Study On Selenium Content And Selenium Form In Human Milk

Objective:Selenium(Se)is an essential trace element for both human and animals,Se is found mostly in the form of selenoprotein and is involved in various biochemical and physiological functions in mammalian systems,such as the enhancement of immunity and oxidation resistance.Several studies on Se-deficiency in infants,especially preterm infants,have reported that the condition might cause growth retardation and acute diseases in childhood along with potentially long-term negative outcomes in adults.Human breast milk is the ideal source of Se intake for infants,relavant nutrients and their composition in which is fundamental reference for the manufacture of infant formula milk.An appropriate recipe for infant formula milk should mimic the composition of human breast milk from healthy,well-nourished women,not only in terms of the content also the chemical form of nutrients whenever possible.Therefore,the total Se content,the main Se compounds and their content of human breast milk from lactating mothers from "Se-adequent region" can provide important scientific basis for the optimal dosage and type selection of Se supplementation in infant formula milk.This study carried out dietary surveys in the lactating mothers from three Se level regions(low-Se,adequent-Se and high-Se),determined Se content in whole blood,total Se content and Se sepeciation in the breast milk of lactating mothers,and conducted a comprehensive analysis.The specific aims include:1)to compare the Se intake from dietary survey with that from the projection of Se in the whole blood;2)to explore variances among different Se level regions on the Se content in dietary intake,in the breast milk and blood respectively,thus to obtain the Se content in the breast milk of lactating mothers from "Se-adequent region" for providing the scientific reference for standard on Se content in infant formula;3)to establish qualitative and quantitative methods for determining the Se in breast milk;4)to explore Se speciation in the breast milk among different Se level regions,thus to obtain the Se speciation and specific compositions of lactating mothers from "adequent-Se region" and to provide scientific basis for the Se compounds and specific content supplemented in infant formula milk;5)to explore the effect of Se intake of lactating mothers on the Se content and Se sepeciation in breast milk.Methods:In this study,a stratified random sampling was used to select 20 lactating women per site,from Liangshan autonomous prefecture in Sichuan Province(low-Se region),Xicheng District in Beijing City(adequent-Se region)and Enshi City in Hubei Province(high-Se region),respectively.Dietary surveys for lactating women was carried out and the dietary nutritiens including Se was determined.Anova analysis was used to compare the differeces among three regions.Blood and milk samples in perinatal period of lactating women were collected along with the dietary survey.The total Se content in whole blood and milk was determined by inductively coupled plasma mass spectrometry,then the dietary Se intake was calculated from Se content in whole blood.Anova analysis was used to compare the differeces between dietary Se intake calculated from dietary survey and Se content in whole blood in three different Se-level regions.Obtain the mean of Se content in milk of lactating mothers from "Se-adequent region".An ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for the quantification of free and protein-binding selenomethionine(SeMet),selenocystine(SeCys2)was established and applied in human breast milk and cow milk.An alkylated-UPLC-MS/MS method was established for the qualitative determination selenocysteine(SeCys),and selenocystamine(SeCysA)in human breast milk and cow milk.The analyte was separated on an Acquity UPLC Amide column with gradient elution.At positive electrospray ionization mode,multiple reaction monitoring of the precursor-product ion transitions of m/z 198.0?181.1(SeMet)and m/z 337.0?248.0(SeCys2)was used for the quantification.Acetonitrile precipitation was used as pretreatment of free Se speciation analysis,and enzyme hydrolysis was used as pretreatment of free and protein-bounding Se speciation analysis.An high performance liquid chromatography-inductively coupled plasma mass spectrometry(HPLC-ICP-MS)method for the quantification of SeMet,SeCys2,methyl selenocysteine(MSC)and inorganic Se was established and applied to the quantification of protein-binding SeMet,SeCys2,MSC and inorganic Se in human breast milk.Milk samples in perinatal period of lactating women were collected and analysed.Anova analysis was used to compare the differeces of Se speciation and its content in human breast milk among three different regions,then the relationship between dietary Se intake and the Se speciation and its content in human breast milk was explored.Results:Se intake of lactating women from Liangshan Autonomous Prefecture of Sichuan,Xicheng district of Beijing and Enshi of Hubei based on dietary survey was X±SD 41.57±21.19 ng/d,51.06±22.60 ng/d and 614.84±177.80 ng/d,respectively(P<0.05).Se intake of lactating women from three regions above based on the projection of Se in the whole blood was X ± SD 31.18±13.28 ng/d,41.58±10.48 ng/d,152.01±121.33 ng/d,respectively(P<0.05).And a significant difference was found between the two methods above(P<0.05).Refered to the 2013 revised RNI and UI values of lactating mothers,results from two methods indicated dietary Se intakes of lactating mothers from Liangshan and Beijing were insufficient.Dietary Se intakes of Enshi(based on the dietary survey)were higher than UI,and dietary Se intake(projection from the total Se in the whole blood)were higher than that of the other two regions,wherein some higher than UI.The total Se content in human breast milk of Liangshan,Beijing and Enshi was 11.73 ± 9.33 ng/mL,25.31 ± 10.22 ng/mL and 44.28 ± 26.92 ng/mL,respectively(P<0.05).Multivariate analysis showed that dietary Se intake had a positive effect on the total Se content in human breast milk(P<0.0001,adjusted R2 = 0.5924).The corresponding mean of total Se content in lactating mother from "adequent-Se region"was 25.31 ?g/L,indicating that 3.616?4.218 p.g/100 kcal was the most appropriate Se content in infant formula milk in China.An UPLC-MS/MS method was well established for the quantification of free and protein-binding SeMet and SeCys2 in human breast milk and cow milk.Since SeCys and SeCysA were instable and easy to be oxidized,an alkylated-UPLC-MS/MS method was established for the qualitative determination,while quantitative analysis was canceled subject to lack of available standards.The analysis of Se speciation in cow milk and human breast milk from different Se-level regions indicated that:1)The Se existed mainly in the form of organic speciation,the most two forms of which were SeMet and SeCys,there was no exsitance of inorganic Se,or somewhat few if any.2)Total SeMet and SeCys2 concentrations in the enzymolyzed human breast milk from Liangshan was 3.98 ± 1.16 ng Se/mL and 3.56 ± 1.96 ng Se/mL,wherein the corresponding proportion of the free SeMet and SeCys2 were 10.41%± 7.85%and 0.84%±1.16%,respectively.Total SeMet and SeCys2 concentrations in the enzymolyzed human breast milk from Beijing was 2.51 ± 1.69 ng Se/mL and 21.41 ±2.02 ng Se/mL,wherein the corresponding proportion of the free SeMet and SeCys2 were 25.86%±20.76%and 1.470%± 0.49%,respectively.Total SeMet and SeCys2 concentrations in the enzymolyzed human breast milk from Enshi was 15.34 ± 10.61 ng Se/mL and 39.50±16.77 ng Se/mL,wherein the corresponding proportion of the free SeMet and SeCys2 were 26.45%±48.11%and 1.48%±1.39%,respectively;3)The contents of different organic Se in human milk were all higher than those in cow milk;4)Free Se compounds accounted for only 2%?3%of the total Se in milk,and existed in the form of protein-binding.;5)The main Se speciation in human breast milk of "adequent-Se lactating mother" were SeMet and SeCys2,and total SeMet and SeCys2 concentrations were 2.51 ng Se/mL and 21.41 ng Se/mL,which accounted 9.94%and 87.79%for the total Se,respectively;6)Multivariate analysis showed that dietary Se intake had a positive effect on the SeMet and SeCys2 content in human breast milk.Conclusion:1)Significances of the dietary Se intake from dietary survey,total Se content in the whole blood and total Se content in human breast milk of lactating mothers from different Se-level regions were found;2)The corresponding mean of total Se content in lactating mother from "adequent-Se region" was 25.31 ?g/L,indicating that 3.616?4.218 ?g/100 kcal was the most suitable Se content in infant formula milk for Chinese infants,meanwhile larger-scale data was still needed;3)Since SeCys and SeCysA were instable and easy to be oxidized,alkylation was an essential step before the qualitative determination;4)Se in human breast milk mainly exsit in the form of organic Se,which was mainly incorporated into protein,and inorganic Se was mere if any;5)The main Se speciation detected in human breast milk of lactating mother from"adequent-Se region" were SeMet and SeCys2,and total SeMet and SeCys2 concentrations were 2.51 ng Se/mL and 21.41 ng Se/mL,which account 9.94%and 87.79%for the total Se,respectively.To date,Se compounds used in infant formula milk(Na2SeO3,Na2SeO4)in most countries are different from those in human breast milk,indicating that they were not the most suitable type of Se supplements in infant formula milk.Organic Se,such as SeMet and SeCys2,should be selected as optimal Se compounds supplemented in infant formula mik.