Chemoenzymatic Synthesis of N-Propionyl-Neuraminic Acid Derivative and Its Application in the Determination of Sialic Acid Content in Poultry Eggs
-
摘要: 为实现禽类蛋黄和蛋清中N-乙酰神经氨酸（N-acetylneuraminic acid，Neu5Ac）的准确定量，消除禽蛋样品中分析物以外的物质产生的基质效应对分析结果造成的影响，本研究以甘露糖胺为底物采用化学酶法合成了非天然唾液酸衍生物5-溴-4-氯-3-吲哚-β-D-半乳糖苷-N-丙酰-唾液酸（5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside-N-propionyl-sialic acid，X-Gal-α-2，6-Neu5Prop），对该衍生物在酸性条件下的水解特性以及稳定性进行研究，并以该衍生物为内标测定了鹌鹑、鹅、珍珠鸡、鸵鸟、鸭、鸽子、火鸡等9种禽类蛋黄和蛋清中Neu5Ac的含量。禽蛋样品和内标物在酸性条件下处理，唾液酸糖缀合物被解离为游离的唾液酸后经荧光标记物邻苯二胺衍生，采用高效液相色谱-荧光检测器对样品进行分析。结果表明，相同浓度的X-Gal-α-2，6-Neu5Ac和X-Gal-α-2，6-Neu5Prop在2 mol/L的醋酸溶液80℃的水解条件下水解程度相当，90 min后能够完全转化为游离的唾液酸。Neu5Ac和Neu5Prop在10 h内仍保持相似的稳定性。蛋清中Neu5Ac的含量在不同物种之间表现出很大的差异。鹌鹑蛋清中Neu5Ac的含量最低（0.13 mg/g），而鸵鸟蛋清中的最高（2.20 mg/g），比鹌鹑蛋清Neu5Ac含量高出近17倍。该方法能够消除生物样品中的基质效应，实现对Neu5Ac的准确定量，且样品前处理简单。因此，适合常规食品中Neu5Ac的检测与分析。
-
关键词:
- 唾液酸 /
- Neu5Ac /
- 蛋清 /
- 蛋黄 /
- 高效液相色谱-荧光检测器(HPLC-FLD)
Abstract: To realize the accurate quantification of N-acetylneuraminic acid(Neu5Ac)in egg yolk and egg white of poultry,and eliminate the matrix effect caused by substances other than analytes in egg samples,5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside-N-propionyl-sialic acid(X-Gal-α-2,6-Neu5Prop)was synthesized by chemical enzymatic method using mannosamine as substrate. The hydrolysis characteristics and stability of the derivative under acidic conditions were studied. The content of Neu5Ac in egg yolk and egg white of quail,goose,guinea fowl,ostrich,duck,pigeon and turkey was determined by using the derivative as internal standard. The glycoconjugates of sialic acid were dissociated into free sialic acid after being treated in acidic condition. The samples were analyzed by high performance liquid chromatography with fluorescence detector(HPLC-FLD).It was found that the hydrolysis rate of X-gal-α-2,6-Neu5aAc and X-gal-α-2,6-Neu5Prop at the same concentration were similar under the condition of 2 mol/L acetic acid solution at 80 ℃,which could be completely converted into free sialic acid after 90 min. The stability of Neu5Ac and Neu5Prop were similar at 10 h. The Neu5Ac content of the egg whites showed a wide variation across species. Quail egg whites had the lowest concentration of Neu5Ac(0.13 mg/g),while the ostrich had the highest(2.20 mg/g),which was seventeen times higher nearly. And Neu5Ac content of chicken egg yolk and albumen was found to be in agreement with previous reports. The method of analysis described herein could eliminate the matrix effect in biological samples and quantify the content of Neu5Ac accurately,with facile sample preparation requirements and was well suited to the routine analysis of foodstuffs. -
[1] Traving C,Schauer R.Structure,function and metabolism of sialic acids[J].Cellular and Molecular Life Sciences,1998,54(12):1330-1349.
[2] Lamari F N,Karamanos N K.Separation methods for sialic acids and critical evaluation of their biologic relevance[J].Journal of Chromatography B,2002,781(1/2):3-19.
[3] Angata T,Varki A.Chemical diversity in the sialic acids and related α-keto acids:An evolutionary perspective[J].Chemical Reviews,2002,102(2):439-470.
[4] Cohen M,Varki A. The sialome——far more than the sum of its parts[J]. Omics:A Journal of Integrative Biology,2010,14(4):455-464.
[5] Okerblom J,Varki A.Biochemical,cellular,physiological,and pathological consequences of human loss of N-glycolylneuraminic acid[J].Chem Bio Chem,2017,18(13):1155-1171.
[6] Schauer R.Sialic acids:Fascinating sugars in higher animals and man[J].Zoology,2004,107(1):49-64.
[7] Siebert H C,Kaptein R,Beintema J J,et al.Carbohydrate-protein interaction studies by laser photo CIDNP NMR methods[J].Glycoconjugate Journal,1997,14(4):531-534.
[8] Zheng F,Du Y M,Lin X S,et al.N-glycosylation plays an essential and species-specific role in anti-infection function of milk proteins using Listeria monocytogenes as model pathogen[J]. Journal of Agricultural and Food Chemistry,2019,67(38):10774-10781.
[9] Wu C X,Xia L,Liu L J,et al.A sensitive and efficient method for determination of N-acetylhexosamines and N-acetylneuraminic acid in breast milk and milk-based products by high-performance liquid chromatography via UV detection and mass spectrometry identification[J].Journal of Chromatography B,2016,1011:14-23.
[10] Bratosin D,Mazurier J,Tissier J P,et al.Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages.A review[J].Biochimie,1998,80(2):173-195.
[11] 刘志东,王荫榆,郭本恒,等.唾液酸的研究进展[J].食品工业科技,2010,31(4):368-373. [12] Wang F,Xie B Y,Wang B,et al.LC-MS/MS glycomic analyses of free and conjugated forms of the sialic acids,Neu5Ac,Neu5Gc and KDN in human throat cancers[J].Glycobiology,2015,25(12):1362-1374.
[13] Wang B,Miller J B,McNeil Y,et al.Sialic acid concentration of brain gangliosides:Variation among eight mammalian species[J].Comparative Biochemistry and Physiology Part A:Molecular & Integrative Physiology,1998,119(1):435-439.
[14] Warren L.The thiobarbituric acid assay of sialic acids[J].The Journal of Biological Chemistry,1959,234(8):1971-1975.
[15] Koketsu M,Sakuragawa E,Linhardt R J,et al.Distribution of N-acetylneuraminic acid and sialylglycan in eggs of the Silky fowl[J].British Poultry Science,2003,44(1):145-148.
[16] Yao H L,Conway L P,Wang M M,et al.Quantification of sialic acids in red meat by UPLC-FLD using indoxylsialosides as internal standards[J].Glycoconjugate Journal,2016,33(2):219-226.
[17] Cai Z,Conway L P,Huang Y Y,et al.Enzymatic synthesis of trideuterated sialosides[J].Molecules,2019,24(7):1368.
[18] Cao C,Wang W J,Huang Y Y,et al.Determination of sialic acids in liver and milk samples of wild-type and CMAH knock-out mice[J].Journal of Visualized Experiments,2017(125):e56030.
[19] Juneja L R,Koketsu M,Nishimoto K,et al.Large-scale preparation of sialic acid from chalaza and egg-yolk membrane[J].Carbohydrate Research,1991,214(1):179-186.
[20] Koketsu M,Juneja L R,Kawanami H,et al.Preparation of N-acetylneuraminic acid from delipidated egg yolk[J]. Glycoconjugate Journal,1992,9(2):70-74.
[21] Hatta H,Kapoor M P,Juneja L R.Bioactive components in egg yolk[M]//Egg Bioscience and Biotechnology.Hoboken,NJ,USA:John Wiley & Sons,Inc.:185-237.
[22] Both P,Riese M,Gray C J,et al.Applications of a highly α2,6-selective pseudosialidase[J].Glycobiology,2018,28(5):261-268.
[23] Reyes Martínez J E,Šardzík R,Voglmeir J,et al.Enzymatic synthesis of colorimetric substrates to determine α-2,3-and α-2,6-specific neuraminidase activity[J].RSC Advances,2013,3(44):21335.
[24] Wang S,Laborda P,Lu A,et al.N-acetylglucosamine 2-epimerase from Pedobacter heparinus:First experimental evidence of a deprotonation/reprotonation mechanism[J]. Catalysts,2016,6(12):212.
[25] Huang K,Wang M M,Kulinich A,et al.Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila[J].Carbohydrate Research,2015,415:60-65.
[26] Maurer U,Neumann U,Šoštaric' K,et al.S19.11 Different distribution of glycosphingolipids in mouse and rabbit skeletal muscle demonstrated by biochemical and immunohistological analyses[J].Glycoconjugate Journal,1993,10(4):337.
[27] Kloepfer A,Quintana J B,Reemtsma T.Operational options to reduce matrix effects in liquid chromatography-electrospray ionisation-mass spectrometry analysis of aqueous environmental samples[J].Journal of Chromatography A,2005,1067(1/2):153-160.
[28] 刘敏,刘戎,王立琦,等.猪肝中β-受体激动剂多残留的样品前处理方法比较及同时检测[J].分析测试学报,2012,31(3):290-295. [29] 贾彦波,王清清,宋海峰.高效液相色谱-串联质谱法(HPLC-MSn)分析生物样品时的基质效应研究[J].军事医学,2011,35(2):149-152. [30] Schauer R.Achievements and challenges of sialic acid research[J].Glycoconjugate Journal,2000,17(7/8/9):485-499.
计量
- 文章访问数:
- HTML全文浏览量:
- PDF下载量:
下载:
