#1669 Gangliosides as biomarkers for early diagnosis of diabetic kidney disease: detection and characterization by high-performance mass spectrometry
Authors: Suteanu-Simulescu Anca , Zamfir Alina Diana , Sarbu Mirela , Ica Raluca , Gadalean Florica , Vlad Adrian , Bob Flaviu , Glavan Mihaela , Balint Lavinia , Ienciu Silvia , Iancu Lavinia , Petrica Ligia 
Published: May 2024

Abstract

Background and Aims
The early identification of diabetic kidney disease (DKD) is crucial as it may facilitate immediate intervention, thus impeding the progression of the condition and the risk of developing end-stage renal disease (ESRD). Enhancing our understanding of the pathophysiology of this microvascular complication as well as developing therapeutic strategies are imperative. Hence, the aim of the current study was to address the problematics of detection and characterization of gangliosides in the urine of type 2 diabetes mellitus (DM) patients with DKD.

Method
In this cross-sectional study, the urine ganglioside content from 24 h collected urine samples of 60 type 2 DM patients (20 with normal to mildly increased albuminuria—A1 group, 20 with moderately increased albuminuria—A2 group, 20 with severe increased albuminuria—A3 group) was compared to that of 20 healthy controls using a promising technique based on high-resolution (HR) mass spectrometry (MS). Furthermore, for structural elucidation of individual compounds, we isolated the ions GT1 (d18:1/18:0), identified at m/z 708.3379 and, GQ1 (d18:1/18:0) at m/z 812.7068, observed only in the screening analysis of A3 patients, and exposed them to Higher-energy collisional dissociation (HCD MS/MS) in the negative ion mode.

Results
Patients with type 2 diabetes mellitus display a considerably greater abundance of different species in their urinary gangliosidome, which varies in either glycan or ceramide structure, in comparison to the control group. Even within the A1 group, a greater variety of structures exhibiting varying degrees of sialylation were observed. A wide range of carbohydrate chains were discovered, ranging from short, monosialylated chains (GM) to complex, pentasialylated chains (GP). Additionally, some ganglioside chains were modified with and O-acetyl (O-Ac) attachments. The gangliosidome of the A3 group comprises a higher variety of structures, compared to A1 and A2, differing in their overall sialic acid content, and more complex structures, including various biologically significant modifications, such as O-fucosylation, O-GalNAc and CH3COO− attachments. Both albuminuria and glomerular filtration rate proved to be correlated with the degree of sialylation of species. Furthermore, significant alterations occur also in the ceramide part, with the most noteworthy being the presence of sphingoid base trihydroxylation, which is shown in both A1 and A3 samples. Additionally, A3 exhibits unusual length of the fatty acid chains. GQ1d(d18:1/18:0), GT1α(d18:1/18:0), and GT1b(d18:1/18:0) were identified as predictive for specific isomers linked to the location of Neu5Ac along the oligosaccharide structure using HCD-based MS/MS.

Conclusion
We concluded that gangliosides may be involved in the development and progression of DKD through the use of mass spectrometry techniques, which provides considerable precision and ultrahigh reproducibility, sensitivity and processing power, and the capability to identify minor elements in complex biological matrices. Thus, a set of promising bioindicators for early detection and progression of DKD was established. Even in the normoalbuminuric stage, the sialylation level of the expressed species appears to be an extremely important biochemical marker of DKD. Moreover, the modifications detected in the composition of ceramide indicate that, alongside the oligosaccharide structure, the lipid component might function as a specific molecular indicator for different stages of DKD. The presence of a few isomers in the A3 group has been discovered to be associated with DKD progression, based on the informative sequence ions obtained from the structural analysis. Moreover, gangliosides might be used as target molecules for the implementation of therapeutic schemes in type 2 DM patients.