Quantitation of picomole levels of N-acetyl- and N-glycolylneuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay |
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| Authors: | L D Powell G W Hart |
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| Affiliation: | 1. Pamukkale University, Faculty of Art and Science, Chemistry Department, Denizli, Turkey;2. Sen Research Group, Biochemistry Department, Faculty of Arts and Science, Dumlupınar University, Evliya Çelebi Campus, 43100 Kutahya, Turkey;1. PhD Program of Environmental and Occupational Medicine & Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan;2. Department of Medicinal and Applied Chemistry, Kaohsiung Medical University (KMU), Kaohsiung City 807, Taiwan;3. Department of Public Health, College of Health Sciences, Kaohsiung Medical University, Kaohsiung City 807, Taiwan;4. Department of Family Medicine, Kaohsiung Medical University Hospital (KMUH), Kaohsiung City 807, Taiwan;5. Ph.D. Program of Aquatic Science and Technology, & Department of Marine Environmental Engineering, College of Hydrosphere Science, National Kaohsiung University of Science and Technology (NKUST), Kaohsiung City 811, Taiwan;6. Department of Medical Research, Kaohsiung Medical University Hospital (KMUH), Kaohsiung city 807, Taiwan;7. Department of Chemistry, National Sun Yat-sen University, Kaohsiung City 804, Taiwan;1. Chemistry Department, University of Manitoba, 144 Dysart Road, Winnipeg, MB R2T 2N2, Canada;2. INSERM UMR 10-64, Institut de Transplantation Urology Nephrology (ITUN), Université de Nantes, France |
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| Abstract: | A simple HPLC adaptation of the periodate-TBA assay for free N-acetyl- and N-glycolylneuraminic acids greatly extends the sensitivity and increases the specificity of this standard colorimetric assay. The method, employing a C18 reverse-phase column eluted isocratically with a phosphoric acid-MeOH buffer, is linear over a range of 2 pmol to 20 nmol. Analyses can be performed directly on cell lysates and digests without prior purification of released sialic acids from contaminating salts and biological materials. Interference from 2-deoxysugars is completely eliminated as the chromophore from these compounds is completely resolved from that derived from sialic acids. The application of the technique to quantify cell-surface and total cellular TBA-reactive sialic acids on the surfaces of a variety of tumor cells is described. Additionally, the extent of desialylation of erythrocytes necessary to expose the T antigen is determined. |
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