Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection

doi:10.1016/S0378-4347(00)80766-5

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 377, 1986, Pages 111-119

https://doi.org/10.1016/S0378-4347(00)80766-5 Get rights and content

Abstract

A simple, rapid and highly sensitive high-performance liquid chromatographic method for the determination of N-acetyl- and N-glycolylneuraminic acids in serum and urine is described. The neuraminic acids, released by hydrolysis of serum and urine, are converted in dilute sulphuric acid with 1,2-diamino-4,5-dimethoxybenzene, a fluorogenic reagent for α-keto acids, to highly fluorescent derivatives. The derivatives are separated isocratically within 8 min by reversed-phase chromatography using a Radial-Pak cartridge C₁₈ column and detected fluorimetrically. The limit of detection is 40 fmol (12 pg) for both neuraminic acids in 10-μl injection volume [0.3 nmol (90 ng)/ml) of serum or urine]. This sensitivity permits the precise determination of the neuraminic acids in 5 μl of serum or urine. The method was applied to the determination of the neuraminic acids in sera from normal subjects and cancer patients, normal urine and rat serum.

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Citation Excerpt :
This is due to its high selectivity for the diketone species formed when sialic acid is hydrolysed under mildly acidic conditions. The method is very specific and is also highly sensitive with studies reporting detection limits of sialic acids as low as 12 pg to as high as 0.46 ng.76,78,80,81 One study reported the lower limit of detection as 0.3 mg but this appears to be due to the fact that this was the lowest size standard sample used by the authors and it appears that further validation with smaller sample quantities could be carried out.79
N-Acetyl neuraminic acid (sialic acid) is a monosaccharide generally found as the terminating unit on glycans, which in turn are found on the surface of cells and glycoproteins. These glycans aid in a variety of biological functions such as cell interactions and immune response. Sialic acid has been identified as a biomarker for cardiovascular disease, diabetes and a range of other inflammatory and degenerative conditions. It has also been identified as a marker for different types of cancer. Sialic acid levels vary depending on the level of inflammation present during the course of an inflammatory disease and it is overexpressed by tumours as a shield against the immune system. Since the discovery of sialic acid, numerous assays have been developed for the identification and quantification of different sialic acid derivative monosaccharides and these assays fall into four main groups: colorimetric, fluorometric, enzymatic and chromatographic/mass spectrometric, with much overlap between these. Given the importance of sialic acids in biological pathways, this review article critically appraises assays that are used to detect and quantify sialic acid and its derivatives. Thus it details the method, sensitivity, specificity and wider scope of a range of assays, and concludes by suggesting some future directions for assay development and application. In this way, insight is provided into assays that allow for the accurate quantitation of sialic acid in biological samples, which may facilitate identification of the roles of sialic acid in healthy and disease pathways.
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Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection

Abstract

J. Biol. Chem.

Virology

Anal. Biochem.

Anal. Biochem.

Methods Enzymol.

FEBS Lett.

J. Chromatogr.

Anal. Biochem.

J. Chromatogr.

Anal. Chim. Acta