儿童铁吸收和利用率研究

发布时间：2018-01-25 10:52

本文关键词： 儿童铁表观吸收率校正吸收率稳定性同位素多接收电感耦合等离子体质谱法利用率红细胞掺入率粪便监测法代谢平衡法血容量　出处：《中国疾病预防控制中心》2012年博士论文　论文类型：学位论文

【摘要】：目的 1稳定同位素铁口服剂的制备及样品中同位素检测方法的建立 (1)利用稳定同位素.57Fe纯品为原料,合成硫酸亚铁口服制剂,为后续铁吸收和利用率研究提供基础； (2)利用多接收电感耦合等离子质谱技术,建立生物样品中稳定同位素铁的检测方法,并进行相关质控评价。 2儿童铁吸收率评价研究 (1)利用稳定同位素示踪技术,以粪便监测法对铁的表观吸收率进行评价,并对吸收率与性别相关性进行探讨； (2)以代谢平衡法测定膳食条件下儿童铁的吸收率； (3)探讨稳定同位素示踪剂与稀土元素的相关性。 3稳定同位素评价儿童铁利用率研究 (1)以稳定同位素示踪技术评价铁的全血利用率,并探讨不同性别铁全血利用率的差异； (2)根据受试者铁利用率情况,对受试者14天红细胞掺入率进行探讨,并比较不同性别受试者的掺入率差异； (3)探讨受试者全血和红细胞中57Fe丰度的变化情况,为获取其生理需要量数据提供基础； (4)采用两种方法计算受试者血容量,并对其计算结果进行比较,以获取最适宜的血容量计算法。方法 1稳定同位素铁口服剂的制备及样品中同位素检测方法的建立 1.1稳定同位素铁口服制剂的制备采用进口纯品稳定同位素57Fe(富集度为94.693%)、稀硫酸为主要原料,以无机化学反应Fe+H2SO4→FeSO4+H2↑为基本反应原理合成57FeS04。先以普通铁粉行模拟试验,通过对FeSO4溶液稳定性、回收率试验和pH值等进行测定,计算最佳稀硫酸用量。以模拟试验得出的硫酸用量与57Fe进行化合,在测定57FeSO4浓度、回收率和pH值后,加入DyCl3和VC,并对其口味进行适度调整,灌装后高压灭菌处理。 1.2稳定同位素57Fe质谱分析方法的建立模拟人血液样品中的无机成分,配置相应的混合标准溶液,以AG MP-1阴离子树脂对Fe进行分离,以Φ3×100(mm)石英交换柱对混合标准溶液进行分离,采用四极杆质谱(ICP-QMS)测量分离所得洗脱液,以达到有效地将铁与其它杂质元素分离；采用IRMM014铁同位素标准物质对测定结果进行校正,在同位素和稀土元素测定前,调谐仪器至最佳工作参数,消除测定环境中常见的同量异位素和多原子离子干扰,以及未知基体干扰。结合人体试验样品浓度,以100μg/L的铁标准应用液和实际试验样品进行IR分析方法的探索。 2儿童铁吸收率评价研究选择10-12岁学生60名进行人体代谢试验,共14天。所有受试者分5天10餐口服稳定同位素示踪铁剂30mg、稀土元素镝3.0mmg,于早、晚餐前30mmin服用,并分别于第1日早餐前和第11日晚餐后口服2粒卡红胶囊,收集代谢期内含示踪剂和稀土元素的全部粪样。代谢期内粪便样品经冷冻干燥、粉碎和硝酸微波消解后,以原子吸收法测定粪便和膳食样品中铁总量,ICP-QMS法测定稀上元素镝的浓度,MC-ICP-MS法测定样品中57Fe/56Fe比值。以粪便监测法计算铁的表观吸收率,根据稀土元素镝的回收率进行校正,并以代谢平衡法计算膳食条件下铁的吸收率。 3稳定同位素评价儿童铁利用率研究男、女性受试者分别于口服稳定同位素铁前和服用后第14天取血并分离全血和红细胞样品,经微波消解后以原子吸收测定总铁浓度,以MC-ICP-MS测定样品中57Fe/56Fe比值。以体成分仪测定受试者总体水量,分别依据受试者的体重和总体水进行血容量推算,并比较受试者经两种方法计算的血容量差异。以全血、红细胞样品中总铁浓度、57Fe/56Fe比值和血容量等数据,计算男、女性受试者57Fe的全血利用率和红细胞掺入率,并对不同性别受试者的全血利用率和红细胞掺入率进行比较。以男、女性受试者第0(基线血)、14、28和90天全血和红细胞中57Fe/56Fe比值,分析样品中的57Fe丰度变化情况。结果 1稳定同位素铁口服剂的制备及样品中同位素检测方法的建立 1.1稳定同位素铁口服制剂的制备合成后的57FeSO4溶液经平行测定6次,回收率为96.60±1.48%,溶液经原子吸收测定浓度为2.68±0.12mg/mL,回收率和浓度符合要求。溶液静置3天内铁元素含量无明显改变,RSD%均8%,且无沉淀现象。经高压蒸汽灭菌锅121℃,20min灭菌后,在培养皿中培养,所有样品均为阴性,微生物学指标合格,可作为口服制剂应用于后续研究。 1.2稳定同位素57Fe质谱分析方法的建立以100μg/L铁标准溶液中行回收率测定,其回收率为102%。在人血液分离实验中,以血液标准物中铁的理论含量做参照,铁元素的回收率达106.49%,回收情况良好。标准品中57Fe/56Fe比值的短期和长期RSD%分别为0.15%和0.24%,代谢样品和血液样品中57Fe/56Fe比值的短期和长期RSD%分别为0.18%和0.26%,稳定性良好,精密度均达到分析要求。以ICP-MS测定Dy标准品溶液和样品中回收率均在98%以上,RSD%3%。 2儿童铁吸收率评价研究以同位素示踪法计算得出的男、女性受试者铁表观吸收率分别为26.71±2.94%和29.76±2.20%,女性表观吸收率显著高于男性,(P0.05),稀土元素Dy平均回收率分别为93.91±8.44%和94.24±6.98%,以Dy回收率校正后铁表观吸收率分别为27.45±2.83%和31.01±2.48%(P0.05)；以代谢平衡法计算得到铁的表观吸收率分别为8.05±0.90%和9.13±0.57%。57Fe与Dy在粪便排泄上均呈正态分布,二者具有良好的相关性(r=0.745,P0.01)。 3稳定同位素评价儿童铁利用率研究利用稳定同位素示踪法计算得出的男、女性受试者14天铁全血利用率分别为20.4+2.0%和22.0+0.8%(Hawkins血容量推算法)、20.4+2.0%和22.0+0.8%(TBW血容量推算法),男性受试者利用率略低于女性受试者,且差异具有统计学意义(P0.05)；男、女性受试者14天红细胞掺入率分别为88.66±2.93%和93.89±2.27%(Hawkins血容量推算法)、90.7±3.12%和91.9±2.63%(TBW血容量推算法),男性受试者的红细胞掺入率略低于女性受试者,且差异具有统计学意义(P0.05)；依据体重和总体水量推算得出的男性BV分别为3.19±0.41L和3.31±0.34L,两种方法计算无显著性差异(P0.05),女性BV分别为3.15±0.29L和3.13±0.20L,两种方法计算结果无显著性差异(P0.05)。男、女性受试者全血中57Fe/56Fe比值均呈现先迅速升高,28天后缓慢降低的趋势,而红细胞中57Fe/56Fe比值在升高后,至第90天才逐渐降低。小结 1稳定同位素铁口服剂的制备及样品中同位素检测方法的建立 1.1稳定同位素铁口服制剂的制备以稳定同位素铁纯品或氧化物为原料,进行硫酸盐的实验室转化,方法安全可靠、操作简便、回收率高。是对同位素口服制剂合成的有力探索,对今后国内该领域有重要的借鉴意义。 1.2稳定同位素57Fe质谱分析方法的建立 (1)本研究采用的MC-ICP-MS和ICP-QMS技术能够较好的消除各种干扰源对数据分析的影响。 (2)本研究以MC-ICP-MS技术建立了生物样品中稳定同位素57Fe/56Fe测定方法,且具较高的精密度和稳定性。 (3)本研究以ICP-QMS技术建立了粪便样品中稀土元素Dy含量的测定方法,具有较高的稳定性和回收率。 2儿童铁吸收率评价研究 (1)稀土元素Dy在消化道中移行的的过程与铁非常相近,可以其回收率作为校正评价粪便回收完整度和铁的吸收率。本研究中稀土元素Dy回收较为完整,在95%-105%范围内。 (2)男、女性受试者空腹服用稳定同位素57Fe吸收率均较高,在25%-35%左右,女性受试者校正后铁吸收率显著高于男性受试者,且差异具有统计学意义。 (3)代谢平衡法测定膳食条件下男、女性受试者吸收率均小于10%,且男性显著低于女性。 3稳定同位素评价儿童铁利用率应用研究 (1)利用餐前大剂量服用稳定同位素57Fe的方法探讨铁的利用率和红细胞掺入率,方法可行,数据可靠,为相关人群的生理需要量研究奠定了基础。 (2)利用稳定同位素57Fe示踪法发现,男性受试者铁的全血利用率和红细胞掺入率均显著低于女性。 (3)男、女性受试者体内全血样品中57Fe/56Fe比值均呈现出先迅速升高,随后缓慢降低的趋势,峰值出现在14-28天左右；而RBC样品中57Fe/56Fe比值在14-28天出现峰值后,升高幅度缓慢,至90天时才呈现下降趋势。 (4)铁的利用率、红细胞掺入率与体内铁的储存状态有关,并与血清铁蛋白含量呈负相关。 (5)两种方法(根据体重和TBW)推算出的血容量结果准确且相近,但根据TBW推算的结果更符合不同体脂构成的人群。
[Abstract]:objective
The preparation of 1 stable isotopic iron oral agent and the establishment of isotopic detection method in the sample
(1) making use of stable isotopes.57Fe as raw materials to synthesize oral ferrous sulfate, which provides a basis for the study of subsequent iron absorption and utilization.
(2) the detection method of stable isotopic iron in biological samples was established by multi receiving inductively coupled plasma mass spectrometry (ICP MS), and the related quality control was evaluated.
Study on the evaluation of iron absorptivity in 2 children
(1) using the stable isotope tracer technique, the apparent absorptivity of iron was evaluated by the stool monitoring method, and the correlation between the absorption rate and the sex was discussed.
(2) the metabolic balance method was used to determine the absorption rate of iron in children under dietary conditions.
(3) the study of the correlation between stable isotope tracers and rare earth elements.
Study on the evaluation of iron utilization rate in children by 3 stable isotopes
(1) the whole blood utilization rate of iron was evaluated with stable isotope tracer technique, and the difference of whole blood utilization rate of different same-sex iron was discussed.
(2) according to the iron utilization rate of the subjects, the rate of erythrocyte incorporation of the subjects for 14 days was discussed, and the difference in the incorporation rate of the subjects of different sexes was compared.
(3) to investigate the changes in the abundance of 57Fe in the whole blood and red blood cells of the subjects, and provide the basis for obtaining the data of their physiological needs.
(4) the blood volume of the subjects was calculated by two methods, and the results were compared in order to obtain the most suitable method of blood volume calculation.
Method
The preparation of 1 stable isotopic iron oral agent and the establishment of isotopic detection method in the sample
Preparation of 1.1 stable isotopic iron oral preparation
Imported pure product of stable isotope 57Fe (enrichment of 94.693%), dilute sulfuric acid as the main raw material, the basic principle of reaction synthesis of 57FeS04. with ordinary iron line simulation test on the inorganic chemical reaction of Fe+H2SO4, FeSO4+H2 increase, through the stability of FeSO4 solution, recovery test and pH value were measured to calculate the optimal amount of dilute sulfuric acid. Such as the amount of sulfuric acid and 57Fe were obtained in the simulation experiment, the concentration of 57FeSO4, the recovery rate and pH value after adding DyCl3 and VC, and the appropriate adjustment to the taste, after filling high-pressure sterilization.
The establishment of 1.2 stable isotope 57Fe mass spectrometry
Simulation of inorganic constituents in blood samples of the people, the allocation of mixed standard solution corresponding to the separation of the Fe AG MP-1 anion exchange resin, to Phi 3 * 100 (mm) of quartz exchange column mixed standard solution were separated by quadrupole mass spectrometry (ICP-QMS) measurements separated from the eluent, in order to effectively and iron other impurity elements separated by IRMM014; iron isotope reference material to correct the measurement results, the isotope and REE was measured by the instrument tuning to the best working parameters, eliminate the determination of environmental hormone and isobaric common multiple primary ion interference, and unknown matrix interference. Combined with the human test sample concentration, to explore the method of IR analysis the application of liquid iron standard 100 g/L and the actual test samples.
Study on the evaluation of iron absorptivity in 2 children
Selection of 60 10-12 years old children of human metabolic test, a total of 14 days. All the subjects were divided into 5 day 10 meals oral iron stable isotope tracer 30mg, dysprosium, 3.0mmg, in the early, taking 30mmin before dinner, and on the first day, before breakfast and eleventh supper after oral administration of 2 grains of red card collection capsule. Metabolic stage intrinsic tracer and rare earth elements in all fecal samples. The metabolic period of fecal samples by freeze drying, crushing and nitrate after microwave digestion, determination of fecal samples and dietary total by atomic absorption spectrometry, determination of the concentration of the rare earth element dy ICP-QMS method, determination of the ratio of 57Fe/56Fe MC-ICP-MS in the sample. By fecal monitoring method of iron the apparent absorption rate, according to the recovery of rare earth elements dysprosium was corrected and metabolic balance method to calculate the iron absorption of dietary conditions.
Study on the evaluation of iron utilization rate in children by 3 stable isotopes
Male and female subjects were orally before and after taking iron isotope fourteenth days of blood and the separation of whole blood and erythrocyte samples after microwave digestion by atomic absorption determination of total iron concentration, as measured by the MC-ICP-MS 57Fe/56Fe ratio in the sample. The subjects were measured with the overall water body composition analyzer, respectively according to the subjects the body weight and overall water blood volume projections, blood volume and compare subjects by two methods. In whole blood, the total iron concentration of red blood cells in the sample, the ratio of 57Fe/56Fe and blood volume data, calculation of male, female subjects 57Fe blood utilization rate and erythrocyte incorporation rate. According to different sex by the utilization rate were compared between subjects with red blood cells and whole blood. The male, female subjects (zeroth 14,28, 57Fe/56Fe ratio of baseline blood) and 90 days of whole blood and red blood cells, analysis of 57Fe abundance variation in the sample.
Result
The preparation of 1 stable isotopic iron oral agent and the establishment of isotopic detection method in the sample
Preparation of 1.1 stable isotopic iron oral preparation
After the synthesis of 57FeSO4 solution by 6 determinations, the recovery rate was 96.60 + 1.48%, the solution by atomic absorption determination of concentration was 2.68 + 0.12mg/mL, recovery rate and concentration of solution to meet the requirements. The static iron content within 3 days no significant change of RSD% was 8%, and no precipitation phenomenon by high pressure steam sterilizer. At 121 20min after sterilization, cultured in a dish, all the samples were negative, microbiological indicators qualified, can be used as an oral formulation in the follow-up study.
The establishment of 1.2 stable isotope 57Fe mass spectrometry
The Bank of China 100 g/L iron standard solution the recovery rate measurement, the recovery rate is 102%. in human blood separation experiment, the content of iron in blood theory as reference standard, the iron recovery rate was 106.49%, the recovery in good condition. The short-term and long-term RSD% standard in 57Fe/56Fe ratio were 0.15% and 0.24%, short-term the RSD% 57Fe/56Fe ratio and long-term metabolic samples and blood samples were 0.18% and 0.26%, good stability and precision can meet the requirements of analysis. Determination of Dy standard solution and the sample recovery rate was above 98% in ICP-MS, RSD%3%.
Study on the evaluation of iron absorptivity in 2 children
The isotope tracer method calculated between male and female subjects the apparent iron absorption rate were 26.71 + 2.94% and 29.76 + 2.20%, female apparent absorption rate was significantly higher than that of male (P0.05), rare earth element Dy and the average recovery rate was 93.91 + 8.44% and 94.24 + 6.98%, the recovery rate of Dy positive after school the apparent iron absorption rate were 27.45 + 2.83% and 31.01 + 2.48% (P0.05); with metabolic balance method is used to calculate the apparent absorption rate of iron were 8.05 + 0.90% and 9.13 + 0.57%.57Fe and Dy in feces were normal distribution, has a good correlation between the two (r=0.745, P0.01).
Study on the evaluation of iron utilization rate in children by 3 stable isotopes
Calculated using stable isotope tracer method of male and female subjects 14 blood utilization rate were 20.4+2.0% iron and 22.0+0.8% (Hawkins algorithm, 20.4+2.0% blood volume) and 22.0+0.8% (TBW blood volume calculation method), male subjects using rate is slightly lower than the female subjects, and the difference was statistically significant (P0.05) male and female subjects; 14 days erythrocyte incorporation rate were 88.66 + 2.93% and 93.89 + 2.27% (Hawkins blood volume calculation method), 90.7 + 3.12% and 91.9 + 2.63% (TBW blood volume calculation method), red blood cells with male subjects into rate slightly lower than female subjects. And the difference was statistically significant (P0.05); according to the calculated weight and total content of male BV were 3.19 + 0.41L and 3.31 + 0.34L calculation, no significant difference between the two methods (P0.05), female BV were 3.15 + 0.29L and 3.13 + 0.20L, the results of the two methods had no significant difference (P0.05) the ratio of 57Fe/56Fe in the whole blood of male and female subjects increased first and then decreased slowly after 28 days, while the 57Fe/56Fe ratio in red blood cells increased gradually to ninetieth days.
Summary
The preparation of 1 stable isotopic iron oral agent and the establishment of isotopic detection method in the sample
Preparation of 1.1 stable isotopic iron oral preparation
Taking stable isotopes, iron or pure oxide or oxide as raw materials, the laboratory transformation of sulfate is safe, reliable, easy to operate, and high recovery. It is a powerful exploration for the synthesis of isotopic oral preparations, and has important reference significance for the future domestic field.
The establishment of 1.2 stable isotope 57Fe mass spectrometry
(1) the MC-ICP-MS and ICP-QMS techniques used in this study can better eliminate the impact of various interference sources on data analysis.
(2) a method for the determination of stable isotope 57Fe/56Fe in biological samples with high precision and stability was established by MC-ICP-MS technology.
(3) a method for the determination of Dy content of rare earth elements in fecal samples with high stability and recovery was established by ICP-QMS technology in this study.
Study on the evaluation of iron absorptivity in 2 children
(1) the migration process of Dy in the digestive tract is very similar to that of iron. The recovery rate can be used as a correction to evaluate the integrity of feces recovery and the absorption rate of iron. In this study, the Dy recovery of rare-earth elements is relatively complete, in the range of 95%-105%.
(2) male and female subjects had high 57Fe absorption rate on fasting stable isotope. At 25%-35%, the iron absorption rate of female subjects was significantly higher than that of male subjects, and the difference was statistically significant.
(3) the metabolic balance method was used to determine the male in the diet, the absorption rate of the female subjects was less than 10%, and the male was significantly lower than that of the female.
Application of 3 stable isotopes in evaluating the utilization rate of iron in children
(1) to explore the utilization rate of iron and the rate of red blood cell incorporation before taking large doses of stable isotope 57Fe before meal. The method is reliable and reliable, which lays a foundation for the physiological needs of the related population.
(2) using the stable isotope 57Fe tracer method, it was found that the total blood utilization rate and the incorporation rate of red blood cells in male subjects were significantly lower than those of women.
(3) male, female subjects 57Fe/56Fe ratio showed first increased rapidly in vivo blood samples, then decreased slowly, the peak appeared in 14-28 days; while the ratio of 57Fe/56Fe in RBC samples peaked at 14-28, increased slowly to 90 days is decreased.
(4) the utilization of iron, the rate of erythrocyte incorporation is related to the storage state of iron in the body, and has a negative correlation with the content of serum ferritin.
(5) the results of the two methods (based on weight and TBW) were accurate and similar, but the results calculated by the TBW were more consistent with people with different body fat.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R153.2

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