本文關(guān)鍵詞： 兒童 鐵 表觀吸收率 校正吸收率 穩(wěn)定性同位素 多接收電感耦合等離子體質(zhì)譜法 利用率 紅細(xì)胞摻入率 糞便監(jiān)測(cè)法 代謝平衡法 血容量　出處：《中國(guó)疾病預(yù)防控制中心》2012年博士論文　論文類型：學(xué)位論文

【摘要】：目的 1穩(wěn)定同位素鐵口服劑的制備及樣品中同位素檢測(cè)方法的建立 (1)利用穩(wěn)定同位素.57Fe純品為原料,合成硫酸亞鐵口服制劑,為后續(xù)鐵吸收和利用率研究提供基礎(chǔ)； (2)利用多接收電感耦合等離子質(zhì)譜技術(shù),建立生物樣品中穩(wěn)定同位素鐵的檢測(cè)方法,并進(jìn)行相關(guān)質(zhì)控評(píng)價(jià)。 2兒童鐵吸收率評(píng)價(jià)研究 (1)利用穩(wěn)定同位素示蹤技術(shù),以糞便監(jiān)測(cè)法對(duì)鐵的表觀吸收率進(jìn)行評(píng)價(jià),并對(duì)吸收率與性別相關(guān)性進(jìn)行探討； (2)以代謝平衡法測(cè)定膳食條件下兒童鐵的吸收率； (3)探討穩(wěn)定同位素示蹤劑與稀土元素的相關(guān)性。 3穩(wěn)定同位素評(píng)價(jià)兒童鐵利用率研究 (1)以穩(wěn)定同位素示蹤技術(shù)評(píng)價(jià)鐵的全血利用率,并探討不同性別鐵全血利用率的差異； (2)根據(jù)受試者鐵利用率情況,對(duì)受試者14天紅細(xì)胞摻入率進(jìn)行探討,并比較不同性別受試者的摻入率差異； (3)探討受試者全血和紅細(xì)胞中57Fe豐度的變化情況,為獲取其生理需要量數(shù)據(jù)提供基礎(chǔ)； (4)采用兩種方法計(jì)算受試者血容量,并對(duì)其計(jì)算結(jié)果進(jìn)行比較,以獲取最適宜的血容量計(jì)算法。 方法 1穩(wěn)定同位素鐵口服劑的制備及樣品中同位素檢測(cè)方法的建立 1.1穩(wěn)定同位素鐵口服制劑的制備 采用進(jìn)口純品穩(wěn)定同位素57Fe(富集度為94.693%)、稀硫酸為主要原料,以無(wú)機(jī)化學(xué)反應(yīng)Fe+H2SO4→FeSO4+H2↑為基本反應(yīng)原理合成57FeS04。先以普通鐵粉行模擬試驗(yàn),通過對(duì)FeSO4溶液穩(wěn)定性、回收率試驗(yàn)和pH值等進(jìn)行測(cè)定,計(jì)算最佳稀硫酸用量。以模擬試驗(yàn)得出的硫酸用量與57Fe進(jìn)行化合,在測(cè)定57FeSO4濃度、回收率和pH值后,加入DyCl3和VC,并對(duì)其口味進(jìn)行適度調(diào)整,灌裝后高壓滅菌處理。 1.2穩(wěn)定同位素57Fe質(zhì)譜分析方法的建立 模擬人血液樣品中的無(wú)機(jī)成分,配置相應(yīng)的混合標(biāo)準(zhǔn)溶液,以AG MP-1陰離子樹脂對(duì)Fe進(jìn)行分離,以Φ3×100(mm)石英交換柱對(duì)混合標(biāo)準(zhǔn)溶液進(jìn)行分離,采用四極桿質(zhì)譜(ICP-QMS)測(cè)量分離所得洗脫液,以達(dá)到有效地將鐵與其它雜質(zhì)元素分離；采用IRMM014鐵同位素標(biāo)準(zhǔn)物質(zhì)對(duì)測(cè)定結(jié)果進(jìn)行校正,在同位素和稀土元素測(cè)定前,調(diào)諧儀器至最佳工作參數(shù),消除測(cè)定環(huán)境中常見的同量異位素和多原子離子干擾,以及未知基體干擾。結(jié)合人體試驗(yàn)樣品濃度,以100μg/L的鐵標(biāo)準(zhǔn)應(yīng)用液和實(shí)際試驗(yàn)樣品進(jìn)行IR分析方法的探索。 2兒童鐵吸收率評(píng)價(jià)研究 選擇10-12歲學(xué)生60名進(jìn)行人體代謝試驗(yàn),共14天。所有受試者分5天10餐口服穩(wěn)定同位素示蹤鐵劑30mg、稀土元素鏑3.0mmg,于早、晚餐前30mmin服用,并分別于第1日早餐前和第11日晚餐后口服2粒卡紅膠囊,收集代謝期內(nèi)含示蹤劑和稀土元素的全部糞樣。代謝期內(nèi)糞便樣品經(jīng)冷凍干燥、粉碎和硝酸微波消解后,以原子吸收法測(cè)定糞便和膳食樣品中鐵總量,ICP-QMS法測(cè)定稀上元素鏑的濃度,MC-ICP-MS法測(cè)定樣品中57Fe/56Fe比值。以糞便監(jiān)測(cè)法計(jì)算鐵的表觀吸收率,根據(jù)稀土元素鏑的回收率進(jìn)行校正,并以代謝平衡法計(jì)算膳食條件下鐵的吸收率。 3穩(wěn)定同位素評(píng)價(jià)兒童鐵利用率研究 男、女性受試者分別于口服穩(wěn)定同位素鐵前和服用后第14天取血并分離全血和紅細(xì)胞樣品,經(jīng)微波消解后以原子吸收測(cè)定總鐵濃度,以MC-ICP-MS測(cè)定樣品中57Fe/56Fe比值。以體成分儀測(cè)定受試者總體水量,分別依據(jù)受試者的體重和總體水進(jìn)行血容量推算,并比較受試者經(jīng)兩種方法計(jì)算的血容量差異。以全血、紅細(xì)胞樣品中總鐵濃度、57Fe/56Fe比值和血容量等數(shù)據(jù),計(jì)算男、女性受試者57Fe的全血利用率和紅細(xì)胞摻入率,并對(duì)不同性別受試者的全血利用率和紅細(xì)胞摻入率進(jìn)行比較。以男、女性受試者第0(基線血)、14、28和90天全血和紅細(xì)胞中57Fe/56Fe比值,分析樣品中的57Fe豐度變化情況。 結(jié)果 1穩(wěn)定同位素鐵口服劑的制備及樣品中同位素檢測(cè)方法的建立 1.1穩(wěn)定同位素鐵口服制劑的制備 合成后的57FeSO4溶液經(jīng)平行測(cè)定6次,回收率為96.60±1.48%,溶液經(jīng)原子吸收測(cè)定濃度為2.68±0.12mg/mL,回收率和濃度符合要求。溶液靜置3天內(nèi)鐵元素含量無(wú)明顯改變,RSD%均8%,且無(wú)沉淀現(xiàn)象。經(jīng)高壓蒸汽滅菌鍋121℃,20min滅菌后,在培養(yǎng)皿中培養(yǎng),所有樣品均為陰性,微生物學(xué)指標(biāo)合格,可作為口服制劑應(yīng)用于后續(xù)研究。 1.2穩(wěn)定同位素57Fe質(zhì)譜分析方法的建立 以100μg/L鐵標(biāo)準(zhǔn)溶液中行回收率測(cè)定,其回收率為102%。在人血液分離實(shí)驗(yàn)中,以血液標(biāo)準(zhǔn)物中鐵的理論含量做參照,鐵元素的回收率達(dá)106.49%,回收情況良好。標(biāo)準(zhǔn)品中57Fe/56Fe比值的短期和長(zhǎng)期RSD%分別為0.15%和0.24%,代謝樣品和血液樣品中57Fe/56Fe比值的短期和長(zhǎng)期RSD%分別為0.18%和0.26%,穩(wěn)定性良好,精密度均達(dá)到分析要求。以ICP-MS測(cè)定Dy標(biāo)準(zhǔn)品溶液和樣品中回收率均在98%以上,RSD%3%。 2兒童鐵吸收率評(píng)價(jià)研究 以同位素示蹤法計(jì)算得出的男、女性受試者鐵表觀吸收率分別為26.71±2.94%和29.76±2.20%,女性表觀吸收率顯著高于男性,(P0.05),稀土元素Dy平均回收率分別為93.91±8.44%和94.24±6.98%,以Dy回收率校正后鐵表觀吸收率分別為27.45±2.83%和31.01±2.48%(P0.05)；以代謝平衡法計(jì)算得到鐵的表觀吸收率分別為8.05±0.90%和9.13±0.57%。57Fe與Dy在糞便排泄上均呈正態(tài)分布,二者具有良好的相關(guān)性(r=0.745,P0.01)。 3穩(wěn)定同位素評(píng)價(jià)兒童鐵利用率研究 利用穩(wěn)定同位素示蹤法計(jì)算得出的男、女性受試者14天鐵全血利用率分別為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),男性受試者利用率略低于女性受試者,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；男、女性受試者14天紅細(xì)胞摻入率分別為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),男性受試者的紅細(xì)胞摻入率略低于女性受試者,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；依據(jù)體重和總體水量推算得出的男性BV分別為3.19±0.41L和3.31±0.34L,兩種方法計(jì)算無(wú)顯著性差異(P0.05),女性BV分別為3.15±0.29L和3.13±0.20L,兩種方法計(jì)算結(jié)果無(wú)顯著性差異(P0.05)。男、女性受試者全血中57Fe/56Fe比值均呈現(xiàn)先迅速升高,28天后緩慢降低的趨勢(shì),而紅細(xì)胞中57Fe/56Fe比值在升高后,至第90天才逐漸降低。 小結(jié) 1穩(wěn)定同位素鐵口服劑的制備及樣品中同位素檢測(cè)方法的建立 1.1穩(wěn)定同位素鐵口服制劑的制備 以穩(wěn)定同位素鐵純品或氧化物為原料,進(jìn)行硫酸鹽的實(shí)驗(yàn)室轉(zhuǎn)化,方法安全可靠、操作簡(jiǎn)便、回收率高。是對(duì)同位素口服制劑合成的有力探索,對(duì)今后國(guó)內(nèi)該領(lǐng)域有重要的借鑒意義。 1.2穩(wěn)定同位素57Fe質(zhì)譜分析方法的建立 (1)本研究采用的MC-ICP-MS和ICP-QMS技術(shù)能夠較好的消除各種干擾源對(duì)數(shù)據(jù)分析的影響。 (2)本研究以MC-ICP-MS技術(shù)建立了生物樣品中穩(wěn)定同位素57Fe/56Fe測(cè)定方法,且具較高的精密度和穩(wěn)定性。 (3)本研究以ICP-QMS技術(shù)建立了糞便樣品中稀土元素Dy含量的測(cè)定方法,具有較高的穩(wěn)定性和回收率。 2兒童鐵吸收率評(píng)價(jià)研究 (1)稀土元素Dy在消化道中移行的的過程與鐵非常相近,可以其回收率作為校正評(píng)價(jià)糞便回收完整度和鐵的吸收率。本研究中稀土元素Dy回收較為完整,在95%-105%范圍內(nèi)。 (2)男、女性受試者空腹服用穩(wěn)定同位素57Fe吸收率均較高,在25%-35%左右,女性受試者校正后鐵吸收率顯著高于男性受試者,且差異具有統(tǒng)計(jì)學(xué)意義。 (3)代謝平衡法測(cè)定膳食條件下男、女性受試者吸收率均小于10%,且男性顯著低于女性。 3穩(wěn)定同位素評(píng)價(jià)兒童鐵利用率應(yīng)用研究 (1)利用餐前大劑量服用穩(wěn)定同位素57Fe的方法探討鐵的利用率和紅細(xì)胞摻入率,方法可行,數(shù)據(jù)可靠,為相關(guān)人群的生理需要量研究奠定了基礎(chǔ)。 (2)利用穩(wěn)定同位素57Fe示蹤法發(fā)現(xiàn),男性受試者鐵的全血利用率和紅細(xì)胞摻入率均顯著低于女性。 (3)男、女性受試者體內(nèi)全血樣品中57Fe/56Fe比值均呈現(xiàn)出先迅速升高,隨后緩慢降低的趨勢(shì),峰值出現(xiàn)在14-28天左右；而RBC樣品中57Fe/56Fe比值在14-28天出現(xiàn)峰值后,升高幅度緩慢,至90天時(shí)才呈現(xiàn)下降趨勢(shì)。 (4)鐵的利用率、紅細(xì)胞摻入率與體內(nèi)鐵的儲(chǔ)存狀態(tài)有關(guān),并與血清鐵蛋白含量呈負(fù)相關(guān)。 (5)兩種方法(根據(jù)體重和TBW)推算出的血容量結(jié)果準(zhǔn)確且相近,但根據(jù)TBW推算的結(jié)果更符合不同體脂構(gòu)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.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R153.2

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