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Journal of the American College of Nutrition, Vol. 23, No. 4, 309-315 (2004)
Published by the American College of Nutrition

Zinc Supplementation Reduces Fractional Zinc Absorption in Young and Elderly Korean Women

Jihye Kim, MS, Hee Young Paik, ScD, Hyojee Joung, PhD, Leslie R. Woodhouse, PhD, Shanji Li, MS and Janet C. King, PhD

Department of Food and Nutrition (J.K., H.Y.P., S.L.)
The School of Public Health (H.J.)
Seoul National University, Seoul, KOREA, USDA/ARS Western Human Nutrition Research Center, Davis (L.R.W.)
Children’s Hospital Oakland Research Institute, Oakland (J.C.K.), California

Address correspondence to Janet C. King, PhD, Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609. E-mail: jking{at}chori.org


   ABSTRACT
TOP
 FOOTNOTES
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Background: Adjustments in zinc losses and absorption are thought to maintain zinc homeostasis with changes in intake, but the capacity to make these adjustments appears to be affected by aging. Zinc status of the individual may also influence adjustments in fractional zinc absorption.

Objective: The purpose of this study was to compare the effects of changes in zinc status due to zinc supplementation on fractional zinc absorption in young and elderly Korean women.

Design: Zinc status and absorption were measured initially in 15 young (20–24 yr) and 15 elderly women (64–75 yr) confined to a metabolic unit and consuming a typical Korean diet. Upon discharge from the unit the women were supplemented with 22 mg zinc/d for 28 days. On d 20, the women returned to the unit for measurement of zinc status and absorption. Fractional zinc absorption (FZA) was estimated from the same typical Korean breakfast at both time points using the dual isotopic tracer method. Zinc stable isotopic ratios were measured in urine samples collected for 3 days following isotope administration.

Results: Plasma and urinary zinc concentrations increased significantly with zinc supplementation in the young but not the elderly women. FZA decreased following zinc supplementation in the young women from 22 to 8% (p < 0.0001) and in the elderly women from 19 to 10% (p < 0.0001). The decline in young women was greater than that in elderly women (p < 0.05).

Conclusions: Both young and elderly women reduced their efficiency of zinc absorption with zinc supplementation. But, plasma and urinary zinc concentrations did not increase in the elderly following zinc supplementation suggesting that age altered the use of the additional zinc.

Key words: Zn supplementation, zinc status, zinc absorption, aging, humans


   INTRODUCTION
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Zinc homeostasis can be maintained over a wide range of intakes in humans primarily due to adjustments in endogenous zinc losses [1,2]. Reductions in endogenous fecal zinc losses occur within several days following changes in zinc intake [3]. Zinc absorption also appears to be altered with changes in zinc intake to maintain homeostasis. However, the studies that were used to support this conclusion are confounded by the fact that zinc absorption was measured from low zinc diets in zinc-depleted individuals and from higher zinc diets in zinc-adequate individuals. Thus, the level of zinc intake may have affected fractional zinc absorption (FZA) independently of zinc status. Zinc absorption from the same diet has not been measured in individuals in two different states of zinc nutriture.

Elderly individuals are thought to be at greater risk of zinc depletion than younger adults [4]. This may reflect a lower intake of zinc [5] and/or a reduced efficiency of zinc absorption with aging. Several [6,7], but not all [8], studies have shown that zinc absorption from a test meal is decreased in the elderly compared with younger people. Turnlund et al. [6] showed that zinc absorption averaged 17% in elderly men receiving a diet containing 15 mg Zn/d, significantly less than average zinc absorption of 31% in young men. August et al. [7] observed that zinc absorptions were 39% and 64% in young subjects receiving an adequate Zn diet (12.8–15 mg Zn/d) and low Zn diet (2.8–5 mg Zn/d); zinc absorptions were 21% and 43% in the elderly subjects fed the same diets. A reduction in zinc absorption with aging may account for the higher prevalence of low plasma zinc concentration in the elderly. De Jong et al. [9] reported that twelve percent of New Zealand older women had serum zinc levels below the cut-off value.

Subclinical zinc depletion, defined as marginal reduction in plasma zinc concentrations (<10.7 µmol/L), has been recognized in population groups in both developing and industrialized countries [10]. The high prevalence of low zinc intakes and plasma zinc concentrations among Koreans suggests that their zinc status may be marginal. Rural adults had lower zinc intakes and higher phytate/zinc molar ratios than urban adults [11]. Zinc intakes average 5.2 mg/d for women and 7.3 mg/d for men living in rural areas where about 60%–70% of the zinc is provided by plant foods that are high in phytate [12] and 45% of the rural adults had plasma zinc concentration below 10.7 µmol/L [13]. Nationwide plasma zinc concentrations averaged 11 µmol/L among Korean adults compared to 13 µmol/L among US adults [13,14]. Serum and dietary intake of zinc among the elderly in Korea were reported to be lower than that of younger adults [15].

The purposes of this study, therefore, were to determine the effect of zinc supplementation on the efficiency of zinc absorption from the same test meal fed before and after supplementation in young and elderly Korean women, and to evaluate the effect of age on the capacity to adjust the efficiency of zinc absorption with changes in zinc status.


   SUBJECTS AND METHODS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects
Fifteen healthy young women (20–24 years) and 15 elderly women (64–75 years) were recruited for the study by word-of-mouth and flyers on the campus of Seoul National University and in neighboring areas. Exclusion criteria included a body mass index of less than 17 or greater than 28 kg/m2; cigarette smoking; chronic use of alcohol, prescription drugs, use of oral contraceptives or vitamin or mineral supplements; a hemoglobin level <105 g/L; the presence of acute disease or chronic disease such as diabetes, gastrointestinal disorder, hyperlipidemia; and a usual dietary zinc intake <5 mg/d or >15 mg/d. Five of the elderly women took diuretics for treating hypertension during study.

All subjects gave their informed consent to participate in the study. The study protocol was reviewed and approved by the Committee on Human Research at the College of Human Ecology at Seoul National University and the Office of Human Research Protection at the University of California, Davis.

Study Design
Plasma and urinary zinc and FZA were measured in the young and elderly women prior to and following supplementation with 22 mg zinc/d for 28 days. The women were housed in a metabolic unit for a 9-d comprehensive study of zinc homeostasis and kinetics during the baseline period (to be reported separately). A controlled diet of common Korean foods was fed during the metabolic study. Two metabolic units were created to do this study in Seoul. The young women were housed at the Faculty House of Seoul National University and the elderly stayed in an apartment near Seoul National University. Upon completion of the metabolic study, the women were discharged with a 22 mg zinc supplement (as zinc gluconate) to take daily for the next 28 days. They were told to take the supplement either at bedtime or between meals while eating their usual diet. On the 20th day of supplementation, they returned to the metabolic unit for measurements of FZA, plasma and urinary zinc concentrations. The elderly women moved into the Metabolic Unit on the 20th day and consumed a controlled diet for a 9-day metabolic period. Since the young women were students and attending classes, they lived at home and consumed their normal diet prior to and following isotope administration on day 20 of supplementation.

The dual isotopic tracer method was used to measure FZA. On the morning of the 5th day of the baseline metabolic period and on the 20th day of zinc supplementation the women received 1 mg of 67Zn (ZnCl2) in orange juice immediately following the typical Korean breakfast test meal that provided 1.3 mg of zinc. Shortly thereafter 0.3 mg of 70Zn, as ZnCl2, was infused intravenously by a registered nurse.

A 24-hr urine sample was collected the day before each isotope study to measure background levels of the stable isotopes and total urinary zinc excretion. Spot urine samples were collected for 3 consecutive days starting on the third day following isotope administration for measurement of FZA. The first AM urinary void was collected from the young women; complete 24-h collections were made in the elderly women. Fasting blood samples were collected on the isotope administration day for measuring plasma zinc concentrations.

Usual zinc intakes were estimated from 3-day diet records completed before each study. Subject height and weight were measured at the time of recruitment; weight was measured again on the isotope-administration day of each metabolic period before the breakfast meal.

Diets
The test meal for estimating FZA prior to and following zinc supplementation consisted of typical Korean breakfast foods (Table 1). The zinc supplement was not given with the meal in the supplementation period. The same test meal was fed at both time points. At baseline, a controlled metabolic diet of typical Korean foods was fed for 4 days prior to measuring FZA in both the young and elderly women. The calcium, zinc, and phytate intakes in the controlled metabolic diet were analyzed from the composites of each meal (Table 2). During supplementation, the women were not confined to the metabolic unit, and they consumed their usual self-selected diet prior to the measurement. Their reported intakes for the 3 days prior to measurement of FZA during supplementation are also in Table 2. Their usual dietary intakes were averaged from 3-day diet records collected during screening and supplementation, and nutrient intakes were estimated using a nutrient database developed by the Korean Nutrition Society [16]. The usual intakes estimated from these records are reported in Table 3.


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  		Table 1. Menu for the Breakfast Test Meal*

 

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  		Table 2. Composition of the Diets Consumed by the Women during the Metabolic Studies before and during Zinc Supplementation1

 

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  		Table 3. Characteristics of Study Participants1

 
Isotope Preparation
Stable isotopic tracers of zinc, highly enriched in 67Zn (89.2% or 89.6% abundance) or 70Zn (95.56% abundance), were purchased as zinc oxide from Trace Sciences Inc., Toronto, Canada. Chloride salts of the isotopes were prepared from oxide using concentrated HCl. Solutions of 67Zn, for oral isotope administration, were diluted to 1 mg Zn/mL with triply deionized water, sterile filtered using Millex-GV 0.22u filters, and stored at 4°C until use [17]. Solutions of 70Zn, for intravenous isotope administration, were diluted to 0.3 mg Zn/mL with triply deionized water and sent to the Parenteral Solution Laboratory at the University of California, San Francisco, for sterile preparation, testing, and packaging into vials. The IV isotopes were stored at 4°C until use.

Sample Collection and Analyses
Two composites of the breakfast test meal were prepared during each metabolic study and aliquots were saved for zinc and phytate analysis. Blood samples for plasma zinc analysis were collected in plastic syringes (Sarstedt 8 mL Monovette syringes with ammonium heparin coated beads, Newton, North Carolina, USA) and were kept on ice for a maximum of 2 h before centrifugation at 3000 rpm for 10 min at 4°C (Hanil micro 17R Plus, Incheon, Korea). After centrifugation, the plasma was transferred to polypropylene tubes using transfer pipettes and stored at –70°C prior to analysis. Hemoglobin was measured at each visit using a HemoCue (HemoCue AB, Angelholm, Sweden). All urine samples were collected in polypropylene containers. After collection, the urine samples were mixed by shaking vigorously, weighed, acidified with 0.4 mL concentrated HCl/100 g urine (Trace metal grade; Merck, Germany), and aliquots were stored at –20°C until analysis for zinc. Precautions against environmental zinc contamination were taken for all diet, blood collections, and analyses. Only new polypropylene containers were used, and samples were prepared with triply deionized water and ultra high-purity reagents. All glassware was acid-washed in 10% nitric acid and rinsed 3 times with triply deionized water.

Diet, plasma, and urinary zinc content were determined by Inductively Coupled Plasma Atomic Emission Spectrophotometry (ICP-AES, Vista, Varian Inc., Walnut Creek, CA). Diet samples were freeze-dried (Bondyro, Ilsin Inc., Seoul, Korea) and ground to homogeneity. Weighed aliquots (0.2–0.4 g) were digested by microwave digestion (MARS 5, CEM Corp., Matthews, NC) prior to determination of zinc by ICP-AES. Plasma samples were diluted with 6% nitric acid (Optima grade; Fisher Scientific; Pittsburgh, PA) and were centrifuged at 4°C for 15 minutes at 3200 rpm (2200 x g) prior to analysis for zinc. Urinary samples were centrifuged at 1000 rpm (230 x g, 4°C, 10 min) using Allegra 6R (Beckman Coulter Inc., Palo Alto, CA) to remove solid material and were diluted with 1% nitric acid (Optima grade; Fisher Scientific; Pittsburgh, PA).

The total phytate content of the test meal was determined by Dionex Liquid Chromatograph System (Dionex Corp., Sunnyvale, CA, USA). Phytate was extracted from freeze-dried diet composites (0.25 g) using 1.25% H2SO4 and eluted with 200 mM NaOH solution.

The ratios of zinc stable isotopes were determined in urine samples by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A detailed description of sample preparation for ICP-MS analysis is described elsewhere [17]. In brief, urine samples were centrifuged at 1000 rpm (230 x g, 4°C, 10 min) using Allegra 6R (Beckman Instruments, Inc., Palo Alto, CA) and the inorganic salts were removed using a chelating resin (Chelex 100 resin; Bio-Rad Laboratories, Hercules, CA). Zinc was separated from the eluent by ion exchange chromatography (type AG1X-8 ion exchange resin; Bio-Rad Laboratories, Hercules, CA). The isotope mass ratios were measured using a Sciex ELAN 6000 ICP-MS instrument (Perkin-Elmer, Norwalk, CT) equipped with a U-6000AT+ ultrasonic nebulizer (Cetac Technologies Inc, Omaha, NE) and a model AS 93+ autosampler (Perkin-Elmer, Norwalk, CT). Tracer-to-tracee ratios were calculated using the natural abundances of the various zinc isotopes and the tracer mass (in mg) was determined [17].

Calculations
Fractional zinc absorption (FZA) was calculated using the following equation [17]:

For the young and elderly women, FZA values were determined from the average of the 24-h urine samples over days 7 through 9 of the baseline period and spot urine samples from young women and 24-h urine samples from the elderly women over days 22 through 24 of the zinc supplementation period.

Statistical Analysis
Results are expressed as means ± standard deviations (SDs). Differences between the two metabolic periods were determined using paired comparison t-tests. Differences due to age were determined using one way ANOVA [18]. All significant differences were defined as a p value < 0.05. Statistical analyses were conducted with SAS 8.2 (SAS institute Inc, NC 27513, USA).


   RESULTS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
The characteristics of the young and elderly women are described in Table 3. All of the women were of normal weight-for-height and had normal hemoglobin values. The usual zinc and phytate intakes of the women did not differ between the two groups.

Plasma zinc concentrations before supplementation were lower in the young compared to elderly women (p < 0.05) (Table 4). But, there were no significant differences in urinary zinc excretion. Supplementation with 22 mg zinc/d for 20 days increased plasma zinc by 31% and urinary zinc by 44% in the young women (p < 0.01). No changes were seen in the plasma or urinary zinc concentrations of the elderly women with supplementation, however. The elderly women taking diuretics for hypertension tended to have lower levels of plasma zinc and higher rates of urinary zinc excretion both prior to and following zinc supplementation, but this effect did not reach statistical significance.


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  		Table 4. Plasma and Urinary Zinc and Fractional Zinc Absorption before and during Zinc Supplementation in Young and Elderly Women1

 
Fractional zinc absorption did not differ between the two groups of women at either time point. At baseline, the young women absorbed about 22% of the zinc in the test meal, and the elderly women absorbed about 19%. After zinc supplementation, FZA declined 63 ± 23% to about 7.5%/d in the young women and 43 ± 20% to about 10% in the elderly; the reduction in FZA with zinc supplementation differed significantly between the two groups (p < 0.05).


   DISCUSSION
 
Although both groups consumed similar amounts of zinc and phytate in their usual diets, the plasma zinc concentrations of the elderly were 20% higher than that of the young women prior to supplementation. Urinary zinc concentrations did not differ between the two groups. FZA was not different between two groups at either time point. Supplementation with 22 mg zinc/d for 20 days increased the plasma and urinary zinc concentrations of young, but not elderly, Korean women. This suggests that the distribution of the additional absorbed zinc from the supplement was used differently in the elderly compared to young women.

The lack on an increase in plasma zinc with supplementation in the elderly women may reflect an increased deposition of absorbed zinc in tissues or an increase in endogenous fecal zinc excretion. Wastney et al. [19] reported that the transport rates of zinc from plasma to muscle and bone increased with age by 60% when the subjects consumed supplemental zinc. This shift in zinc metabolism of older women with supplementation was similar to that seen in our studies of men during severe zinc depletion [20]. In severe zinc depletion, the men also sequestered zinc in slowly turning over tissues. This tendency to sequester absorbed zinc in tissues may reflect the changes in protein turnover that occur with aging. Zinc tends to be found in tissues bound to proteins. A reduction in the turnover of those proteins would lead retention of zinc in the tissue and thereby prevent a rise in plasma zinc with supplementation. Short et al. reported that muscle protein metabolism declines with age [21].

Zinc supplementation lowered FZA in both young and elderly women, but the decline in the young women was greater than that of the elderly women (p < 0.05). The observed changes in FZA and the increase in plasma zinc and urinary zinc excretion suggest that the zinc status of the young women improved as a result of taking supplemental zinc for 20 days. Changes in plasma and urinary zinc with supplementation were not seen in the elderly women. The decline in FZA was presumably due to a down-regulation in zinc absorption by the gastrointestinal tract with zinc supplementation. Several studies have reported that FZA is increased with low zinc intakes in either young [22] or elderly [7]. But, in all of those studies, a lower zinc diet was fed during the low-zinc periods. In the present study, the zinc content of the two test meals prior to and following zinc supplementation was the same. This suggests some factor reflecting the zinc status of the individual adjusted the efficiency of zinc absorption with changes in status. This factor is unknown. Some have proposed that metallothionein (MT), a zinc binding protein, plays a role in regulating zinc absorption [23–25]. Supplemental zinc may increase the synthesis of MT in the intestine, which in turn blocks further absorption of zinc [26]. The metallothionein content of the pancreas may also be involved in the regulation of zinc absorption [27]. If the ability to synthesize MT in response to zinc status declines with age, the ability to regulate the amount of zinc absorbed may also decline in the elderly [19,28]. Age may affect MT metabolism by altering the regulation of gene expression, zinc binding ability, or turnover rate [19].

Prior to supplementation, the young and elderly women absorbed about 20% of the zinc from the test meal. This is similar to the amount of zinc absorbed by a group of young American women who consumed beans as a test meal (15%) [29]. Possibly, the two values of FZA are similar because the phytate/zinc molar ratios of the test meals were high in both studies. In the present study, we used a high phytate/zinc test meal, 23, because we predicted that phytate intakes would be high in the diets of Koreans based on food consumption data (phytate/zinc molar ratio = 16) [11]. But, the phytate/zinc molar ratios in the reported usual diet from the Korean women studied were comparable to that seen in Americans with omnivorous diets (4–9 for men, 5–12 for women) [30]. In recent years, the diet of Koreans has changed from traditional foods to more western-style foods. A recent survey showed that the phytate/zinc molar ratio in the diet of Koreans in an urban area was about 10 [31]. These data are thought to be more accurate than previous data since the phytate/zinc molar ratios were estimated from 24-hour recalls rather than household food consumption. Men who consumed diets with phytate/zinc molar ratios of about 20 absorbed similar amounts of zinc as did the Korean women in this study; FZA averaged 23 and 20% in young and elderly men, respectively [8]. When American women were fed similar amounts of zinc, 7 mg zinc/d, from a diet with a lower phytate/zinc molar ratio (<5), the fractional zinc absorption was higher, 30% [32].


   CONCLUSIONS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 CONCLUSIONS
ACKNOWLEDGMENTS
 REFERENCES
 
Both young and elderly women reduced their efficiency of zinc absorption following 20 days of supplementation with zinc, but the decline was greater in the younger than the elderly women. Although plasma and urinary zinc increased significantly following supplementation in the young women, with no change occurred with supplementation among the older women. This suggests that the additional zinc absorbed from the supplement is used differently by elderly women in comparison to younger women. Further studies of zinc kinetics and zinc homeostasis are needed in the elderly to better understand the changes in zinc regulation with age.


   ACKNOWLEDGMENTS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
REFERENCES
 
We are grateful to study participants and all staff in the Human Nutrition Laboratory at Seoul National University in Seoul, Korea, Tuan Q. Nguyen at Children’s Hospital Oakland Research Institute, Oakland, CA, Erik R. Gertz at USDA/ARS Western Human Nutrition Research Center, Davis, CA, and Dr. Ross Welch, for phytate analysis, at Cornell University. This research was supported by the Korea Research Foundation and USDA/ARS Western Human Nutrition Research Center.


   FOOTNOTES
TOP
 FOOTNOTES
ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Supported by Korea Research Foundation and USDA/ARS Western Human Nutrition Research Center.

Received October 30, 2003. Accepted March 3, 2003.


   REFERENCES
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 

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