Glycoconjugate Journal

, Volume 33, Issue 2, pp 219–226 | Cite as

Quantification of sialic acids in red meat by UPLC-FLD using indoxylsialosides as internal standards

  • Hong L. Yao
  • Louis P. Conway
  • Mao M. Wang
  • Kun Huang
  • Li LiuEmail author
  • Josef VoglmeirEmail author
Original Article


Herein we describe a UPLC-FLD-based method for the quantification of the sialic acid content of red meat, using a synthetic neuraminic acid derivative as an internal standard. X-Gal-α-2,6-N-propionylneuraminic acid was synthesized via a chemoenzymatic pathway and its hydrolytic stability was characterized. Known quantities of this compound were incubated with samples of red meat under sialic acid-releasing conditions. The released sialic acids were derivatized, analyzed by UPLC-FLD, and the Neu5Ac/Neu5Gc content of the meat sample was determined by comparison with the internal standard. A number of red meats were analyzed by this method with the following results (Neu5Ac μg/g tissue, Neu5Gc μg/g tissue ± s.d.): pork (68 ± 3, 15.2 ± 0.7), beef (69 ± 8, 36 ± 5), lamb (46 ± 2, 33 ± 1), rabbit (59 ± 2, 0.4 ± 0.4), and hare (50 ± 4, 1 ± 1). We envisage that this methodology will find application in investigating the health effects of dietary Neu5Gc.

Graphical abstract


Sialic acid Neu5Gc N-glycolylneuraminic acid, Neu5Ac red meat UPLC-FLD 



cytidyl 5′-monophosphate




Escherichia coli neuraminic acid synthase


electrospray ionization


fluorescence detector


α-2,6-keto-3-deoxy-d-glycero-d-galactononic acid


matrix-assisted laser desorption ionization


mass spectrometry


N-acetylneuraminic acid


N-glycolylneuraminic acid


N-propylneuraminic acid




Neisseria meningitidis CMP-sialic acid synthase


Photobacterium damselae α-2,6-sialyltransferase




ultra high performance liquid chromatography





The authors would like to thank Prof. Akemi Suzuki (Hiratsuka, Japan) for helpful discussions on sialic acids.

Compliance with ethical standards

Source of funding

This work was supported in parts by the Natural Science Foundation of China (grant number 31,471,703 to L.L. and J.V., A0201300537 to J.V. and L.L.), Natural Science Foundation of the Jiangsu Province Higher Education Institutions (grant number 13KJD230002 to L.H.Y.), and the 100 Foreign Talents Plan (grant number JSB2014012 to J.V.).

Supplementary material

10719_2016_9659_MOESM1_ESM.pdf (167 kb)
ESM 1 (PDF 166 kb)


  1. 1.
    Angata T., Varki A.: Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem. Rev. 102, 439–469 (2002)CrossRefPubMedGoogle Scholar
  2. 2.
    Schauer R.: Achievements and challenges of sialic acid research. Glycoconj. J. 17, 485–499 (2000)CrossRefPubMedGoogle Scholar
  3. 3.
    Chen X., Varki A.: Advances in the biology and chemistry of sialic acids. ACS Chem. Biol. 5, 163–176 (2010)CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lacomba R., Salcedo J., Alegria A., Barbera R., Hueso P., Matencio E., Lagarda M.J.: Effect of simulated gastrointestinal digestion on sialic acid and gangliosides present in human milk and infant formulas. J. Agric. Food Chem. 59, 5755–5762 (2011)CrossRefPubMedGoogle Scholar
  5. 5.
    Lacomba R., Salcedo J., Alegria A., Jesus Lagarda M., Barbera R., Matencio E.: Determination of sialic acid and gangliosides in biological samples and dairy products: a review. J Pharmaceut Biomed. 51, 346–357 (2010)CrossRefGoogle Scholar
  6. 6.
    Nie H., Li Y., Sun X.L.: Recent advances in sialic acid-focused glycomics. J. Proteome. 75, 3098–3112 (2012)CrossRefGoogle Scholar
  7. 7.
    Chou H.H., Takematsu H., Diaz S., Iber J., Nickerson E., Wright K.L., Muchmore E.A., Nelson D.L., Warren S.T., Varki A.: A mutation in human CMP-sialic acid hydroxylase occurred after the homo-Pan divergence. P Natl Acad Sci USA. 95, 11751–11756 (1998)CrossRefGoogle Scholar
  8. 8.
    Lamari F.N., Karamanos N.K.: Separation methods for sialic acids and critical evaluation of their biologic relevance. J. Chromatogr. B. 781, 3–19 (2002)CrossRefGoogle Scholar
  9. 9.
    Rehan I.F., Ueda K., Mitani T., Amano M., Hinou H., Ohashi T., Kondo S., Nishimura S.-I.: Large-scale glycomics of livestock: discovery of highly sensitive serum biomarkers indicating an environmental stress affecting immune responses and productivity of Holstein dairy cows. J. Agric. Food Chem. 63, 10578–10590 (2015)CrossRefPubMedGoogle Scholar
  10. 10.
    Wang B., McVeagh P., Petocz P., Brand-Miller J.: Brain ganglioside and glycoprotein sialic acid in breastfed compared with formula-fed infants. Am J Clin Nutr. 78, 1024–1029 (2003)PubMedGoogle Scholar
  11. 11.
    Wang B.: Molecular mechanism underlying sialic acid as an essential nutrient for brain development and cognition. Adv Nutr. 3, 465S–472S (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gulesserian T., Engidawork E., Fountoulakis M., Lubec G.: Manifold decrease of sialic acid synthase in fetal down syndrome brain. Amino Acids. 32, 141–144 (2007)CrossRefPubMedGoogle Scholar
  13. 13.
    Uslu E, Guzey FK, Oguz E, Guzey D The effects of ageing on brain tissue sialic acid contents following cold trauma. Acta Neurochir. 146:1337–1340 discussion 1340 2004Google Scholar
  14. 14.
    Wang B., Miller J.B., McNeil Y., McVeagh P.: Sialic acid concentration of brain gangliosides: variation among eight mammalian species. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 119, 435–439 (1998)CrossRefPubMedGoogle Scholar
  15. 15.
    Wang B., Brand-Miller J.: The role and potential of sialic acid in human nutrition. Eur. J. Clin. Nutr. 57, 1351–1369 (2003)CrossRefPubMedGoogle Scholar
  16. 16.
    Wang B., Brand-Miller J., McVeagh P., Petocz P.: Concentration and distribution of sialic acid in human milk and infant formulas. Am J Clin Nutr. 74, 510–515 (2001)PubMedGoogle Scholar
  17. 17.
    Morgan B.L., Winick M.: Effects of administration of N-acetylneuraminic acid (NANA) on brain NANA content and behavior. J Nutr. 110, 416–424 (1980)PubMedGoogle Scholar
  18. 18.
    Samraj A.N., Laubli H., Varki N., Varki A.: Involvement of a non-human sialic acid in human cancer. Front Oncol. 4, 33 (2014)PubMedPubMedCentralGoogle Scholar
  19. 19.
    Diaz S.L., Padler-Karavani V., Ghaderi D., Hurtado-Ziola N., Yu H., Chen X., der Linden EC B.-v., Varki A., NM V.: Sensitive and specific detection of the non-human sialic acid N-glycolylneuraminic acid in human tissues and biotherapeutic products. PLoS One. 4, e4241 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Nguyen D.H., Tangvoranuntakul P., Varki A.: Effects of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cells. J. Immunol. 175, 228–236 (2005)CrossRefPubMedGoogle Scholar
  21. 21.
    der Linden EC B.-v., Sjoberg E.R., Juneja L.R., Crocker P.R., Varki N., Varki A.: Loss of N-glycolylneuraminic acid in human evolution. Implications for sialic acid recognition by siglecs. J Biol Chem. 275, 8633–8640 (2000)CrossRefGoogle Scholar
  22. 22.
    Tangvoranuntakul P., Gagneux P., Diaz S., Bardor M., Varki N., Varki A., Muchmore E.: Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. P Natl Acad Sci USA. 100, 12045–12050 (2003)CrossRefGoogle Scholar
  23. 23.
    Hedlund M., Padler-Karavani V., Varki N.M., Varki A.: Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression. P Natl Acad Sci USA. 105, 18936–18941 (2008)CrossRefGoogle Scholar
  24. 24.
    Pham T., Gregg C.J., Karp F., Chow R., Padler-Karavani V., Cao H., Chen X., Witztum J.L., Varki N.M., Varki A.: Evidence for a novel human-specific xeno-auto-antibody response against vascular endothelium. Blood. 114, 5225–5235 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Lofling J.C., Paton A.W., Varki N.M., Paton J.C., Varki A.: A dietary non-human sialic acid may facilitate hemolytic-uremic syndrome. Kidney Int. 76, 140–144 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Byres E., Paton A.W., Paton J.C., Lofling J.C., Smith D.F., Wilce M.C., Talbot U.M., Chong D.C., Yu H., Huang S., Chen X., Varki N.M., Varki A., Rossjohn J., Beddoe T.: Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. Nature. 456, 648–652 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Svennerholm L.: Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim. Biophys. Acta. 24, 604–611 (1957)CrossRefPubMedGoogle Scholar
  28. 28.
    Warren L.: The thiobarbituric acid assay of sialic acids. J Biol Chem. 234, 1971–1975 (1959)PubMedGoogle Scholar
  29. 29.
    Hurum D.C., Rohrer J.S.: Determination of sialic acids in infant formula by chromatographic methods: a comparison of high-performance anion-exchange chromatography with pulsed amperometric detection and Ultra-high-performance liquid chromatography methods. J. Dairy Sci. 95, 1152–1161 (2012)CrossRefPubMedGoogle Scholar
  30. 30.
    Ito M., Ikeda K., Suzuki Y., Tanaka K., Saito M.: An improved fluorometric high-performance liquid chromatography method for sialic acid determination: an internal standard method and its application to sialic acid analysis of human apolipoprotein E. Anal. Biochem. 300, 260–266 (2002)CrossRefPubMedGoogle Scholar
  31. 31.
    Huang K., Wang M.M., Kulinich A., Yao H.L., Ma H.Y., Martinez J.E., Duan X.C., Chen H., Cai Z.P., Flitsch S.L., Liu L., Voglmeir J.: Biochemical characterisation of the neuraminidase pool of the human gut symbiont akkermansia muciniphila. Carbohydr. Res. 415, 60–65 (2015)CrossRefPubMedGoogle Scholar
  32. 32.
    Knorst M., Fessner W.D.: CMP-sialate synthetase from Neisseria meningitidis - overexpression and application to the synthesis of oligosaccharides containing modified sialic acids. Adv. Synth. Catal. 343, 698–710 (2001)CrossRefGoogle Scholar
  33. 33.
    Martinez J.E.R., Sardzik R., Voglmeir J., Flitsch S.L.: Enzymatic synthesis of colorimetric substrates to determine alpha-2,3-and alpha-2,6-specific neuraminidase activity. RSC Adv. 3, 21335–21338 (2013)CrossRefGoogle Scholar
  34. 34.
    Lapidot Y., Rappoport S., Wolman Y.: Use of esters of N-hydroxysuccinimide in the synthesis of N-acylamino acids. J. Lipid Res. 8, 142–145 (1967)PubMedGoogle Scholar
  35. 35.
    Anumula K.R.: Rapid quantitative determination of sialic acids in glycoproteins by high-performance liquid chromatography with a sensitive fluorescence detection. Anal. Biochem. 230, 24–30 (1995)CrossRefPubMedGoogle Scholar
  36. 36.
    Terada T, Kitazume S, Kitajima K, Inoue S, Ito F, Troy FA, Inoue Y Synthesis of CMP-deaminoneuraminic acid (CMP-KDN) using the CTP:CMP-3-deoxynonulosonate cytidylyltransferase from rainbow trout testis. Identification and characterization of a CMP-KDN synthetase. Synthesis of CMP-deaminoneuraminic acid (CMP-KDN) using the CTP:CMP-3-deoxynonulosonate cytidylyltransferase from rainbow trout testis. Identification and characterization of a CMP-KDN synthetase 268:2640–8 1993Google Scholar
  37. 37.
    Spichtig V., Michaud J., Austin S.: Determination of sialic acids in milks and milk-based products. Anal. Biochem. 405, 28–40 (2010)CrossRefPubMedGoogle Scholar
  38. 38.
    Muthing J., Maurer U., Sostaric K., Neumann U., Brandt H., Duvar S., Peter-Katalinic J., Weber-Schurholz S.: Different distributions of glycosphingolipids in mouse and rabbit skeletal muscle demonstrated by biochemical and immunohistological analyses. J. Biochem. 115, 248–256 (1994)PubMedGoogle Scholar
  39. 39.
    Nakamura K., Ariga T., Yahagi T., Miyatake T., Suzuki A., Yamakawa T.: Interspecies comparison of muscle gangliosides by two-dimensional thin-layer chromatography. J. Biochem. 94, 1359–1365 (1983)PubMedGoogle Scholar
  40. 40.
    Müthing J., Maurer U., Šoštarié K., Neumann U., Brandt H., Duvar S., Peter-Katalinié J., Weber-Schurholz S.: Different distributions of glycosphingolipids in mouse and rabbit skeletal muscle demonstrated by biochemical and immunohistological analyses. J. Biochem. 115, 248–256 (1994)PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and TechnologyNanjing Agricultural UniversityNanjingChina
  2. 2.Food Science DepartmentJingling Institute of TechnologyNanjingChina

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