对碘量法测定日粮、肠道食糜及排泄物中半胱胺盐酸盐含量的改进研究
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摘要
试验旨在探讨碘量法测定日粮、食糜及排泄物中半胱胺盐酸盐(cysteamine hydrochloride,CSH)含量的改进方法,为评估包膜CSH在动物胃肠道内释放过程中CSH的定量提供参考。试验分3部分:试验一:检验包膜CSH的上样量对碘标准液消耗量的线性关系,以确定碘量法检验包膜产品的准确度,采用单因素完全随机设计,CSH的上样量设6个水平,分别为28.28、113.12、226.24、339.36、452.48和565.61mg;试验二:考察H2SO4预处理对消除日粮、食糜和排泄物对碘量法测定CSH含量的影响,采用单因素完全随机设计,日粮、十二指肠食糜、空肠食糜、回肠食糜及排泄物样品中CSH(试剂级)的含量均设3个水平,即30、100和300mg/2g,形成15个含有CSH的样品,H2SO4溶液(6mol/L)设3个水平,即0.1、0.5和1mL,每个处理设3个重复;试验三:考察传统碘量法与试验二建立的改进方法在测定日粮、食糜及排泄物中包膜CSH含量的差异,采用两样本试验设计,日粮、十二指肠食糜、空肠食糜、回肠食糜及排泄物中CSH的含量均设28.73、84.84、141.47和200.56mg/2g。结果表明:①CSH的上样量对碘标准液消耗量及CSH测定值均呈显著的线性关系(P<0.01,R2=0.9999)。②在含有CSH的日粮中,H2SO4溶液处理后由碘量法测定的CSH质量对实际质量的回归均符合Y=X(P>0.05)。在含有CSH的十二指肠食糜和回肠食糜中,所有H2SO4溶液处理下CSH质量对实际质量的回归均不符合Y=X(P<0.05),但0.5和1.0mL比0.1mL H2SO4溶液处理使测定值更接近实际值。在含有CSH的空肠食糜及排泄物中,0.5和1.0mL H2SO4溶液处理使测定值对实际值的回归符合Y=X(P>0.05),而0.1mL H2SO4溶液处理下CSH测定值显著低于实际值(P<0.01)。③在含有CSH的日粮、十二指肠食糜、空肠食糜、回肠食糜及排泄物样品中,常规方法测定的CSH对实际质量均偏离Y=X(P<0.05),而1.0mL H2SO4溶液处理后的测值对实际质量均符合Y=X(P>0.05)。由此可见,采用适量H2SO4溶液进行处理后,可以排除日粮、食糜及排泄物对CSH测定的干扰。
This experiment was conducted to improve the method of iodimetry for the determination of cysteamine hydrochloride(CSH)in diet,digesta,and excreta,which will be used to assay the CSH for evaluating the releasing process of encapsulated cysteamine in gastro-intestine of animal.Experiment 1 was to examine the linear relationship between iodine consumption on the amount of CSH with the iodimetry.Six levels of 28.28,113.12,226.24,339.36,452.48,565.61 mg of CSH was adopted in a single-factor completely randomized design.Experiment 2 was to investigate the effect of pretreatment on eliminating interference from diet,digesta and excreta in the determination of CSH with iodimetry.Fifteen samples were composed that in each 2 g of diet,duodenal digesta,jejunaldigesta,ilealdigesta or excreta of chicken,the amounts of CSH(reagent grade)were 30,100 and 300 mg.The volume of 0.1,0.5 or 1 mL of 6 mol/L sulphuric acid solution was adopted in single-factor completely randomized design to pretreat each sample.Experiment 3 was to test the differences between conventional iodimetry and the improved method established in the experiment 2 to determine CSH in diet,digesta and excreta.The amounts of CSH(provided by the encapsulated product)of 28.73,84.84,141.47 and 200.56 mg were mixed in each 2 g of diets,duodenal digesta,jejunaldigesta,ilealdigesta,and excreta of chicken.A twosample experimental design was adopted to compare 2 methods to determine the content of CSH.The results showed as follows:(1)There was a significant linear relationship between the iodine consumption or the determined CSH on actual amount of CSH(P<0.01,R2=0.9999);(2)In the diet mixed with CSH,sulfuric acid pretreatment for iodimetry made the regression of determined on actual amount of CSH consistent with Y=X(P>0.05).In the duodenal or ilealdigesta mixed with CSH,sulfuric acid pretreatment for iodimetry didn't made the regression of determined on actual amount of CSH consistent with Y=X(P<0.05),however,the pretreatment with 0.5 or1 mL sulfuric acid made the determinred value closer to the actual value than the pretreatment with 0.1 mL.In the jejunaldigesta or excreta mixed with CSH,sulfuric acid pretreatment made the regression of determined on actual amount of CSH consistent with Y=X(P>0.05),whereas,the pretreatment with 0.1 mL sulfuric acid made the determined value significantly lower than actual value of CSH(P<0.01).(3)In diet,duodenal,ileal and jejunaldigesta or excreta mixed with CSH,conventional method made the regression of determined on actual amount of CSH deviate from Y=X(P<0.05),whereas,pretreatment with 1.0 mL sulfuric acid made the regression of determined on actual amount of CSH consistent with Y=X(P>0.05).In conclusion,pretreatment with an appropriate amount of sulphuric acid solution cculd improve the determination of CSH by eliminating the interference from diet,digesta or excreta.
This experiment was conducted to improve the method of iodimetry for the determination of cysteamine hydrochloride(CSH)in diet,digesta,and excreta,which will be used to assay the CSH for evaluating the releasing process of encapsulated cysteamine in gastro-intestine of animal.Experiment 1 was to examine the linear relationship between iodine consumption on the amount of CSH with the iodimetry.Six levels of 28.28,113.12,226.24,339.36,452.48,565.61 mg of CSH was adopted in a single-factor completely randomized design.Experiment 2 was to investigate the effect of pretreatment on eliminating interference from diet,digesta and excreta in the determination of CSH with iodimetry.Fifteen samples were composed that in each 2 g of diet,duodenal digesta,jejunaldigesta,ilealdigesta or excreta of chicken,the amounts of CSH(reagent grade)were 30,100 and 300 mg.The volume of 0.1,0.5 or 1 mL of 6 mol/L sulphuric acid solution was adopted in single-factor completely randomized design to pretreat each sample.Experiment 3 was to test the differences between conventional iodimetry and the improved method established in the experiment 2 to determine CSH in diet,digesta and excreta.The amounts of CSH(provided by the encapsulated product)of 28.73,84.84,141.47 and 200.56 mg were mixed in each 2 g of diets,duodenal digesta,jejunaldigesta,ilealdigesta,and excreta of chicken.A twosample experimental design was adopted to compare 2 methods to determine the content of CSH.The results showed as follows:(1)There was a significant linear relationship between the iodine consumption or the determined CSH on actual amount of CSH(P<0.01,R2=0.9999);(2)In the diet mixed with CSH,sulfuric acid pretreatment for iodimetry made the regression of determined on actual amount of CSH consistent with Y=X(P>0.05).In the duodenal or ilealdigesta mixed with CSH,sulfuric acid pretreatment for iodimetry didn't made the regression of determined on actual amount of CSH consistent with Y=X(P<0.05),however,the pretreatment with 0.5 or1 mL sulfuric acid made the determinred value closer to the actual value than the pretreatment with 0.1 mL.In the jejunaldigesta or excreta mixed with CSH,sulfuric acid pretreatment made the regression of determined on actual amount of CSH consistent with Y=X(P>0.05),whereas,the pretreatment with 0.1 mL sulfuric acid made the determined value significantly lower than actual value of CSH(P<0.01).(3)In diet,duodenal,ileal and jejunaldigesta or excreta mixed with CSH,conventional method made the regression of determined on actual amount of CSH deviate from Y=X(P<0.05),whereas,pretreatment with 1.0 mL sulfuric acid made the regression of determined on actual amount of CSH consistent with Y=X(P>0.05).In conclusion,pretreatment with an appropriate amount of sulphuric acid solution cculd improve the determination of CSH by eliminating the interference from diet,digesta or excreta.
引文
[1]MCELWAIN K V,ESTIENNE M J,BARB C R.Effect of cysteamine hydrochloride on secretion of growth hormone in male swine[J].Life Sciences,1999,64(24):2233-2238.
[2]MCLEOD K R,HARMON D L.Cysteamine-induced depletion of somatostatin in sheep:Time course of depletion and changes in plasma metabolites,insulin,and growth hormone[J].Journal of Animal Science,1995,73(1):77-87.
[3]BROWN M R,FISHER L A,SAWCHENKO P E,et al.Biological effects of cysteamine:Relationship to somatostatin depletion[J].Regulatory Peptides,1983,5(2):163-179.
[4]唐好文,李辉,李婷.半胱胺的动物营养研究进展[J].中国畜牧兽医,2008,1:33-36.TANG H W,LI H,LI T.Advances in animal nutrition research of cysteamine[J].China Animal Husbandry&Veterinary Medicine,2008,1:33-36.(in Chinese)
[5]ZHOU P,LUO Y,ZHANG L,et al.Effects of cysteamine supplementation on the intestinal expression of amino acid and peptide transporters and intestinal health in finishing pigs[J].Animal Science Journal,2017,88(2):314-321.
[6]秦龙山,邢月腾,张杨,等.半胱胺对宁乡猪血清生化指标和肝脏脂肪代谢的影响[J].动物营养学报,2017,29(11):3987-3993.QIN L S,XING Y T,ZHANG Y,et al.Effect of cysteamine on serum biochemical parameters and liver fat metabolism in Ningxiang pigs[J].Chinese Journal of Animal Nutrition,2017,29(11):3987-3993.(in Chinese)
[7]ZHOU P,ZHANG L,LI J,et al.Effects of dietary crude protein levels and cysteamine supplementation on protein synthetic and degradative signaling in skeletal muscle of finishing pigs[J].PLoS One,2015,10(9):e0139393.
[8]朱宇旌,王浩然,李方方,等.半胱胺螯合锌对仔猪生长性能、血清生化指标、养分消化率及粪中微生物菌群的影响[J].动物营养学报,2015,27(10):3225-3232.ZHU Y J,WANG H R,LI F F,et al.Effects of cysteamine chelated zinc on growth performance,serum biochemical parameters,nutrient digestibility and microbial flora of feces in piglets[J].Chinese Journal of Animal Nutrition,2015,27(10):3225-3232.(in Chinese)
[9]徐雪松,吴金节,李健,等.半胱胺对仔猪生长性能及小肠内容物消化酶活性的影响[J].中国兽医学报,2008,9:1088-1091.XU X S,WU J J,LI J,et al.Effects of cysteamine on growth performance and digestive enzyme activity of small intestine content in piglets[J].Chinese Journal of Veterinary Science,2008,9:1088-1091.(in Chinese)
[10]吴建设,黄建国,李军.半胱胺对中华宫廷黄鸡生长及相关生理生化指标影响的研究[J].动物营养学报,2001,13(4):24-27.WU J S,HUANG J G,LI J.Effects of cysteamine on the growth and related physiological and biochemical indexes of Chinese Court Yellow chicken[J].Chinese Journal of Animal Nutrition,2001,13(4):24-27.(in Chinese)
[11]LIU G M,WANG Z S,WU D,et al.Effects of dietary cysteamine supplementation on growth performance and whole-body protein turnover in finishing pigs[J].Livestock Science,2009,122(1):86-89.
[12]马现永,林映才,周桂莲,等.半胱胺添加水平对黄羽肉鸡生长性能、肉质及机体抗氧化能力的影响[J].动物营养学报,2009,21(6):916-923.MA X Y,LIN Y C,ZHOU G L,et al.Effects of cysteamine levels on growth performance,meat quality and antioxidant capacity of Yellow Feather broilers[J].Chinese Journal of Animal Nutrition,2009,21(6):916-923.(in Chinese)
[13]朱玉萍,周平,李蛟龙,等.低蛋白氨基酸平衡日粮添加半胱胺对生长猪肉质和相关基因表达的影响[J].畜牧兽医学报,2017,48(4):660-668.ZHU Y P,ZHOU P,LI J L,et al.Effects of cysteamine supplementation on growth pork quality and related gene expression in low protein amino acid balanced diets[J].ActaVeterinaria et Zootechnica Sinica,2017,48(4):660-668.(in Chinese)
[14]秦龙山,邢月腾,张杨,等.半胱胺对宁乡猪胴体性状和肉品质的影响[J].动物营养学报,2017,29(9):3325-3330.QIN L S,XING Y T,ZHANG Y,et al.Effect of cysteamine on carcass traits and meat quality of Ningxiang pigs[J].Chinese Journal of Animal Nutrition,2017,29(9):3325-3330.(in Chinese)
[15]张献新,张益禅,邹芳.缓释包膜半胱胺对生长肥育猪生长性能及胴体品质的影响[J].农业现代化研究,2014,3:326-328.ZHANG X X,ZHANG Y C,ZOU F.Effect of sustained-release coated cysteamine on growth performance and carcass quality of growing-finishing pigs[J].Research of Agricultural Modernization,2014,3:326-328.(in Chinese)
[16]张昌如,周立明,徐利明.缓释包膜半胱胺对育肥猪生产性能的影响[J].上海畜牧兽医通讯,2003,1:22.ZHANG C R,ZHOU L M,XU L M.Effect of sustained-release coated cysteamine on performance of finishing pigs[J].Shanghai Journal of Animal Husbandry and Veterinary,2003,1:22.(in Chinese)
[17]中国国家标准管理委员会.GB/T 24832-2009饲料添加剂.半胱胺盐酸盐β环糊精微粒[S].北京:中国标准出版社,2010.CHINA NATIONAL STANDARDS MANGAGE-MENT COMMITTEE.GB/T 24832-2009Feed additives.Cysteamine hydrochloride beta cyclodextrinparticles[S].Beijing:China Standard Press,2010.(in Chinese)
[18]STACHOWICZ M,LEHMANN B,TIBI A,et al.Determination of total cysteamine in human serum by a high-performance liquid chromatography with fluorescence detection[J].Journal of Pharmaceutical and Biomedical Analysis,1998,17(4-5):767-773.
[19]周禾,李方实.半胱胺(盐酸盐)的测定方法[J].广西师范大学学报(自然科学版),2003,21(4):184-186.ZHOU H,LI F S.Method for determination of cysteamine(hydrochloride)[J].Journal of Guangxi Normal University(Natural Science Edition),2003,21(4):184-186.(in Chinese)
[20]中国兽药典委员会.中华人民共和国兽药典[M].北京:中国农业出版社,2015.CHINESE VETERINARY PHARMACOPOEIACOMMISSION.People’s Republic of China Veterinary Pharmacopoeia[M].Beijing:China Agriculture Press,2015.(in Chinese)
[21]ZHAO F,XIE M L,LI H,et al.Developing a silicon tshaped cannula to sample intestinal digesta in ducks[J].Animal Feed Science and Technology,2016,219:175-180.
[22]廖飞,杨晓,康格非,等.紫外吸收碘量法测定微量维生素C和还原型谷胱甘肽[J].重庆医科大学学报,2003,28(3):372-373.LIAO F,YANG X,KANG G F,et al.Determination of trace vitamin C and reduced glutathione by ultraviolet absorption iodometry[J].Journal of Chongqing Medical University,2003,28(3):372-373.(in Chinese)
[23]朱小云,李生兵,郭睿,等.紫外吸收碘量法快速测定体液维生素C[J].重庆医科大学学报,2004,29(6):841-842.ZHU X Y,LI S B,GUO R,et al.Rapid determination of body fluid vitamin c by ultraviolet absorption iodometry[J].Journal of Chongqing Medical University,2004,29(6):841-842.(in Chinese)
[2]MCLEOD K R,HARMON D L.Cysteamine-induced depletion of somatostatin in sheep:Time course of depletion and changes in plasma metabolites,insulin,and growth hormone[J].Journal of Animal Science,1995,73(1):77-87.
[3]BROWN M R,FISHER L A,SAWCHENKO P E,et al.Biological effects of cysteamine:Relationship to somatostatin depletion[J].Regulatory Peptides,1983,5(2):163-179.
[4]唐好文,李辉,李婷.半胱胺的动物营养研究进展[J].中国畜牧兽医,2008,1:33-36.TANG H W,LI H,LI T.Advances in animal nutrition research of cysteamine[J].China Animal Husbandry&Veterinary Medicine,2008,1:33-36.(in Chinese)
[5]ZHOU P,LUO Y,ZHANG L,et al.Effects of cysteamine supplementation on the intestinal expression of amino acid and peptide transporters and intestinal health in finishing pigs[J].Animal Science Journal,2017,88(2):314-321.
[6]秦龙山,邢月腾,张杨,等.半胱胺对宁乡猪血清生化指标和肝脏脂肪代谢的影响[J].动物营养学报,2017,29(11):3987-3993.QIN L S,XING Y T,ZHANG Y,et al.Effect of cysteamine on serum biochemical parameters and liver fat metabolism in Ningxiang pigs[J].Chinese Journal of Animal Nutrition,2017,29(11):3987-3993.(in Chinese)
[7]ZHOU P,ZHANG L,LI J,et al.Effects of dietary crude protein levels and cysteamine supplementation on protein synthetic and degradative signaling in skeletal muscle of finishing pigs[J].PLoS One,2015,10(9):e0139393.
[8]朱宇旌,王浩然,李方方,等.半胱胺螯合锌对仔猪生长性能、血清生化指标、养分消化率及粪中微生物菌群的影响[J].动物营养学报,2015,27(10):3225-3232.ZHU Y J,WANG H R,LI F F,et al.Effects of cysteamine chelated zinc on growth performance,serum biochemical parameters,nutrient digestibility and microbial flora of feces in piglets[J].Chinese Journal of Animal Nutrition,2015,27(10):3225-3232.(in Chinese)
[9]徐雪松,吴金节,李健,等.半胱胺对仔猪生长性能及小肠内容物消化酶活性的影响[J].中国兽医学报,2008,9:1088-1091.XU X S,WU J J,LI J,et al.Effects of cysteamine on growth performance and digestive enzyme activity of small intestine content in piglets[J].Chinese Journal of Veterinary Science,2008,9:1088-1091.(in Chinese)
[10]吴建设,黄建国,李军.半胱胺对中华宫廷黄鸡生长及相关生理生化指标影响的研究[J].动物营养学报,2001,13(4):24-27.WU J S,HUANG J G,LI J.Effects of cysteamine on the growth and related physiological and biochemical indexes of Chinese Court Yellow chicken[J].Chinese Journal of Animal Nutrition,2001,13(4):24-27.(in Chinese)
[11]LIU G M,WANG Z S,WU D,et al.Effects of dietary cysteamine supplementation on growth performance and whole-body protein turnover in finishing pigs[J].Livestock Science,2009,122(1):86-89.
[12]马现永,林映才,周桂莲,等.半胱胺添加水平对黄羽肉鸡生长性能、肉质及机体抗氧化能力的影响[J].动物营养学报,2009,21(6):916-923.MA X Y,LIN Y C,ZHOU G L,et al.Effects of cysteamine levels on growth performance,meat quality and antioxidant capacity of Yellow Feather broilers[J].Chinese Journal of Animal Nutrition,2009,21(6):916-923.(in Chinese)
[13]朱玉萍,周平,李蛟龙,等.低蛋白氨基酸平衡日粮添加半胱胺对生长猪肉质和相关基因表达的影响[J].畜牧兽医学报,2017,48(4):660-668.ZHU Y P,ZHOU P,LI J L,et al.Effects of cysteamine supplementation on growth pork quality and related gene expression in low protein amino acid balanced diets[J].ActaVeterinaria et Zootechnica Sinica,2017,48(4):660-668.(in Chinese)
[14]秦龙山,邢月腾,张杨,等.半胱胺对宁乡猪胴体性状和肉品质的影响[J].动物营养学报,2017,29(9):3325-3330.QIN L S,XING Y T,ZHANG Y,et al.Effect of cysteamine on carcass traits and meat quality of Ningxiang pigs[J].Chinese Journal of Animal Nutrition,2017,29(9):3325-3330.(in Chinese)
[15]张献新,张益禅,邹芳.缓释包膜半胱胺对生长肥育猪生长性能及胴体品质的影响[J].农业现代化研究,2014,3:326-328.ZHANG X X,ZHANG Y C,ZOU F.Effect of sustained-release coated cysteamine on growth performance and carcass quality of growing-finishing pigs[J].Research of Agricultural Modernization,2014,3:326-328.(in Chinese)
[16]张昌如,周立明,徐利明.缓释包膜半胱胺对育肥猪生产性能的影响[J].上海畜牧兽医通讯,2003,1:22.ZHANG C R,ZHOU L M,XU L M.Effect of sustained-release coated cysteamine on performance of finishing pigs[J].Shanghai Journal of Animal Husbandry and Veterinary,2003,1:22.(in Chinese)
[17]中国国家标准管理委员会.GB/T 24832-2009饲料添加剂.半胱胺盐酸盐β环糊精微粒[S].北京:中国标准出版社,2010.CHINA NATIONAL STANDARDS MANGAGE-MENT COMMITTEE.GB/T 24832-2009Feed additives.Cysteamine hydrochloride beta cyclodextrinparticles[S].Beijing:China Standard Press,2010.(in Chinese)
[18]STACHOWICZ M,LEHMANN B,TIBI A,et al.Determination of total cysteamine in human serum by a high-performance liquid chromatography with fluorescence detection[J].Journal of Pharmaceutical and Biomedical Analysis,1998,17(4-5):767-773.
[19]周禾,李方实.半胱胺(盐酸盐)的测定方法[J].广西师范大学学报(自然科学版),2003,21(4):184-186.ZHOU H,LI F S.Method for determination of cysteamine(hydrochloride)[J].Journal of Guangxi Normal University(Natural Science Edition),2003,21(4):184-186.(in Chinese)
[20]中国兽药典委员会.中华人民共和国兽药典[M].北京:中国农业出版社,2015.CHINESE VETERINARY PHARMACOPOEIACOMMISSION.People’s Republic of China Veterinary Pharmacopoeia[M].Beijing:China Agriculture Press,2015.(in Chinese)
[21]ZHAO F,XIE M L,LI H,et al.Developing a silicon tshaped cannula to sample intestinal digesta in ducks[J].Animal Feed Science and Technology,2016,219:175-180.
[22]廖飞,杨晓,康格非,等.紫外吸收碘量法测定微量维生素C和还原型谷胱甘肽[J].重庆医科大学学报,2003,28(3):372-373.LIAO F,YANG X,KANG G F,et al.Determination of trace vitamin C and reduced glutathione by ultraviolet absorption iodometry[J].Journal of Chongqing Medical University,2003,28(3):372-373.(in Chinese)
[23]朱小云,李生兵,郭睿,等.紫外吸收碘量法快速测定体液维生素C[J].重庆医科大学学报,2004,29(6):841-842.ZHU X Y,LI S B,GUO R,et al.Rapid determination of body fluid vitamin c by ultraviolet absorption iodometry[J].Journal of Chongqing Medical University,2004,29(6):841-842.(in Chinese)
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