【作者】 陈科；

【导师】 李廷玉；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 博士

【摘要】 第一部分人群维生素A营养状况与铁代谢稳态相关性研究目的:横断面调查学龄前儿童维生素A营养状况与铁代谢动态平衡及机体总铁含量之间的关系,并评估不同维生素A营养状况下贫血与储铁不足的患病风险。方法:利用整群抽样和分层抽样相结合的方法从巴南区鱼洞镇共20多所幼儿园随机抽取其中4所进行调查,同时问卷调查其人口学资料、社会经济状况以及饮食习惯等。利用高效液相色谱法测定血清维生素A浓度,酶联免疫法测定血清铁蛋白(serum ferritin, SF)水平、微粒子增强透射免疫法检测可溶性转铁蛋白受体(serum transferrin receptor, sTfR)水平、透射免疫比浊法测定血清C-反应蛋白(C- reactive protein, CRP)水平以及高铁氰化法测定血红蛋白(hemoglobin, HB)浓度,并计算sTfR-SF指数(TFR-F指数)以及机体总铁含量。结果:共471名学龄前儿童纳入研究,其中男童236名,女童235名,年龄范围为2～7岁,平均年龄为(4.0±0.85)(均数±标准差)岁。HB水平为(115.8±9.2)g/L ,贫血患病率为23.5 %; SF水平为(24.75±14.71)μg/L,sTfR水平为(1.28±0.33)mg/L,储铁不足发生率为45.1%。血清维生素A水平为(1.21±0.36)μmol/L,维生素A缺乏(vitamin A deficiency, VAD)患病率为6.5%,可疑亚临床维生素A缺乏( suspect sub-clinic vitamin A deficiency, SSVAD)患病率为26.0%。TFR-F指数0.9595 (0.7257,1.2226)[中位数(P25,P75)] ,机体铁含量8.868 (6.986,10.470) mg/kg。校正儿童年龄、性别、不同幼儿园来源、家庭经济状况以及膳食模式等因素后,血清维生素A与HB、SF对数浓度、sTfR水平、TFR-F指数以及机体铁含量的偏相关系数(radjust)分别是0.16(p<0.001), -0.13 (p=0.037), 0.17 (p=0.0011), -0.013 (p=0.7935)以及-0.05 (p=0.3652)。Logitic回归分析显示,VAD是导致储铁不足(以SF水平作为判断标准)的独立危险因素[优势比(odds ratio, OR)(95% CI): 1.88(1.01, 2.97)。若以TFR-F为判断指标,VAD则不是储铁不足患病危险因素[OR(95% CI): 1.365(0.286,6.513)]。VAD儿童患贫血的危险性[OR (95% CI): 2.56(1.15,5.70)]是NVA儿童贫血患病风险的2.56倍;若对SF和sTfR校正以后,VAD儿童贫血患病风险则明显下降[OR (95% CI): 1.53(1.01,1.88)]。结论:1学龄前儿童血清维生素A水平与反映机体铁储备和铁利用的生化指标密切相关,而与铁吸收及机体总铁含量无明显关系。2 VAD是储铁不足患病的独立危险因素,其可能主要通过影响铁储备和铁利用产生作用,而对铁吸收影响不大。3 VAD是贫血患病的独立危险因素,除主要影响铁代谢外,还可能通过其他途径产生作用。第二部分维生素A等多种微量营养素干预对铁代谢稳态的影响目的:观察单独维生素A补充、维生素A+铁以及多种微量营养素联合补充等干预方式对铁代谢相关生化指标的影响,并评估不同干预方式对贫血以及储铁不足发病的保护作用。方法:本研究为随机、对照、盲法现场干预试验,对象来源于第一部分研究。从该地区共20多所幼儿园中随机选取3所中的全部226名2-7岁学龄前儿童纳入计划干预对象。对其分别补充100%RDA的维生素A(Ⅰ组),维生素A加铁(Ⅱ组)以及维生素A,铁,维生素B1,核黄素,叶酸,尼克酸,锌以及钙(Ⅲ组)。干预前后分别测定利用高效液相色谱法测定血清维生素A浓度,酶联免疫法测定血清铁蛋白(serum ferritin, SF)水平、微粒子增强透射免疫法检测可溶性转铁蛋白受体(serum transferring receptor, sTfR)水平、透射免疫比浊法测定血清C-反应蛋白(C- reactive protein, CRP)水平以及高铁氰化法测定血红蛋白(hemoglobin, HB)浓度,并计算sTfR-SF指数(TFR-F指数)及机体总铁含量。同时利用问卷方式调查这些儿童的人口学治疗、社会经济状况以及饮食习惯等。结果:三种干预方式均可使HB水平均较干预前明显升高(P<0.0001),但各干预组间无显著差异(p>0.05)。干预后各组维生素A水平均有所提高,而以维生素A+铁组以及多种微量营养素补充组最为明显(p<0.05)。干预后各组SF值均较干预前明显下降(P<0.01),而维生素A+铁补充组以及多种微量营养素补充组下降最为明显(P<0.05)。干预后各组sTfR水平均出现明显降低(P<0.01),尤以单独维生素A补充组最明显(P<0.05)。单独维生素A补充组其TFR-F指数以及机体总铁含量于干预前后并无明显变化(P>0.05)。在校正了儿童性别、年龄、被动吸烟、社会经济状况以及膳食模式以后,三种干预方式对贫血发病的保护作用并无明显差异(P>0.05)。若以铁蛋白作为储铁不足乏判断的指标,三种干预方式对储铁不足保护作用也无差异(P>0.05);但若以TFR-F指数作为判定指标,则相对于单独维生素A补充而言,维生素A+铁以及多种微量营养素的联合补充是储铁不足更好的保护因素[相对危险度(relative risk, RR)(95% CI): 0.410(0.218,0.992)]。结论:1维生素A干预对铁储备及铁利用过程均有影响。2维生素A干预对TFR-F指数以及机体总铁含量无明显影响,表明维生素A对铁在肠道的吸收无明显作用。3维生素A联合铁剂等多种微量营养素的补充可能是改善储铁不足状态的较好干预方式。第三部分维生素A营养状况对铁调节模式的影响及其相关机制目的:了解不同维生素A营养状况下大鼠贫血的性质及其对铁效应元件-铁调节蛋白作用模式(iron responsive element-iron regulated protein, IRE-IRP模式)和肝调素-膜铁转运蛋白作用模式(hepcidin- ferroportin,Hepc-FPN模式)各组件在肝脏mRNA和蛋白水平表达的影响。方法:清洁级健康雌性Wistar大鼠30只随机分为3组,饲以相应饲料,3周后与健康雄性Wistar大鼠交配,1:2雌雄合笼::(1)正常维生素A饲喂组母鼠(normal vitamin A status, NVA)(10只),给予维生素A充足饲料(含维生素A 6500 U/kg),随机从10窝中每窝各抽取雌雄仔鼠各1只共计20只用于本实验,仔鼠饲料同母鼠;(2)低维生素A饲喂组母鼠(low vitamin A status, LVA)(10只),给予低维生素A饲料(含维生素A 400IU/kg) ,随机从10窝中每窝各抽取雌雄仔鼠各1只共计20只用于本实验,仔鼠饲料同母鼠,其幼鼠为维生素A缺乏鼠(vitamin A deficiency, VAD);(3)生后4周干预组母鼠(vitamin A intervention, VAI)(10只),给予低维生素A饲料饲喂至分娩,随机从10窝中每窝各抽取雌雄仔鼠各1只共计20只用于本实验,仔鼠饲料同母鼠,4周龄大小后改饲维生素A充足饲料。各组仔鼠于8周龄处死。用高效液相色谱法(HPLC)检测血清维生素A浓度,利用全自动血细胞分析仪测定全血红细胞指数,荧光定量PCR法、Western blotting法以及酶联免疫法分别测定铁调节IRE-IRP模式和Hepc-FPN模式各组件在肝脏mRNA和蛋白水平表达的水平。结果:①仔鼠在第八周时,NVA组和VAD组大鼠外周血红细胞计数、血红蛋白浓度以及红细胞压积均无显著差异(p>0.05),而VAI大鼠上述指标均显著高于前两组(p<0.05)。VAD组红细胞平均容积最低(p<0.05),NVA组和VAI组大鼠之间则无显著差异(p>0.05);VAI组大鼠红细胞分布宽度显著高于NVA组和VAD组(p<0.05),后两组该值无显著差异(p>0.05)。三个组大鼠平均血红蛋白含量组间无差异(p>0.05),而VAI组平均血红蛋白浓度则显著低于VAN组和MVAD组(p<0.05),后两组该指标之间无显著差异(p>0.05)。②Hepc-FPN模式中肝调素、骨形成蛋白-4以及白细胞介素-6在不同维生素A营养状况下其在肝脏的mRNA及蛋白水平的表达均出现差异。③IRE-IRP模式中铁调节蛋白-1、膜铁转运蛋白-1、铁蛋白轻链以及转铁蛋白受体-1在不同维生素A营养状况下在肝脏的mRNA及蛋白水平的表达均出现差异,而二价金属离子转运体-1的表达则差异不明显。结论:1维生素A缺乏时外周血主要为小细胞低色素表现,补充维生素A可使上述改变有所恢复;维生素A缺乏对平均血红蛋白量以及平均血红蛋白浓度影响不大;2不同维生素A营养状况对铁代谢Hepc-FPN模式具有明显影响,而其可能途径是从转录水平调节IL-6和BMP-4生成从而影响Hepcidin的合成的;3不同维生素A营养状况对铁代谢IRE-IRP模式具有明显影响,而其可能途径之一是通过从转录水平调节IRP-1的合成起作用的。更多还原

【Abstract】 PartⅠCorrelation between Vitamin A Nutritional Status and Iron Metabolic HomeostasisObjective: To investigate the correlation between vitamin A nutritional status of preschool children and iron metabolic homeostasis and the total body iron content, as well as to evaluate the odds ratio of anemia and deficient iron storage by vitamin A status with cross-sectional descriptive study..Subjects and methods: Preschool children in four pre-school institutions were randomly chosen from the 20 kindergartens in Yudong Town, Banan District with cluster and stratified sampling method. The total children in one institution were planed to recruitment into present study as a whole. Simultaneously, children’s demographic data, socio-economic status and eating habits, etc. were investigated by questionnaires. The concentration of serum vitamin A was measured by high-performance liquid chromatography (HPLC), serum ferritin (SF) by enzyme-linked immunosorbent assay (ELISA), serum transferring receptor (sTfR) by microparticle-enhanced immunoassay, C-reactive protein (CRP) by particle-enhanced immunoturbidimetry and hemoglobin (HB) by hemiglobincyanide. The sTfR-SF index (TFR-F index) and total body iron content were computed, respectively.Result: A total of 471 preschool children, 236 for boys and 235 for girls, were included in the cross-sectional study with age range from 2 to 7 years old with average age (4.0±0.85) (means±standard deviation) yrs. The concentrations of HB, SF, sTfR, serum vitamin A, TFR-F index and total body iron content were (115.8±9.2)g/L, (24.75±14.71)μg/L, (1.28±0.33)mg/L, (1.21±0.36)μmol/L, 0.9595(0.7257,1.2226) [media (P25, P75)] and 8.868 (6.986,10.470) mg/kg, respectively. The prevalence of anemia, deficient iron storage, vitamin A deficiency (VAD) and suspect sub-clinic vitamin A deficiency (SSVAD) were 23.5%, 45.1%, 6.5% and 26.0, respectively. After adjustment for the age, gender, different sources of kindergarten, family economic situation and dietary pattern, etc. the partial correlation coefficient (radjust) between vitamin A and HB, log SF, sTfR, TFR-F index and total body iron content were 0.16 (p<0.001), -0.13 (p=0.037), 0.17 (p=0.0011), -0.013 (p=0.7935) and -0.05 (p=0.3652), respectively. With a multiple logistic regression model, VAD was an independent risk factor for prevalence of deficient iron storage [(odds ratio, OR) (95% CI): 1.88(1.01, 2.97)] defined by SF but not a risk factor [OR (95% CI): 1.365(0.286, 6.513)] when defined by TFR-F index. Also, VAD was a independent risk factor for the prevalence of anemia [OR (95% CI): 2.56(1.15, 5.70)], however, when adjusting for SF and sTfR, the OR value decreased [OR (95% CI): 1.53(1.01,1.88)].Conclusion:1 Serum vitamin A levels of preschool children was closely correlated with biochemical index reflecting body iron reserves and mobilization, while not iron absorption and body total iron contents.2 VAD is an independent risk factor for prevalence of deficient iron storage mainly by its impact on iron reserves and mobilization, but seldom by iron absorption.3 VAD is an independent risk factor for the prevalence of anemia, mainly by its impact on iron metabolism, while there may exist other ways.PartⅡEffects of Vitamin A, Vitamin A plus Iron and Multiple Micronutrient-Fortified Seasoning Powder on Iron metabolic HomeostasisObjective: To evaluate the effect of vitamin A, vitamin A plus iron and multiple micronutrient-fortified seasoning powder on iron metabolic homeostasis, as well as to evaluate the relative risk of anemia and iron deficient disease by interventional styles.Subjects and methods: This was a randomized, control and blinded interventional field trial. A total of 226 2-7 years old preschool children were recruited from three nurseries in the area, and they were randomly assigned into three different fortified diet groups for 6 months. The Group I was fortified with vitamin A; Group II and III were fortified with vitamin A plus iron and vitamin A plus iron, thiamine, riboflavin, folic acid, niacinamide, zinc and calcium, respectively. The concentration of serum vitamin A was measured by high-performance liquid chromatography (HPLC), serum ferritin (SF) by enzyme-linked immunosorbent assay (ELISA), serum transferring receptor (sTfR) by microparticle-enhanced immunoassay, C-reactive protein (CRP) by particle-enhanced immunoturbidimetry and hemoglobin (HB) by hemiglobincyanide and sTfR-SF index (TFR-F index) and total body iron content were computed, respectively before and after intervention. Simultaneously, children’s demographic data, socio-economic status and eating habits, etc were investigated by questionnaires.Result: The levels of HB and serum vitamin A significantly increased after intervention in all groups (p<0.05) but no marked difference was observed between groups (p>0.05) for HB while vitamin A levels in group Ⅱand groupⅢwere higher than that of groupⅠ. The levels of SF and sTfR significantly decreased after intervention in all groups (p<0.05) especially in groupⅡand groupⅢfor SF (p<0.05) and groupⅠfor sTfR (p<0.05). No marked change of TFR-F index and total body iron contents was observed in groupⅠ(p>0.05), while TFR-F index decreased and total body iron contents increased in groupⅡand groupⅢ(p<0.05) after intervention. After adjustment for the age, gender, different sources of kindergarten, family economic situation, passive smoking exposure and dietary pattern, etc, no interventional styles was a prominent protective factor of the risk for anemia and deficient iron storage defined by SF (p<0.05). Compared to vitamin A intervention, vitamin A + iron and multiple micronutrients supplementation were significant protective factor for deficient iron storage defined by TFR-F index [(relative risk, RR) (95% CI): 0.410(0.218, 0.992)].Conclusion:1 Vitamin A intervention has significant effect on iron storage and mobilization.2 Vitamin A intervention has no significant effect on TFR-F index and total body iron contents which prompted the possibility of seldom effect of vitamin A on iron absorption in small intestine.3 The combination of vitamin A and other micronutrients might be a better intervention for the improvement of iron deficiency for preschool-children.PartⅢEffect of Vitamin A Status on Rat Liver Iron Metabolic mode and Its MechanismObjective: To evaluate the effect of vitamin A status on erythrocyte index and to measure the expressions of the components of iron responsive element-iron regulated protein mode (IRE-IRP mode) and hepcidin- ferroportin (Hepc-FPN mode) in liver on mRNA and protein levels by vitamin A status.Method: About 30 healthy female Witster rats were randomly divided into vitamin A normal feeding group (NVA group, n = 10), vitamin A deficient feeding group (VAD group, n = 10) and vitamin A interventional group (VAI group, n=10) on which were fed forage including vitamin A 6500 IU/Kg and 400 IU/Kg, respectively from 3 weeks before and throughout pregnancy and lactation. In VAI group the mothers received VAD diet till post partum, and then received normal diet. The offspring were given low-dose vitamin A from postnatal and received normal diet after 4 weeks. The pups received the same diets as their mothers both before and after weaning until 8 weeks and 20 pups were randomly chosen from the three groups (two pups in each clutch with equal male and female) to measure the concentration of serum retinol by HPLC, levels of hemoglobin (HB), red blood cell (RBC), hematocrit (Hct), red cell distribution width (RDW), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) by automatic blood cytoanalyze. Moreover, the expressions of the components of IRE-IRP mode and Hepc-FPN mode in liver with mRNA and protein levels by fluorescent quantitation PCR, western blotting and euzymelinked immunosorbent assay (ELISA).Result: At eight week, the levels of HB, RBC and Hct in VAI group were significantly higher than that of NVA and VAD group (p<0.05). And the value of MCV was markedly lower than that of the other two group (p<0.05) and RDW was higher in VAI group (p<0.05), MCHC lower in VAI group (p<0.05). No significant difference of MCH was found among the three groups (p>0.05). Moreover, hepcidin, bone morphogenetic protein-4 and interleukin-6, iron regulatory protein-1, ferroportin-1, ferritin light chain and transferrin receptor-1 about components of iron metabolism presented different expression of mRNA and protein levels in liver by vitamin A status but not divalent metal transporter-1.Conclusion:1 Peripheral blood presented hypochromic microcytic by vitamin A deficiency which could be improved by vitamin A intervention and vitamin A deficiency showed no effect on MCH and MCHC.2 Iron Hepc-FPN mode in liver could be affected by vitamin A status by transcriptional regulation of IL-6 and BMP-4 on hepcidin expression.3 Iron IRP-IRE mode in liver could be affected by vitamin A status by transcriptional regulation on IRP-1 expression.更多还原