementation on blood zinc concentrations and urinary zinc excretion
Plasma zinc concentrations increased significantly over the 6 mo of supplementation in the groups that received 15 and 30 mg Zn/d; no significant changes were observed in the placebo group (Table 1). Furthermore, plasma zinc concentrations increased significantly with zinc supplementation when the 3 groups of subjects were compared (Table 1). Baseline plasma zinc concentrations correlated significantly with total dietary zinc intake (diet + supplement: r = 0.381, P < 0.01; n = 48). Nevertheless, erythrocyte zinc concentration and urinary zinc excretion were not significantly different between groups (Table 1).
Effect of zinc supplementation on exchangeable zinc pools according to compartmental modeling
Data for a representative subject after intravenous 70Zn infusion are shown in Figure 3. The curve reflects the response generated by the model shown in Figure 1. The pattern of the curve showed a rapid disappearance of the tracer during the first 8 h, which was followed by a slow decline that extended through >240 h. The kinetic patterns were significantly different between the zinc-supplemented and -nonsupplemented groups (data not shown). The kinetic indexes, the masses of the exchangeable zinc pools, and the half-lives of these pools are listed in Table 2. Pool 1 is composed of plasma zinc, pool 2 is a fast pool composed of zinc found mainly in erythrocytes but also in parts of the liver, and pool 3 is a slow pool composed of zinc found in the liver and in bone (13).
FIGURE 3.. Plasma data for one late-middle-aged subject after 6 mo of zinc supplementation. Compartmental modeling of the data was performed with the use of the SAAM II (stimulation, analysis, and modeling) software package (SAAM Institute Inc, Seattle, WA; 14).
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TABLE 2. Kinetic indexes, exchangeable zinc pool mass (M), and half-lives (t1/2) of the exchangeable zinc pool masses, calculated by using a 3-compartmental model, in the placebo and zinc-supplemented groups1
M1 was significantly increased with zinc supplementation (Table 2). M2, M3, and the sum of the 3 pools (tM) tended to increase with zinc supplementation (P < 0.1 for tM), but this was not statistically significant because of the large intravariability in the group who received the highest zinc dose (Table 2). Moreover, M1, M2, and tM correlated significantly and M3 only tended to be correlated (P = 0.078) with total dietary zinc intake (diet + supplement; Table 3). When these pool masses were adjusted for FFM, M1/FFM and tM/FFM increased significantly with zinc supplementation (P < 0.01 and P < 0.05, respectively), whereas M2/FFM and M3/FFM only tended to increase (P < 0.1; Table 2). Surprisingly, M1/FFM, M2/FFM, and M3/FFM were not correlated with plasma zinc concentrations and only EZP/FFM showed a trend toward a correlation (r = 0.241, P = 0.0993; n = 48; Table 3).
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TABLE 3. Linear regression coefficients between exchangeable zinc pool masses (M) and zinc intake and plasma zinc concentrations1
Fractional transport rates of zinc between pools, irreversible loss from pool 1, flux(0,1) and flux(3,1), and plasma turnover were not significantly different between the 3 subject groups. Plasma zinc flux and flux(2,1) significantly increased with zinc supplementation (Table 2) and were positively correlated with zinc intake (Table 3). The irreversible loss of zinc from pool 1 [k(0,1)] was negatively correlated with zinc intake (P < 0.05; Table 3). In addition, the half-lives of the pools were not significantly different between the 3 groups (Table 2). Finally, plasma zinc flux correlated with plasma zinc concentrations (r = 0.474, P < 0.001; n = 47; Table 3).
Effect of zinc supplementation on the exchangeable zinc pools according to the Miller model
The EZP and the EZP adjusted for FFM increased significantly with zinc supplementation (Table 4). Moreover, the EZP correlated significantly with total dietary zinc intake (diet + supplement; Table 3) and tended to correlate with plasma zinc concentrations (r = 0.236, P = 0.1067; n = 48). In addition, the EZP correlated strongly with M3 (r = 0.591, P < 0.001), tM (r = 0.751, P < 0.001; n = 48), and almost with M2 (r = 0.282, P = 0.0547; n = 48). The half-life and turnover time of the EZP were not significantly different between the 3 groups of subjects. Nevertheless, the EZP turnover tended to correlate with zinc intake (r = 0.259, P = 0.076; n = 48). In addition, the flow-out rate was not significantly different. However, the EZP correlated significantly with the half-life of 70Zn (r = 0.660, P < 0.001; n = 48) and with turnover time (r = 0.658, P < 0.001; n = 48).
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TABLE 4. Effect of 6 mo of zinc supplementation on exchangeable zinc pools (EZPs) in men in the placebo and zinc-supplemented groups according to the Miller model1
Effect of age on the exchangeable zinc pool masses
Both tM and EZP correlated negatively with age in the nonsupplemented group (for tM: r = –0.584, P < 0.05; n = 16; for EZP: r = –0.518, P < 0.05; n = 16), and M3 tended to correlate with age (r = –0.473, P = 0.064; n = 16). When expressed in FFM, this correlation was attenuated; tM/FFM was correlated with age (r = –0.575, P < 0.05; n = 16) but this was not the case for EZP/FFM or M3/FFM. Moreover, plasma turnover was correlated with age in the nonsupplemented group (r = 0.504, P < 0.05; n = 16). Zinc supplementation improved exchangeable zinc pool masses in the supplemented subjects, which canceled out the negative correlation between the exchangeable zinc pool masses and age that was observed in the placebo group.
DISCUSSION
In the present study, we examined the effect of long-term supplementation with 2 moderate doses of zinc (15 and 30 mg/d) on exchangeable zinc pool masses in late-middle-aged men. Kinetic analyses of zinc metabolism were performed over the past 2 decades with the use of radioactive 65Zn as a tracer (13, 19, 20). These studies have provided new insight into the mechanisms of zinc metabolism regulation and zinc homeostasis. Methods that incorporated the use of a stable isotope were validated with improved analytic techniques and produced zinc kinetic data similar to the data obtained with 65Zn (21). Large and elaborate compartmental models have been proposed to explore the underlying physiology of zinc (15, 16, 21, 22), but these models require numerous blood, urinary, and fecal samples and complex mathematical modeling. In our study, a simple 3-compartmental model, as proposed by Pinna et al (7), was applied instead of the more complex multicompartmental models mentioned above. We tested whether a simple model would be sufficient to determine the effect of zinc supplementation on zinc status. The model used in our study was found to be adequate because the plasma concentration of the zinc isotope fitted well to the sum of 3 exponentials. It is difficult to identify a physiologic location for each zinc pool used in the present study, but it has been speculated from animal (23) and human (13, 20) studies that pool 1 represents plasma zinc, whereas pools 2 and 3 represent zinc in erythrocytes, liver, intestine, and bone. In our study, the mass of pool 1 was fixed to achieve the best possible computer fit of the data by calculating the mass on the basis of plasma zinc concentration and plasma volume. This was possible because Yokoi et al (24) showed that the mass of pool 1 is highly correlated with plasma zinc concentrations (P = 0.009). Wastney et al (13) estimated that total body zinc is 1.6 g, 6% of which exchanges rapidly with the plasma and the remaining 94% of which is located largely in muscle and bone. Our results agree with this hypothesis because the exchangeable zinc pool masses were <10% of the total body zinc in our late-middle-aged subjects.
The characteristics of the combined EZP masses, which exchange with plasma zinc within 48 h of tracer administration (11), were also examined. This approach, the use of plasma tracer enrichment data between day 3 and day 7, is simpler than the frequent early sampling that is required for the compartmental analysis. This method, however, is accurate only if the following 2 conditions are met: 1) the loss of tracer from EZP occurs at a monoexponential rate from the moment of tracer administration to the end of the measurement period and 2) the tracer is homogeneously mixed throughout the EZP during the measurement period (20). This method thus overestimates the EZP mass because the initial rapid loss of the tracer from plasma is not accounted for by extrapolation of the monoexponential loss rate during the measurement period and because it is extremely unlikely that the tracer exists at equal concentrations in all compartments of the EZP at a given time (11). In fact, when estimates from both approaches used in the present study were compared, the EZP approach overestimated the EZP mass by 15–20% compared with the mass estimated by the multicompartmental model. In a previous study, in which we determined exchangeable magnesium pool masses by compartmental analysis and by the Miller approach (11), we observed that the exchangeable magnesium pool was overestimated by 45–50% (18). The higher overestimation of magnesium was undoubtedly due to differences in the homeostasis mechanisms of zinc and magnesium in humans.
We observed that the exchangeable zinc pools were 33% lower in our late-middle-aged population than in healthy adults (7). In our study, the masses of these pools were negatively correlated with age in the placebo group. Because FFM is lower in elderly subjects and because zinc is an integral part of the protein mass in lean tissue, the lower exchangeable zinc pool masses observed in our late-middle-aged subjects may have been the result of a lower FFM. However, when expressed as FFM, the exchangeable zinc pool masses determined with the kinetic method were still negatively correlated with age. Moreover, no correlation was observed between FFM and exchangeable zinc pool mass. The low dietary zinc intake in the nonsupplemented subjects may also explain the lower exchangeable zinc pool mass. Indeed, although the dietary zinc intake of our late-middle-aged subjects was only 6 mg/d, their plasma zinc concentrations were normal [>10.7 µmol/L (25)]. The recommended daily intake of zinc in France is 8 mg/d for elderly people who eat a diet rich in animal products (26), whereas in the United States the recommended intake is 11 mg/d for the same population (27).
Zinc supplementation for 6 mo significantly increased exchangeable zinc pool masses, irrespective of the approach used to estimate the pool masses. Very few studies have examined the effect of moderate dietary zinc intake on zinc tracer kinetics, and in some cases the zinc supplementation was very high [100 mg Zn/d for 9 mo (13, 19) compared with 15 or 30 mg Zn/d for 6 mo in our study]. In these complex kinetic models, rapidly exchangeable zinc pool masses increased by 26% (19) or up to 85% (13) when oral zinc intake was increased 11-fold. Moreover, the plasma zinc mass increased by 37% (19). In our study, we showed that the exchangeable zinc pool mass increased by 10–15% with a moderate zinc supplementation of 30 mg/d. Several studies have explored the effects of zinc depletion alone and the effects of zinc depletion and repletion in humans (6-11). Although low exchangeable zinc pool masses were observed with severe zinc depletion, no modification was observed with marginal zinc depletion. Moreover, zinc pool masses were not totally restored after 1 mo of zinc repletion. Therefore, it is possible that the exchangeable zinc pool mass changes only when there is a critical loss of whole-body zinc or after long-term zinc supplementation when a new steady state is reached. A human study indicated that zinc losses and zinc absorption may be adjusted to match zinc intake over a 10-fold range (28). The major sites of whole-body zinc homeostasis are in the gastrointestinal tract, and homeostatic mechanisms involve both zinc absorption and excretion of endogenous zinc in the feces (29). However, mechanisms that regulate total absorbed zinc are limited and may result in exchangeable zinc pool masses that vary directly with the quantity of zinc ingested and absorbed (30). In our study, exchangeable zinc pool masses correlated strongly with zinc intake, as previously observed in a zinc-depletion study (9), in a zinc depletion and repletion study (11), and in a study in breastfed infants (31).
No significant changes in the plasma distribution rate constants were detected, as previously observed with acute zinc depletion (6), which suggests that the determination of zinc pool kinetics is not an effective means of establishing zinc status. In addition, the half-lives of the zinc pools did not change significantly with zinc supplementation. Plasma zinc flux was significantly increased with zinc supplementation and correlated strongly with zinc intake, and previous studies have reported that plasma zinc flux declines with poor zinc status (6, 7). This kinetic parameter appears to be an interesting marker of zinc status, as suggested by Yokoi et al (32). Because plasma turnover was not modified with zinc supplementation, the increased plasma zinc flux can be explained by the increase in plasma zinc concentrations after zinc supplementation. As observed in more complex kinetic studies (13, 15), the plasma zinc pool exchanged 140 times/d.
It is useful to consider whether kinetic markers provide a greater insight into zinc status than do biochemical indicators. As observed in acute zinc depletion (6), plasma zinc concentrations appear to be a better indicator of zinc status than do total exchangeable zinc pool masses. In fact, although plasma zinc concentrations increased up to 22% with zinc supplementation in the present study, zinc concentrations of the total exchangeable zinc pool mass barely increased to 15% with the kinetic method and barely increased to 10% with the EZP method. However, a regression analysis showed that of all the variables measured (both kinetic and biochemical), M1/FFM had the strongest relation to dietary intake, followed by tM/FFM, tM, EZP/FFM, and plasma zinc concentration. This finding is different from what was observed with zinc depletion, where plasma zinc concentrations followed by plasma zinc flux were the parameters with the best relations to dietary intake (5). Another important point is that there are many physiologic and pathological conditions that may increase or decrease plasma zinc concentrations independent of the intake or intestinal absorption of zinc, whereas exchangeable zinc pool masses may not be as affected under these conditions.
In conclusion, our data showed that long-term supplementation with 2 moderate doses of zinc is an efficient way of increasing exchangeable zinc pool masses in late-middle-aged men. The changes in exchangeable zinc pool masses were smaller than the changes in plasma zinc concentration; thus, exchangeable zinc pool masses may not be as sensitive as are plasma zinc concentrations for the assessement of zinc status. Moreover, these exchangeable zinc pool masses decreased with age and correlated with total dietary zinc intake. This may mean that the actual recommended intake of zinc in humans is insufficient in France and should be increased to agree with the recommended intake in the United States.
ACKNOWLEDGMENTS
We thank L Morin, N Mathieu, and G Manlhiot from the Human Nutrition Unit of the Clermont-Ferrand Hospital for caring for the study subjects; S Corny and V Miranda from the Clermont-Ferrand Hospital pharmacy for preparing the stable isotopes for human use; and L Jaffrelo and S Thien for technical assistance. We acknowledge the subjects for their cooperation and their contribution.
CF-C participated in the data analysis and the writing of the manuscript. NM participated in the subject selection, experiment design, and data collection. MR and JCT participated in the data collection and analysis. MB-B participated in the dietary zinc estimation. MA, AM, and KDC provided significant advice. CC participated in the experimental design and the writing of the manuscript. None of the authors had any financial or personal conflicts of interest.
REFERENCES