Serum N-glycan profiling predicts prognosis in patients undergoing hemodialysis

Abstract

Background: The aim of this study is to evaluate the usefulness of serum N-glycan profiling for prognosis in hemodialysis patients.

Methods: Serum N-glycan analysis was performed in 100 hemodialysis patients in June 2008 using the glycoblotting method, which allows high-throughput, comprehensive, and quantitative N-glycan analysis. All patients were longitudinally followed up for 5 years. To evaluate the independent predictors for prognosis, patients' background, blood biochemistry, and N-glycans intensity were analyzed using Cox regression multivariate analysis. Selected N-glycans and independent factors were evaluated using the log-rank test with the Kaplan-Meier method to identify the predictive indicators for prognosis. Each patient was categorized according to the number of risk factors to evaluate the predictive potential of the risk criteria for prognosis.

Results: In total, 56 N-glycan types were identified in the hemodialysis patients. Cox regression multivariate analysis showed cardiovascular events, body mass index, maximum intima media thickness, and the serum N-glycan intensity of peak number 49 were predictive indicators for overall survival. Risk classification according to the number of independent risk factors revealed significantly poor survival by increasing the number of risk factors.

Conclusions: Serum N-glycan profiling may have a potential to predict prognosis in patients undergoing hemodialysis.

Figure 1

General protocol for the integrated glycoblotting technique and workflow for glycoblotting-based high-throughput clinical glycan analysis. Serum samples of 10 μL (a) were applied to the “Sweet Blot” automated machine for glycoblotting. After enzymatic cleaving from serum protein, the total serum N-glycans released in the digest mixture (b) were directly mixed with BlotGlyco H beads (Sumitomo Bakelite, Co., Tokyo, Japan) to capture N-glycans (c). After the beads had been separated from other molecules by washing (d), sialic acid was methyl esterified (e). The processed N-glycans were then labeled with benzyloxyamine (BOA) and released from the BlotGlyco H beads (f). Mass spectra of BOA-labeled N-glycans were acquired with an Ultraflex III instrument (Bruker Daltonics, Germany) (g).

Figure 2

N-Glycan symbols of internal standard and significant N-glycans selected by logistic regression analysis. O-Benzyl hydroxylamine hydrochloride (BOA)-labeled amidated sialic acids (A2 amide), which do not exist in nature (peak number 35), were used as the internal standard (a). Putative structures of significant N-glycans selected using Cox regression analysis are presented. Peak number 49 (P49) was selected for overall survival (b).

Figure 3

Patients' parameters significantly associated with overall survival. Selected parameters for overall survival were presence of DM (a), BMI (b), CRP (c), max-IMT (d), ACI (e), and intensity of P49 (f).

Figure 4

Receiver operating characteristic (ROC) curves. Areas under the curves (AUCs) in BMI, max-IMT, and intensity of P49 were 0.66, 0.67, and 0.67, respectively.

Figure 5

Overall survival from the cutoff points. Significantly poor overall survival was observed in patients with BMI < 22.0 (a), max-IMT > 1.6 (b), and P49 > 0.226 (c) using the log-rank test and the Kaplan-Meier method. Overall survival in patients with cardiovascular events showed not significant but boundary effects on prognosis (P = 0.0623) (d).

Figure 6

Risk classification according to the number of independent risk factors for overall survival. Patients were categorized into 3 groups according to the number of independent risk factors for overall survival: the low-risk group (patients with no risk factors), the intermediate-risk group (1 or 2 risk factors), and the high-risk group (3 or 4 risk factors). Risk classification revealed significantly poor prognosis by increasing the number of risk factors (*P < 0.001). The patients with high-risk group showed significantly poor prognosis compared with those in the other groups.