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Eleven male and thirteen female rural elderly people (aged 60-70 years) were recruited for this study. All the subjects were in good health. In particular, accurate examinations were carried out to exclude subjects with anemia, gastrointestinal diseases, chronic metabolic diseases, or abnormal Zn nutrition status (plasma Zn < 10.7 μmol/L and C-reactive protein > 10 mg/L).
The trial was registered in the Chinese Clinical Trial Registry (No. ChiCTR-OOB-15007070). All the procedures involving human subjects were approved by the Ethical Committee of the Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention. All subjects gave their written informed consent to participate in this study.
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According to the basic data of the typical rural dietary pattern obtained from the China National Nutrition and Health Survey 2010-2013 (CHNNS 2010-2013) and market supply, the varieties and quantities of the foods in the experimental diet were assorted to design a 3-day recipe and repeatedly served during the whole experimental period to each subject. The food met the principles of nutrient balance, and the energy values were distributed for each meal (breakfast, lunch, and supper) according to the energy requirement. Table 1 shows the 3-day recipe used in this study.
Meal Time Day 1 Day 2 Day 3 Breakfast Steam bread Fried dough cake Steam bread Stir-fried cauliflower Stir-fried potato chips Stir-fried lettuces Egg Egg Egg Millet congee Millet congee Rice porridge with bean Lunch Stir-fried pork slices with mushroom Stir-fried pork slices with Chinese mushroom Stir-fried pork with green pepper Supper Stir-fried potato chips Stir-fried spinach Home-style bean curd Stir-fried egg with zucchini Stir-fried egg with tomato Stir-fried mushroom Steam bread Steam bread Steam bread Rice porridge Cabbage soup Tomato egg soup Apple Banana Orange Stir-fried chicken Stir-fried pork with green pepper Stir-fried chicken Stir-fried pork with long bean Stir-fried mushroom Stir-fried zucchini Stir-fried cabbage Stir-fried cabbage Stir-fried potato chips Steam bread Baked pancake Baked pancake Rice porridge with bean Rice porridge with bean Rice porridge with bean Snack Bun Bun Bun Table 1. The Three-day Recipe in the Study
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Zn stable isotopes were obtained from Trace Sciences International as oxide powder (Richmond Hill, ON, Canada), and 67Zn abundance was 88.55%. 67ZnSO4 for oral administration was converted by 67ZnO powder that was dissolved in H2SO4 (0.5 mol/L) and diluted with ultrapure water to a concentration of 0.5 mg/L. This solution was sterilized to remove pyrogens and sent to the Institute of Drug Analysis to test if the preparation was safe and edible.
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The study flow chart is shown in Figure 1. After a 3-day adaptation period, 3 mg 67Zn was administered with porridge and divided into three meals on the fourth day of the test period, which could meet the test requirements[11]. Simultaneously, the rare earth element dysprosium (Dy 0.4 mg Dy per meal), which follows the same excretory pattern as unabsorbed Zn, was given along with the isotope to check the completeness of the fecal samples[11-13]. On the fourth day and the eleventh day, capsules of carmine red dye were given to subjects before breakfast to determine the initiation and end of feces collection. The actual intake of food served for the elderly subjects every day was recorded. A duplicate dietary sample was collected for 3 days, and the concentrations of the primary macronutrients, energy, and phytic acid in the food were examined using the related routine protocols of the National Standard of the People's Republic of China (GB 5009.5-2010, GB/T 5009.6-2003, GB/T 5009.153-2003).
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The fecal samples were homogenized in a blender, and the powder was digested in a microwave oven (Anton Paar, Austria). Total Zn contents of the samples were measured by atomic absorption spectrophotometer, and Dy was analyzed by inductively coupled plasma mass spectrometry. Zn from 0.3 mL the digested samples was isolated by heating (120 ℃) until all of the liquid had evaporated, and then the samples were redissolved in 1 mL of 3 mol/L HCl. The samples were again heated until dry. The digested fecal residue was then redissolved in 1 mL of 3 mol/L HCl before anion exchange chromatography. Columns were first washed three times with 7 mL of 0.5 mol/L HNO3 and 2 mL ultrapure water and then conditioned three times using 2 mL of 3 mol/L HCl. The redissolved fecal samples were passed through the conditioned columns. Zn was eluted from the column five times using 2 mL ultrapure water. The final eluent was dried and reconstituted with 2 mL of 2% HNO3 before mass spectrometric analyses. All the acids used in this experiment were of ultrapure grade. The Zn isotope ratio was detected by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS; Isoprobe, GV, England).
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The phytic acid-to-zinc molar ratio (PZR) was calculated using the following Equation:
$$ {\rm{PZR}} = \frac{{\left( {{\rm{A}}/660.04} \right)}}{{\left( {{\rm{B}}/65.38} \right)}} $$ (1) where A is the intake of phytic acid, and B is the intake of zinc.
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Fractional Zn absorption (FZA) was calculated using the following Equation:
$$ {\rm{PZA}}\left( \% \right) = \frac{{^{67}{\rm{ZnI}}{ - ^{67}}{\rm{ZnM^{\rm s}}}}}{{^{{\rm{67}}}{\rm{ZnI}}}} $$ (2) where 67ZnI is the oral 67Zn intake (mg), and 67ZnMs is the unabsorbed 67Zn in the feces (mg), which were determined as described previously[11]. 67Zn absorption (%) = FZA/dysprosium recovery.
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The most widely used method for estimating dietary Zn requirements is the factorial approach[14]. The estimated average requirement (EAR) is derived by dividing the mean physiologic requirement of absorbed Zn by the estimated average absorption of Zn. The RNI for dietary Zn intakes is thus calculated as the EAR plus two times the CV and is equivalent to 120% of the EAR.
$$ {\rm{EAR}} = \frac{{\rm{A}}}{{\rm{B}}} $$ (3) where A is the physiologic requirement of absorbed Zn, and B is the estimated average absorption of Zn
$$ {\rm{RNI}} = {\rm{EAR}} \times 1.2 $$ (4) -
Results were expressed as mean ± standard deviation (SD). Data were analyzed using SPSS 17.0 software (SPSS Inc, Chicago, IL). Student's t-test was used to detect the presence of statistically significant differences between the male and female groups. P value < 0.05 was considered as statistically significant.
Subjects
Experimental Diet
Stable Isotope Labels
Administration of Meals and Sample Collection
Detection of Zn in the Fecal Samples
Calculation of the Phytic Acid-to-zinc Molar Ratio
Calculation of Zn Absorption
Calculation of Zn RNI
Statistical Analysis
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Eleven male and thirteen female rural elderly people were recruited in this study, but one male individual was excluded during the study because of abnormal Zn absorption data. Anthropometric measurements of the subjects are described in Table 2. Except height, no statistically significant differences were found in the measured physical characteristics.
Characteristics Mean SD Male Female P Mean SD Mean SD Age (years) 63.9 3.6 65.2 2.8 62.9 3.9 0.13 Weight (kg) 66.1 10.3 67.5 9.1 65.0 12.0 0.59 Height (cm) 160.6 7.2 165.1 6.1 157.2 6.1 0.01* BMI (kg/m2) 25.5 3.1 24.7 2.5 26.2 3.3 0.27 Note.*P < 0.05 represented statistically significant difference between males and females. Table 2. Subjects' Basic Characteristics
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Intake of the primary macronutrients, energy, Zn, phytic acid, and the PZR are shown in Table 3. The mean (± SD) Zn intake from the representative Chinese diet was 10.6 ± 1.5 mg/d. The zinc intake amounts in males and females were 10.7 ± 1.5 and 10.5 ± 1.5 mg/d, respectively. The PZR in the diet was 6.4, and it was significantly higher in males than in females (6.6 ± 0.4 vs. 6.2 ± 0.4, P = 0.03). There were no statistically significant differences in terms of protein, fat, carbohydrate, energy, Zn, and phytic acid intakes between the male and female groups.
Nutrient Mean SD Male Female P Mean SD Mean SD Energy (kcal) 3147.8 509.5 3240.5 518.6 3076.4 490.6 0.47 Protein (g) 99.9 16.7 102.5 17.0 97.9 16.3 0.53 Fat (g) 137.6 23.0 141.4 25.9 134.7 20.1 0.51 Carbohydrate (g) 375.4 67.2 387.3 62.3 366.2 69.4 0.48 Zinc (mg) 10.6 1.5 10.7 1.5 10.5 1.5 0.73 Phytic acid (mg) 686.3 115.0 715.8 89.0 663.7 126.9 0.30 Phytic acid/Zn 6.4 0.5 6.6 0.4 6.2 0.4 0.03* Note.*P < 0.05 represented statistically significant difference between the male and female groups. Table 3. Daily Intakes of Major Nutrients
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FZA, dysprosium recovery, and Zn absorption are shown in Table 4. The absorption rate of 67Zn was 27.9% ± 9.2%. The absorption rates of 67Zn in males and females were 30.9% ± 8.3% and 25.5% ± 9.2%, respectively. There were no significant differences in FZA (P = 0.20), dysprosium recovery (P = 0.33), and absorption rate of 67Zn (P = 0.18) between the male and female groups. The absorption of Zn was categorized according to the WHO standards as follows: high ( > 30%-50%), moderate (15%-30%), and low ( < 15%)[15]. As shown in Table 4, the Zn absorption was categorized as high for males and moderate for females.
Items Mean SD Male Female P Mean SD Mean SD FZA (%) 24.3 7.4 26.7 6.8 22.5 7.3 0.20 Dysprosium recovery (%) 88.4 6.2 86.9 5.0 89.5 6.7 0.33 Zn absorption (%) 27.9 9.2 30.9 8.3 25.5 9.2 0.18 Table 4. FZA, Dysprosium Recovery, and Zinc Absorption
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Zn physiologic requirements as recommended by International Zinc Nutrition Consultative Group (IZiNCG) for adult males and females were 2.69 and 1.96 mg/d[8, 16]. Zn EAR and RNI are shown in Table 5. Calculating by the Zn absorption rate of the elderly people in this study, the EAR and RNI of Zn were 8.7 and 10.4 mg/d, respectively, in the elderly men and 7.7 and 9.2 mg/d, respectively, in the elderly women.
Items Male Female Recommendeda Calculatedb Recommendeda Calculatedb Zn EAR 10.4 8.7 6.1 7.7 Zn RNI 12.5 10.4 7.5 9.2 Note. aZn EAR and RNI in Chinese Dietary Reference Intakes (2013). bThe values were calculated by the zinc absorption rate of the elderly in this study. EAR, estimated average requirement. RNI, recommended nutrient intakes. Table 5. Zn Estimated Average Requirement and Recommended Nutrient Intake