Increased levels of acidic free-N-glycans, including multi-antennary and fucosylated structures, in the urine of cancer patients.

We recently reported increased levels of urinary free-glycans in some cancer patients. Here, we focused on cancer related alterations in the levels of high molecular weight freeglycans. The rationale for this study was that branching, elongation, fucosylation and sialylation, which lead to increases in the molecular weight of glycans, are known to be up-regulated in cancer. Urine samples from patients with gastric cancer, pancreatic cancer, cholangiocarcinoma and colorectal cancer and normal controls were analyzed. The extracted free-glycans were fluorescently labeled with 2-aminopyridine and analyzed by multi-step liquid chromatography. Comparison of the glycan profiles revealed increased levels of glycans in some cancer patients. Structural analysis of the glycans was carried out by performing chromatography and mass spectrometry together with enzymatic or chemical treatments. To compare glycan levels between samples with high sensitivity and selectivity, simultaneous measurements by reversed-phase liquid chromatography-selected ion monitoring of mass spectrometry were also performed. As a result, three lactose-core glycans and 78 free-N-glycans (one phosphorylated oligomannose-type, four sialylated hybrid-type and 73 bi-, tri- and tetra-antennary complex-type structures) were identified. Among them, glycans with a1,3-fucosylation ((+/- sialyl) Lewis X), triply a2,6-sialylated tri-antennary structures and/or a (Man3)GlcNAc1-core displayed elevated levels in cancer patients. However, simple a2,3-sialylation and a1,6-core-fucosylation did not appear to contribute to the observed increase in the level of glycans. Interestingly, one tri-antennary free-N-glycan that showed remarkable elevation in some cancer patients contained a unique Glcß1-4GlcNAccore instead of the common GlcNAc2-core at the reducing end. This study provides further insights into free-glycans as potential tumor markers and their processing pathways in cancer. [ABSTRACT FROM AUTHOR]