Presentation Information
[P01-117]A Rapid RP-Ion Pairing HPLC-UV Method for Quantification of Sialic Acid in Cancer Cell Matrices to Evaluate Sialyltransferase Inhibition
○Stephan M Levonis1, Stephanie S Schweiker1 (1. Bond University (Australia))
Keywords:
HPLC,Ion-Pairing,Sialyltransferase,Neu5Ac,Sialylation
[Purpose]
Sialylation is a critical post-translational modification of cell surface glycoconjugates and plays an essential role in numerous biological processes, including cell signaling, immune recognition, and tumor progression. Sialyltransferases, the enzymes responsible for transferring sialic acid residues to glycoproteins and glycolipids, are frequently overexpressed in various cancers and contribute to tumor invasiveness, metastasis, and immune evasion. Consequently, sialyltransferases have emerged as promising therapeutic targets in anticancer drug development. Reliable analytical methods capable of quantifying sialic acid levels in complex biological matrices are therefore important for evaluating enzyme activity and screening potential inhibitors. Although several analytical approaches have been reported for sialic acid determination, many rely on complex derivatization procedures, expensive instrumentation, or have limited applicability to heterogeneous cellular matrices. Furthermore, the quantification of both free and protein-bound sialic acids within intact cancer cell systems remains insufficiently explored.
[Method]
In this study, a simple, rapid, and cost-effective analytical method was developed to detect and quantify sialic acids in cancer cell matrices for the evaluation of sialyltransferase activity. The method employs reverse-phase ion-pairing high-performance liquid chromatography coupled with ultraviolet detection (RP-IP-HPLC-UV). Triisopropanolamine was utilized as the ion-pairing reagent in combination with a C18 stationary phase to enable effective retention and separation of N-acetylneuraminic acid (Neu5Ac), the most common form of sialic acid in mammalian systems. Human cervical carcinoma (HeLa) and human cholangiocarcinoma (HuCCT1) cells were treated with deoxycholic acid, which has previously been reported to inhibit sialyltransferase activity, the sialylation of these cell lines were then analysed using this method.
[Results]
Under the optimized chromatographic conditions, Neu5Ac was successfully separated with a retention time of 6.344 min at a flow rate of 0.4 mL/min, providing rapid analysis while maintaining adequate resolution and reproducibility. A statistically significant decrease in Neu5Ac levels was observed in both cell lines following treatment, indicating reduced sialylation consistent with enzyme inhibition.
[Consideration]
The proposed method was validated according to the AOAC (2013) guidelines. Key validation parameters including linearity, precision, accuracy, sensitivity, and reproducibility were evaluated and demonstrated acceptable performance. The method showed robust detection of Neu5Ac in complex cellular environments without the need for extensive sample derivatization. Importantly, the analytical workflow allows the quantification of both free and protein-bound Neu5Ac present in cancer cell matrices, providing a more comprehensive assessment of overall sialylation levels.
[Conclusion]
These results confirm that the proposed method is capable of detecting biologically relevant changes in cellular sialic acid content associated with modulation of sialyltransferase activity. Overall, this work presents a relatively simple, rapid, and economical analytical approach for quantifying Neu5Ac in complex cancer cell matrices.
Sialylation is a critical post-translational modification of cell surface glycoconjugates and plays an essential role in numerous biological processes, including cell signaling, immune recognition, and tumor progression. Sialyltransferases, the enzymes responsible for transferring sialic acid residues to glycoproteins and glycolipids, are frequently overexpressed in various cancers and contribute to tumor invasiveness, metastasis, and immune evasion. Consequently, sialyltransferases have emerged as promising therapeutic targets in anticancer drug development. Reliable analytical methods capable of quantifying sialic acid levels in complex biological matrices are therefore important for evaluating enzyme activity and screening potential inhibitors. Although several analytical approaches have been reported for sialic acid determination, many rely on complex derivatization procedures, expensive instrumentation, or have limited applicability to heterogeneous cellular matrices. Furthermore, the quantification of both free and protein-bound sialic acids within intact cancer cell systems remains insufficiently explored.
[Method]
In this study, a simple, rapid, and cost-effective analytical method was developed to detect and quantify sialic acids in cancer cell matrices for the evaluation of sialyltransferase activity. The method employs reverse-phase ion-pairing high-performance liquid chromatography coupled with ultraviolet detection (RP-IP-HPLC-UV). Triisopropanolamine was utilized as the ion-pairing reagent in combination with a C18 stationary phase to enable effective retention and separation of N-acetylneuraminic acid (Neu5Ac), the most common form of sialic acid in mammalian systems. Human cervical carcinoma (HeLa) and human cholangiocarcinoma (HuCCT1) cells were treated with deoxycholic acid, which has previously been reported to inhibit sialyltransferase activity, the sialylation of these cell lines were then analysed using this method.
[Results]
Under the optimized chromatographic conditions, Neu5Ac was successfully separated with a retention time of 6.344 min at a flow rate of 0.4 mL/min, providing rapid analysis while maintaining adequate resolution and reproducibility. A statistically significant decrease in Neu5Ac levels was observed in both cell lines following treatment, indicating reduced sialylation consistent with enzyme inhibition.
[Consideration]
The proposed method was validated according to the AOAC (2013) guidelines. Key validation parameters including linearity, precision, accuracy, sensitivity, and reproducibility were evaluated and demonstrated acceptable performance. The method showed robust detection of Neu5Ac in complex cellular environments without the need for extensive sample derivatization. Importantly, the analytical workflow allows the quantification of both free and protein-bound Neu5Ac present in cancer cell matrices, providing a more comprehensive assessment of overall sialylation levels.
[Conclusion]
These results confirm that the proposed method is capable of detecting biologically relevant changes in cellular sialic acid content associated with modulation of sialyltransferase activity. Overall, this work presents a relatively simple, rapid, and economical analytical approach for quantifying Neu5Ac in complex cancer cell matrices.
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