Application of a sensitive HPLC-based fluorometric assay to determine the sialic acid content of human gonadotropin isoforms

The human pituitary gonadotropins, follitropin (hFSH) and lutropin (hLH) are glycoproteins which are microheterogeneous in terms of their charge and molecular size, as well as their in vitro and in vivo bioactivities. The aim of this study was to determine the contribution of variations in sialic acid (N-acetyl neuraminic acid) content to the structural heterogeneity of these glycoproteins. Sialic acid (Neu5Ac) was released by partial acid hydrolysis (0.1 M TFA, 80°C, 1 h) and derivatised with the fluorescent label DMB (1,2-diamino-4,5-methylenedioxybenzene) in conjunction with an internal standard (N-glycoyl-neuraminic acid). The derivatives were then separated by reversed-phase HPLC. This method allowed quantitation of the sialic acid content over a range of 5-100 pmol with between assay variation of < 6% for sialic acid released from approximately 100 ng (3 pmol) of hFSH or hLH. Comparison of the sialic acid contents of standard sialylated glycoproteins by either DMB-derivatisation or high-performance anion-exchange chromatography with pulsed amperometric detection yielded similar results, confirming the reliability of the fluorescence detection method. The sialic acid contents of 9 hFSH isoforms varied between 1.5-13.7 mol Neu5AC/mol FSH, whilst a range of 1.1-9.1 mol Neu5AC/mol LH was observed for 12 hLH isoforms. The sialic acid content of the hFSH isoform was also observed to be related to the hormonal specific activity in a radioreceptor assay, confirming that alterations in the carbohydrate structure can influence the FSH-receptor interaction. In contrast, the sialic acid content of the hLH isoforms was found to be not related to specific activity at the receptor level.