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Previous studies have suggested that the ratio of iron in the blood to total iron in the body, known as the iron circulation rate, remains roughly constant over a short observation period[6]. Therefore, we assumed that the variation in isotope abundance in the blood is consistent with that throughout the body. A previous study showed that the percentage of erythrocyte iron-57 (57Fe) incorporation in children increases starting 14 days after oral administration, reaching a peak on day 60[7]. Diluted by the body’s natural iron isotope, this percentage slowly declines and eventually reaches a relative steady state. The stationary transition of abundance is known as the “steady period” [8] marking the point when the 57Fe is fully integrated into the entire body. Studies have shown that after reaching a steady state, the total amount of iron usually remains constant [9-10], and the physiological requirement in individuals with no significant change in weight is equal to iron loss[6,11]. In the present study, we monitored the isotopic abundance for 2 years to ensure that it had reached the steady state, calculated the iron loss by accurately measuring the change in the abundance of 57Fe, and measured the iron circulation rate. In this way, the physiological requirement for iron was obtained.
Fifty-seven healthy children (30 boys and 27 girls) aged 10 to 12 years from Beijing were randomly recruited to participate in this study. Those who meet the following criteria will be excluded: 1) Menarche or secondary sexual characteristics have appeared; 2) Anemia or other iron metabolic diseases; 3) Unable to complete the required contents of the study; 4) Those who have recently transferred to another school or are unable to stay in school continuously for other reasons; 5) Unwilling to participate in the study. The trial was registered at the Chinese Clinical Trial Registry (No. ChiCTR-OCH-14004302) and approved by the Ethical Committee of the National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention. Informed consent was provided by at least one parent of each participant.
The study flow chart is shown in Figure 1. During the first 2 weeks of the trial, all children stayed in arranged accommodations and were fed a low-iron diet. Diet, stool, and blood samples were collected to determine the iron absorption rate and iron circulation rate, and these results were previously published [7, 12]. In total, 30 mg of 57Fe was administered over a 5-day period. The preparation of stable iron and the specific course of administration were also described in the above-mentioned report[7,12].
Venous blood samples (10 mL) were collected from each child to measure the change in the total iron concentration and 57Fe abundance at day 0, 14, 28, 60, 90, 180, and 360 after the start of the trial and every 90 days thereafter. During follow-up, the children’s health was closely monitored to ensure that they did not exhibit symptoms associated with blood loss or abnormal iron metabolism.
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Blood samples were acid-digested with 70% nitric acid solution using a microwave digestion system. The total iron concentration was quantitatively determined by atomic absorption spectrometry, and the abundance of 57Fe was analyzed by inductively coupled multicollector plasma mass spectrometry. The specific operation method was described by Cai in 2020[13].
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Previous studies have shown that after the 57Fe absorbed during the trial has been completely mixed with the iron of the study subjects and reaches a stable state, the daily iron loss can be calculated by the change in the abundance of 57Fe over a period of time (assuming day i to day i+t)[6]. The formula, which was derived from our previously published work [14], was as follows:
$$ {{R}}={{T}}\;\times\;{{V}}\;\times\;({{P}}_{{i}}-{{P}}_{{{i}}+{{t}}}) \div {{t}}\div [({{P}}_{{i}}-{{P}}_{{{i}}+{{t}}})/2-{\rm{N}}{\rm{A}}] \div {{C}} $$ (1) where R is the daily loss or intake of iron (mg), T is the total iron concentration in the blood (mg/L), Pi is the isotopic abundance on day i, NA is the natural abundance of 57Fe, t is the total study period (days), and V is the blood volume (L), which was estimated by the sex and body weight as described by Etcheverry in 2007[15]:
$$ {\rm{boys}}: {{V}}=0.0753\;\times\;{\rm{BW}}\; ({\rm{kg}})\;-0.05$$ (2) $$ {\rm{girls}}: {{V}}=0.0753\;\times\; {\rm{BW}}\;({\rm{kg}})+0.01\;$$ (3) where C is the iron circulation rate (ratio of iron in the blood to total iron in the body), the results were published in our previous works [16]. Each time venous blood was collected, the individual’s body weight was measured.
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The normality of the data was assessed using the Kolmogorov-Smirnoff test. Variables that conformed to a normal distribution are presented as mean ± standard deviation (SD). Repeated-measures analysis of variance was used for comparisons between groups. All of the statistical analyses were performed using SPSS Version 13.0 software (SPSS Inc., Chicago, IL, USA), and significance was set at the α = 0.05 error rate.
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Both groups had comparable characteristics at baseline (Table 1). The mean age of boys at enrollment was 10.6 ± 0.2 years, and that of girls was 10.4 ± 0.2 years. Boys and girls had mean hemoglobin levels of 133.1 ± 4.9 g/L and 130.8 ± 6.1 g/L, respectively. The mean log-transformed serum ferritin concentration in boys and girls was 15.0 ± 5.1 ng/mL and 14.4 ± 4.8 ng/mL, respectively, and the amount of 57Fe given orally was 27.5 ± 0.3 mg and 27.3 ± 0.2 mg, respectively.
Table 1. Baseline characteristics of the subjects
Gender n Age
(years)BMI
(kg/cm2)Hb
(g/L)SF
(ng/mL)57Fe given orally
(mg)Boys 30 10.6 ± 0.2 19.2 ± 4.4 133.1 ± 4.9 15.0 ± 5.1 27.5 ± 0.3 Girls 27 10.4 ± 0.2 17.9 ± 2.6 130.8 ± 6.1 14.4 ± 4.8 27.3 ± 0.2 Note. BMI: stands for body mass index; Hb: stands for hemoglobin; SF: stands for serum transferrin. -
The iron circulation rate was calculated and published in our previous work [16]. The abundance of isotopes in the circulation peaked on day 60 in all subjects; therefore, the iron circulation rate was calculated using the data at the peak. The mean iron circulation rate for boys and girls was 81.2% ± 2.6% and 82.7% ± 2.9%, respectively, with a significant difference between the sexes (P < 0.05).
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Repeated-measures analysis of variance was used to determine that continuous and complete data were required for the steady period. Thirty-seven subjects (20 boys and 17 girls) with complete blood samples were selected from all subjects at each time point.
The trend of the change in 57Fe abundance over time was basically the same between boys and girls, but the abundance in boys was lower than that in girls at all time points. At about 450 days, the change in 57Fe abundance in the blood had gradually stabilized, as shown in Figure 2. The isotopic abundance data of all subjects at any time from 14 to 720 days were statistically analyzed after natural logarithmic conversion. The results showed a linear trend (Table 2). After 360 days, the quadratic term began to gradually decrease, and after 450 days, it plateaued; this was considered the steady period.
Table 2. P values of polynomial contrast by repeated measure analysis
Ln (57Fe) Time_1 Time_2 Start (day) 14 < 0.0001 0.0089 End (day) 720 Start (day) 60 < 0.0001 0.0238 End (day) 720 Start (day) 90 < 0.0001 0.0387 End (day) 720 Start (day) 180 0.0066 0.0440 End (day) 720 Start (day) 360 0.0289 0.0506 End (day) 720 Start (day) 450 0.0723 0.1980 End (day) 720 Start (day) 540 0.115 0.3720 End (day) 720 Note. Time_N represents the nth degree polynomial contrast for time. -
Factorial modeling and the iron balance are used to determine the physiological requirements for iron. According to the “factor model” method, the physiological need for iron can be divided into three aspects according to function: basal losses, menstrual losses, and accretion. Because the subjects in this study were children who had not yet reached menstruation, menstrual losses could be ignored; only basal losses and accretion were considered. When calculating accretion, children’s growth and development needs for iron should be fully taken into account, especially the influence of the increase in blood volume.
We found that the blood volume and iron content of all children gradually increased over time (Table 3). However, neither the blood volume nor iron content significantly increased from day 450 to 540, day 540 to 630, or day 630 to 720 in either sex. Therefore, the influence of accretion on the iron requirement can be largely ignored in the calculation process. It can be concluded that in the steady period, the physiological requirement for iron is basically equal to basal losses.
Table 3. The blood volume and iron content from day 14 to 720
Days Boys (n = 20) Girls (n = 17) BV (mL) Fecirc (g) BV (mL) Fecirc (g) 14 3.19 ± 0.41 2.01 ± 0.12 3.15 ± 0.29 1.89 ± 0.15 28 3.19 ± 0.38 2.00 ± 0.15 3.15 ± 0.33 1.89 ± 0.15 60 3.19 ± 0.45 2.01 ± 0.15 3.15 ± 0.33 1.89 ± 0.14 90 3.18 ± 0.57 2.01 ± 0.13 3.15 ± 0.40 1.90 ± 0.15 180 3.19 ± 0.34 2.02 ± 0.17 3.15 ± 0.31 1.90 ± 0.13 360 3.19 ± 0.45 2.01 ± 0.15 3.16 ± 0.35 1.90 ± 0.18 450 3.20 ± 0.44 2.02 ± 0.15 3.16 ± 0.36 1.90 ± 0.15 540 3.20 ± 0.41 2.02 ± 0.11 3.16 ± 0.32 1.91 ± 0.17 630 3.20 ± 0.53 2.03 ± 0.16 3.16 ± 0.30 1.91 ± 0.16 720 3.20 ± 0.46 2.03 ± 0.14 3.16 ± 0.34 1.91 ± 0.13 Day 450 to 540, day 540 to 630, and day 630 to 720 were selected as observation time points, and the subjects’ data were included in the linear regression formula to calculate the mean daily basal loss of iron (i.e., physiological requirement). The mean physiological requirement for iron was 16.88 ± 7.12 μg/kg per day in boys and 18.40 ± 8.81 μg/kg per day in girls, calculated from day 450 to 720. The physiological requirement for iron was significantly lower in boys than in girls (P < 0.01) (Table 4). Based on the mean weight of 20 boys (42.8 ± 6.9 kg) and 17 girls (38.5 ± 7.0 kg) measured from day 450 to 720, the physiological requirement for iron in boys and girls was calculated as 722.46 ± 8.43 and 708.40 ± 7.55 μg/day, respectively.
Table 4. Physiologic requirement for iron calculated by different time points (μg/kg per day)
Gender n Day 450 to 540 Day 540 to 630 Day 630 to 720 Day 450 to 720 Boys 20 16.75 ± 6.72 17.14 ± 6.20 16.95 ± 6.94 16.88 ± 7.12 Girls 17 18.11 ± 7.05 18.46 ± 8.07 18.50 ± 6.85 18.40 ± 8.81 t 2.993 2.465 3.017 2.894 P < 0.01 < 0.01 < 0.01 < 0.01 According to the mean physiological requirements for iron, we also determined the estimated average requirement, which was calculated using the standard weight (35.5 kg for boys and 34 kg for girls) and the iron absorption rate (about 10%) for a 10-year-old child. This value was 6.0 mg/day for boys and 6.2 mg/day for girls.
doi: 10.3967/bes2022.090
Estimation of Iron Physiological Requirement in Chinese Children using Single Stable Isotope Tracer Technique
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Abstract:
Objective This study is to obtain precise data on iron physiological requirements in Chinese children using single stable isotope tracer technique. Methods Thirty boys (10.6 ± 0.2 years) and 27 girls (10.4 ± 0.2 years) were received oral 6 mg 57Fe each day for 5 consecutive days. Venous blood samples were subsequently drawn to examine the change of total iron concentration and 57Fe abundance at day 0, 14, 28, 60, 90, 180, 360, 450, 540, 630, 720. The iron physiological requirement was calculated by iron loss combined with iron circulation rate once 57Fe abundance stabilized in human body. Results The iron physiological requirement was significantly lower in boys than those values in girls (16.88 ± 7.12 vs. 18.40 ± 8.81 μg/kg per day, P < 0.05). Correspondingly, the values were calculated as 722.46 ± 8.43 μg/day for boys and 708.40 ± 7.55 μg/day for girls, respectively. Considering nearly 10% iron absorption rate, the estimated average iron physiological requirement was 6.0 mg/day in boys and 6.2 mg/day in girls. Conclusion This study indicate that iron physiological requirement could require more daily iron intake in girls as compare with the values in boys having the same body weight. These findings would be facilitate to the new revised dietary reference intakes. -
Table 1. Baseline characteristics of the subjects
Gender n Age
(years)BMI
(kg/cm2)Hb
(g/L)SF
(ng/mL)57Fe given orally
(mg)Boys 30 10.6 ± 0.2 19.2 ± 4.4 133.1 ± 4.9 15.0 ± 5.1 27.5 ± 0.3 Girls 27 10.4 ± 0.2 17.9 ± 2.6 130.8 ± 6.1 14.4 ± 4.8 27.3 ± 0.2 Note. BMI: stands for body mass index; Hb: stands for hemoglobin; SF: stands for serum transferrin. Table 2. P values of polynomial contrast by repeated measure analysis
Ln (57Fe) Time_1 Time_2 Start (day) 14 < 0.0001 0.0089 End (day) 720 Start (day) 60 < 0.0001 0.0238 End (day) 720 Start (day) 90 < 0.0001 0.0387 End (day) 720 Start (day) 180 0.0066 0.0440 End (day) 720 Start (day) 360 0.0289 0.0506 End (day) 720 Start (day) 450 0.0723 0.1980 End (day) 720 Start (day) 540 0.115 0.3720 End (day) 720 Note. Time_N represents the nth degree polynomial contrast for time. Table 3. The blood volume and iron content from day 14 to 720
Days Boys (n = 20) Girls (n = 17) BV (mL) Fecirc (g) BV (mL) Fecirc (g) 14 3.19 ± 0.41 2.01 ± 0.12 3.15 ± 0.29 1.89 ± 0.15 28 3.19 ± 0.38 2.00 ± 0.15 3.15 ± 0.33 1.89 ± 0.15 60 3.19 ± 0.45 2.01 ± 0.15 3.15 ± 0.33 1.89 ± 0.14 90 3.18 ± 0.57 2.01 ± 0.13 3.15 ± 0.40 1.90 ± 0.15 180 3.19 ± 0.34 2.02 ± 0.17 3.15 ± 0.31 1.90 ± 0.13 360 3.19 ± 0.45 2.01 ± 0.15 3.16 ± 0.35 1.90 ± 0.18 450 3.20 ± 0.44 2.02 ± 0.15 3.16 ± 0.36 1.90 ± 0.15 540 3.20 ± 0.41 2.02 ± 0.11 3.16 ± 0.32 1.91 ± 0.17 630 3.20 ± 0.53 2.03 ± 0.16 3.16 ± 0.30 1.91 ± 0.16 720 3.20 ± 0.46 2.03 ± 0.14 3.16 ± 0.34 1.91 ± 0.13 Table 4. Physiologic requirement for iron calculated by different time points (μg/kg per day)
Gender n Day 450 to 540 Day 540 to 630 Day 630 to 720 Day 450 to 720 Boys 20 16.75 ± 6.72 17.14 ± 6.20 16.95 ± 6.94 16.88 ± 7.12 Girls 17 18.11 ± 7.05 18.46 ± 8.07 18.50 ± 6.85 18.40 ± 8.81 t 2.993 2.465 3.017 2.894 P < 0.01 < 0.01 < 0.01 < 0.01 -
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