【作者】 马文强；

【导师】 许梓荣； 冯杰；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2010， 博士

【摘要】 氨基酸铁是第三代铁源添加剂,具有生物学效价高、吸收率高、化学结构稳定、增强免疫力、利于环保等特点,是当前国内外研制和开发应用的热点。医学研究表明,氨基酸螯合铁在动物体内具有很高的生物学利用率,相当于同水平硫酸亚铁的125-185%。甘氨酸为分子量最小的氨基酸,甘氨酸亚铁在体内应更容易被吸收利用。研究表明,氨基酸铁螯合物高生物学效价与其高效的吸收转运机制有关,但其吸收转运机制仍尚未明确。本研究对甘氨酸亚铁螯合物在缺铁大鼠模型体内铁调控特点、体外肠上皮细胞模型(Caco-2)的吸收转运特点及其在断奶仔猪、肉仔鸡的生物学效应进行了探讨。主要研究内容和结果如下：1、建立了缺铁SD大鼠模型。结果表明,21日龄SD大鼠饲喂2周低铁日粮后,血液血红蛋白水平下降到107.67g/L,血清铁及肝脏铁水平大幅下降,表明SD大鼠缺铁模型已建立。同时研究发现,与对照组相比,大鼠饲喂2周缺铁日粮后体重极显著减轻,体重降低18.03%(P<0.01)；血清TIBC提高了29.37%(P<0.01),SF含量降低了27.71%(P<0.01),CAT含量降低了12.40%(P<0.05)；肝脏Hepcidin相对表达量大幅下调,仅为无机铁组的6%；缺铁组大鼠十二指肠DMT1相对表达量大幅提高,是无机铁组的2.55倍；FP1相对表达量也显著增加,为无机铁组的2.79倍;而PepTl相对表达量没有明显差异。2、研究了甘氨酸亚铁对缺铁SD大鼠生长发育、铁代谢指标及机体铁调控的影响。选用体重为66.43±6.86g的缺铁SD大鼠90只(公母各半),随机分为3组,无机铁组(FeSO4,添加Fe 35 mg/kg,对照组)、甘氨酸组(FeSO4+Glycine,添加Fe 35 mg/kg,摩尔比Fe:AA=1:2)和有机铁组(Fe-Gly,添加Fe 35 mg/kg),试验期为2周。结果表明,与无机铁组相比,甘氨酸亚铁组大鼠体重增加幅度最大,增加了4.05%(P>0.05)；甘氨酸亚铁添加促进大鼠的肝脏、脾脏及肾脏发育,其器官指数显著提高(P<0.05)；甘氨酸亚铁组大鼠血清、肝脏及脾脏铁含量分别提高了26.76%(P<0.01)、34.58%(P<0.01)、26.72%(P<0.01)；甘氨酸亚铁组的大鼠血清SF及CAT分别提高22.72%及81.00%(P<0.01), TIBC及XOD分别降低20.42%和23.05%(P<0.05)；甘氨酸组及甘氨酸亚铁组大鼠肝脏Hepcidin相对表达量显著提高,为无机铁组的3.27倍和5.65倍,大鼠十二指肠DMT1相对表达量大幅降低,仅为无机铁组的33%和23%；FP1相对表达量也显著降低,为无机铁组的38%和22%；PepTl相对表达量同时显著提高,为无机铁组的2.55倍和6.27倍。与硫酸亚铁添加相比,甘氨酸亚铁添加能明显快速改善动物机体缺铁状态,同时发现大鼠十二指肠PepTl表达量有了明显提高,可能PepT1在甘氨酸亚铁整体转运中起到了重要作用。3、Caco-2细胞转运模型的构建。Caco-2细胞在微孔滤膜上培养21d后,形成致密的单层,跨膜电阻值达到稳定的值,为466.75±50.48Ω·cm2,荧光素钠在150min内总透过率为0.85%,细胞肠腔侧碱性磷酸酶活性显著高于基底侧酶活性。构建的Caco-2细胞模型细胞单层生长形态良好、具有良好致密性、细胞极性分化完全,符合吸收转运模型的标准,可用作小肠吸收的体外细胞模型。4、采用Caco-2细胞吸收模型研究甘氨酸亚铁螯合物(Fe-Gly)的吸收机制,分别考察了浓度(0.5-20μmol/L)、转运方向(AP→BL,BL→AP)、时间(0-120min)及温度(37℃,4℃)对转运过程的影响。Fe-Gly和FeSO4从AP→BL方向跨Caco-2细胞单层转运呈浓度和时间依赖型,转运受温度影响明显。Fe-Gly的Papp为0.15-10.40×10-6 cm/s,FeS04的Papp为0.18-4.70×10-6cm/s。37℃孵育条件下,Fe-Gly跨细胞单层的转运率显著高于FeS04(P<0.05),且Fe-Gly的转运率随着浓度的升高而降低,0.5μmol/L时转运率为23.32%,而20μmol/L时则为7.97%。FeSO4及Fe-Gly在Caco-2细胞中可能是经过主动转运吸收,其中FeSO4是通过DMT1介导主动转运吸收,而Fe-Gly可能存在特定或非特定的肠道转运系统,Fe-Gly在Caco-2细胞中的转运率明显高于FeSO4。5、研究了不同因素对甘氨酸亚铁螯合物及硫酸亚铁跨Caco-2细胞转运的影响。将含有10μmol/L不同影响因素(铁吸收促进剂—维生素C及铁吸收抑制剂—草酸钠)和10μmol/L铁(以Fe-Gly、FeS04形式)的D’Hanks液加入细胞单层肠腔侧(AP),37℃培养,在不同时间点(30、60、90、120min)从细胞单层基底侧(BL)吸取200μl样进行铁含量分析。Vc对Fe-Gly在Caco-2细胞中的转运量没有产生显著的影响(P>0.05),但显著增加了FeSO4在Caco-2细胞中的转运量(P<0.05)；草酸钠对Fe-Gly在Caco-2细胞中的转运量没有产生显著的影响(P>0.05),而显著抑制了FeSO4在Caco-2细胞中的转运(P<0.05)。说明不同来源的铁在吸收时所受的影响存在差异,无机铁FeSO4相对于Fe-Gly来说较易受到各种因素的干扰。6、研究了Fe-Gly和FeSO4对断奶仔猪生长性能、免疫机能和肉色的影响。选择180头体重7.81±0.72kg的“杜长大”三元杂交仔猪,按饲养试验要求分为6组,每组设三个重复,每个重复10头(组内公母各半)。对照组饲喂基础日粮,试验1、2、3、4组在基础日粮的基础上分别添加30、60、90、120 mg/kg Fe-Gly(以铁计),试验5组在基础日粮的基础上添加120 mg/kg FeSO4(以铁计)。试验期35天,结果表明,断奶仔猪日粮中添加60、90、120 mg/kg Fe-Gly分别使仔猪日增重提高了9.69%(P<0.05)、11.08%(P<0.05)和9.97 (P<0.05); 60、90、120 mg/kg Fe-Gly和120 mg/kg FeSO4添加明显提高了断奶仔猪胸腺指数(P＜0.05); 90、120 mg/kg Fe-Gly添加提高了ConA诱导的B-淋巴细胞刺激指数(P<0.05); 90 mg/kg Fe-Gly添加使全血中血红蛋白、红细胞数及红细胞压积分别提高了13.08%(P<0.05)、14.31%(P<0.05)及20.53%(P<0.05)；日粮中添加60、90和120 mg/kg Fe-Gly均提高了断奶仔猪背最长肌肌红蛋白含量(P<0.05),改善了肉色红度值(a*值)；添加90 mg/kg Fe-Gly大幅度提高了仔猪肝脏SOD及SDH酶活(P<0.05);90、120mg/kg Fe-Gly及120 mg/kg FeSO4的添加使血清铁、心脏铁、肝脏铁及脾脏铁含量显著提高(P＜O.05); 120 mg/kg Fe-Gly或FeSO4添加使仔猪粪便铁残留量明显增加(P<0.05); 60、90、120 mg/kg Fe-Gly添加降低了血尿氮及总胆固醇含量(P<0.05或P<0.01),提高了碱性磷酸酶的活性(P<0.05)。以上结果表明,90 mg/kg Fe-Gly可明显促进断奶仔猪生长；提高断奶仔猪胸腺指数,促进免疫器官发育；提高血红蛋白及血清铁含量,促进B淋巴细胞正常增殖,提高仔猪的免疫力；提高Mb含量,改善肉色；促进机体组织铁沉积。7、研究了Fe-Gly及FeSO4对肉仔鸡生产性能、免疫机能及抗氧化指标的影响。360只1日龄AA肉鸡随机分为6组,每组设3个重复,每个重复20只。以饲喂基础日粮组为对照组,试验组在基础日粮基础上分别添加40、80、120、160 mg/kg甘氨酸亚铁及160 mg/kg硫酸亚铁(以铁计)。试验期为42天,研究表明,添加120、160 mg/kg甘氨酸亚铁显著提高了42日龄肉仔鸡体重及22-42日龄日增重(P<0.05)。日粮添加80、120 mg/kg甘氨酸亚铁显著提高了21日龄及42日龄肉仔鸡胸腺指数(P＜0.05); 120、160 mg/kg甘氨酸亚铁添加显著增强了脂多糖刺激的21日龄肉仔鸡全血T淋巴细胞增殖(P＜.05); 80、120、160 mg/kg甘氨酸亚铁添加提高了21日龄肉仔鸡血清IgG及IgM含量(P<0.05); 120、160 mg/kg甘氨酸亚铁或160 mg/kg硫酸亚铁添加提高了21日龄及42日龄肉仔鸡血清、肝脏、胸肌铁沉积及粪便铁残留量,且80、120、160 mg/kg甘氨酸亚铁或160 mg/kg硫酸亚铁添加提高了21日龄及42日龄肉仔鸡胫骨铁沉积；添加120及160 mg/kg甘氨酸亚铁能显著性的提高21日龄肉仔鸡血清中SOD和CAT酶的活性(P<0.05),降低MDA的酶活性(P＜.05); 80、120、160 mg/kg甘氨酸亚铁或160 mg/kg硫酸亚铁添加提高了42日龄肉仔鸡CAT的活性(P<0.05)。以上结果表明,120 mg/kg甘氨酸亚铁可显著改善肉鸡的生长性,提高肉鸡的免疫器官指数,增加血清中免疫球蛋白IgM及IgG的含量,促进T淋巴细胞增殖,增强机体的抗氧化能力。综上所述,机体缺铁时,大鼠肝脏铁调素Hepcidin相对表达量大幅下调,十二指肠DMT1及FP1相对表达量显著提高；补饲甘氨酸亚铁后,与添加无机铁相比,机体缺铁状态改善迅速,且十二指肠PepT1相对表达量显著上调,PepT1可能在甘氨酸亚铁的整体吸收转运中发挥了重要作用；甘氨酸亚铁跨Caco-2细胞膜转运量显著高于同浓度硫酸亚铁,且转受温度影响较大,同时受外界(铁吸收促进剂及抑制剂)干扰较小,表明甘氨酸亚铁可能存在一个特定或非特定的肠道主动转运系统；同时,适量添加甘氨酸亚铁可明显改善断奶仔猪生产性能、免疫机能及肉色,也可改善肉仔鸡生产性能、免疫机能及抗氧化指标。更多还原

【Abstract】 With the benefits on absorption rate, growth, immunity and environment, iron amino acid chelate has been paid more attention in animal nutrition. Studies showed that chelated or proteinated sources of Fe have 125-185% relative availability compared with ferrous sulfate. The better bioavailability of iron amino acid chelate is maybe mainly due to its highly efficient absorption, but its absorption mechanism is still not clear. Iron glycine chelate (Fe-Gly) could be more easily absorbed compared with other Fe sources. In this study, research on manipulation of Fe-Gly uptake in iron-deficiency rat model, absorption mechanism of Fe-Gly in Caco-2 cell model and its application in weanling piglets and broilers were carried out to reveal the possible mechanism of absorption and biological effects of Fe-Gly.The main contents and results are as follows:1) Iron-deficiency SD rat model was established. The results showed that blood hemoglobin level fell to 107.67 g/L, serum iron and liver iron levels decreased significantly when 3-w old SD rats fed a low iron diet after 2 weeks. This indicated that SD rats with iron-deficiency model had been initially established. Compared with the control group, iron deficiency significantly reduce body weight of rat by18.03% (P＜0.01), serum TIBC increased by 29.37% (P＜0.01), SF content and CAT level decreased by 27.71% (P<0.01) and 12.40% (P<0.05). Iron deficiency greatly reduced relative expression of Hepcidin to 0.06-fold in liver; and increased duodenal DMT1 and FP1 relative expression level by 2.55-fold and 2.79-fold. There is no significant difference in relative expression of PepTl.2) Iron-deficiency rat model was conducted to determine the effects of Fe-Gly on growth, iron metabolism and iron regulation. Ninety iron-deficiency rats (initial weight of 66.43±6.86 g) were allotted to 3 treatments based on live weight and sex. Treatments consisted of:FeSO4 group (35mg Fe/kg diet from FeSO4, control group); glycine group (35 mg Fe/kg diet from FeSO4, and the molar ratio of Fe:glycine= 1:2); Fe-Gly group (35 mg Fe/kg diet from Fe-Gly). After 2-w feeding trial, the results showed that rat liver, spleen and kidney index was significantly increased (P<0.05) when rat was fed Fe-Gly. Supplemental Fe-Gly in diets increased serum, liver and spleen iron content by 26.76%(P<0.01),34.58% (P＜0.01) and 26.72% (P＜0.01). Addition with Fe-Gly enhanced serum SF and CAT levels by 22.72% and 81.00% (P＜0.01), and reduced TIBC, XOD levels by 20.42% and 23.05% (P＜0.05) compared with the control. Compared with the control, the relative expression levels of liver Hepcidin in glycine group and Fe-Gly group increased to 3.27-fold and 5.65-fold, duodenum PepTl enhanced to 2.55-fold and 6.27-fold, duodenum DMT1 decreased to 0.33-fold and 0.23-fold, duodenum FP1 reduced to 0.38-fold and 0.22-fold, respectively. The result indicated that Fe-Gly could improve the body iron status quickly and also found PepTl maybe play a key role in intestinel absorption of Fe-Gly.3) The Caco-2 cell transport model was established. After 21-days culture, the Caco-2 cell model has formed a tight monolayer, with a steady TEER value 466.75±50.48Ω·cm2 and a transportation percentage of fluorescein sodium at 0.85% in 150 min. The AKP activity in the apical side (AP) is greatly higher than the basolateral side (BL), which means the Caco-2 cell has polarity by 21-days differentiation. Therefore, the Caco-2 cell model established in this study can be used as an in vitro intestinal absorption model with accepted standards.4) The transports of Fe-Gly and FeSO4 in Caco-2 cell monolayers were conducted from AP to BL and BL to AP, respectively. The effects of concentration (0.5-20μmol/L of Fe), time (0-120 min) and temperature (37℃and 4℃) on transport Fe-Gly and FeSO4 were investigated. Transports of Fe-Gly and FeSO4 across Caco-2 monolayers both from AP to BL and BL to AP direction were concentration-and time-dependent. There are more amounts of Fe-Gly and FeSO4 transport under 37℃than those under 4℃from AP to BL direction. The apparent permeability coefficient (Papp) of Fe-Gly was between 0.15×10-6 cm/s and 10.40×10-6 cm/s, which decreased with the increased concentration. Papp of FeSO4 was between 0.18×10-6 cm/s and 4.70×10-6 cm/s. Fe-Gly transport across Caco-2 cell monolayers was significantly higher than FeSO4 (P＜0.05) when incubation temperature under 37℃. The transport rate of Fe-Gly decreased with increasing supplemental levels. Therefore, it can be deduced that the absorption of FeSO4 and Fe-Gly in Caco-2 cells is mainly through active transport. It is known that the intestinal absorption of FeSO4 is through DMT 1-mediated active transport, while the intestinal absorption of Fe-Gly may be through a specific or non-specific intestinal active transit system. The transport mounts of Fe-Gly accros Caco-2 cell monolayers were significantly higher than FeSO45) The study was carried out to determine the effects of different factors on Fe-Gly and FeSO4 transport across Caco-2 cell monolayers. Transport of 10μmol/L of Fe in the form of Fe-Gly or FeSO4 were conducted with 10μmol/L vitamin C (iron absorption enhancer) or sodium oxalate (iron absorption inhibitor), respectively. The studies shown that vitamin C had no great effect on transport of Fe-Gly (P>0.05). However, FeSO4 transport was significantly enhanced by supplemental vitamin C (P＜0.05). Sodium oxalate did not affect the transport of Fe-Gly (P>0.05), but greatly reduced FeSO4 transport (P＜0.05). These results indicated that FeSO4 was easily affected by dietary factors compared to Fe-Gly.6) The study was conducted to determine the effects of Fe-Gly on growth, immunological characteristics and meat color in weanling pigs. One hundred and eighty pigs (initial weight of 7.81±0.72 kg) were allotted to six treatments based on live weight and litter origin. Treatments consisted of 0,30,60,90, and 120 mg/kg Fe-Gly groups (calculated with Fe) and 120 mg/kg FeSO4 group (calculated with Fe). Compared with the control, ADG was enhanced (P＜0.05) when pigs fed diets containing 60,90 or 120 mg/kg Fe-Gly. Supplemental 60,90 or 120 mg/kg Fe-Gly or 120 mg/kg FeSO4 greatly increased thymus gland index (P＜0.05) compared with the control. Lymphocytes from whole blood of experimental pigs had a higher proliferative response to ConA (P<0.05) when diet supplemental 90,120 mg/kg Fe as Fe-Gly. The hemoglobin, RBC and PCV were increased by 13.08% (P＜0.05),14.31% (P＜0.05) and 20.53% (P＜0.05) when pigs fed 90 mg/kg Fe-Gly. Myoblobin concentrations of M. longissimus dorsi were enhanced with addition of Fe-Gly from 60 to 120 mg/kg. SOD and CAT activities were increased when pigs fed 90 mg/kg Fe-Gly.90,120 mg/kg Fe-Gly or 120 mg/kg FeSO4 also enhanced serum, heart, liver and spleen Fe concentration (P＜0.05 or P＜0.01) compared with the control.120 mg/kg Fe as Fe-Gly or FeSO4 enhanced Fe concentration in feces compared with the control. SUN and SUL contents increased and AKP activity decreased when pigs fed 60,90 or 120 mg/kg Fe-Gly. Those results indicated that 90 mg/kg Fe-Gly had benefits on improving growth, immulogical functions and meat color of weanling pigs.7) The study was carried out to determine the effects of Fe-Gly on growth performance, immunological characteristics and antioxidant index of broiler chickens. Three hundred and sixty 1-d old commercial broiler chicks (Ross×Ross) were randomly allotted to six dietary treatments. Treatments consisted of 0,40,80,120, and 160 mg/kg Fe-Gly groups (calculated with Fe) and 160 mg/kg FeSO4 group (calculated with Fe). Feeding trial included 0-21d and 22-42d period. The results showed that compared with the control,120 and 160 mg/kg Fe-Gly improved 6-w body weight and 22-42-d ADG of broiler chickens. Thymus gland index was increased (P＜0.05) when chicks fed 80,120 mg/kg Fe as Fe-Gly at d 21 and d 42. Lymphocytes from whole blood of experimental chickens had a higher proliferative response to LPS (P＜0.05) when diet supplemental 120,160 mg/kg Fe as Fe-Gly at d 21.80,120 mg/kg Fe-Gly enhanced IgM (P＜0.05) and IgG (P＜0.05) contents at d 21 and d 42. The concentration of Fe was significantly increased (P＜0.05) in serum, liver, breast muscle and feces of chicks fed diets supplemented with 120,160 mg/kg Fe as Fe-Gly or 160 mg/kg Fe as FeSO4 at d 21 and d 42. In addition, tibia Fe storage was improved (P＜0.05) when chicks were fed 80,120, 160 mg/kg Fe as Fe-Gly or 160 mg/kg Fe as FeSO4 at d 21 and d 42. Feeding 120,160 mg/kg Fe as Fe-Gly greatly enhanced serum SOD and CAT activities (P＜0.05), and decreased MDA activity of 21-d chicks. Serum CAT activity was increased when chicks fed 80,120,160 mg/kg Fe as Fe-Gly or 160 mg/kg Fe as FeSO4 at d 42. These results indicated that 120 mg/kg Fe as Fe-Gly has better effects on improving growth, increasing development of immune organs, enhancing serum IgM and IgG levels, promoting T lymphocyte proliferation, and enhancing antioxidant capacity of broiler chickens.In summary, the relative expression of liver hepcidin significantly reduced and relative expression of duodenal DMT1 and FP1 greatly improved when SD rat was in iron deficiency. Addition with iron glycine chelate could improve iron status of iron-deficiency rat quickly, and the relative expression of duodenal PepTl was significantly raised compared to FeSO4. PepTl may be played a key role in the intestine absorption of Fe-Gly. The transport mounts of Fe-Gly across Caco-2 cell monolayers were significantly higher than FeSO4, while FeSO4 was easily affected by dietary factors relative to Fe-Gly. Those indicated that there may be a specific or non-specific intestinal active transport system to transport Fe-Gly. In addition, dietary Fe-Gly was beneficial to growth, immune function and meat color of weanling pigs; also improve growth, immune function and antioxidation of broiler chickens.更多还原