High-Throughput and Site-Specific N-Glycosylation Analysis of Human Alpha-1-Acid Glycoprotein Offers a Great Potential for New Biomarker Discovery.

Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases, and some changes appear to be disease-specific; thus, it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers, and enable better understanding of AGP's role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 µl of bloodplasma in a 96-well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography-based solid-phase extraction and analyzed by reversed-phase-liquid chromatography-electrospray ionization-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in three time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP's second glycosylation site and lower sialylation of N-glycans on AGP's third and AGP1's fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.

Correction to: Increased plasma N-glycome complexity is associated with higher risk of type 2 diabetes.

The authors regret that Nish Chaturvedi's name was spelt incorrectly in the author list. The details given in this correction are correct.

Increased plasma N-glycome complexity is associated with higher risk of type 2 diabetes.

AIMS/HYPOTHESIS: Better understanding of type 2 diabetes and its prevention is a pressing need. Changes in human plasma N-glycome are associated with many diseases and represent promising diagnostic and prognostic biomarkers. Variations in glucose metabolism directly affect glycosylation through the hexosamine pathway but studies of plasma glycome in type 2 diabetes are scarce. The aim of this study was to determine whether plasma protein N-glycome is changed in individuals who are at greater risk of developing type 2 diabetes. METHODS: Using a chromatographic approach, we analysed N-linked glycans from plasma proteins in two populations comprising individuals with registered hyperglycaemia during critical illness (increased risk for development of type 2 diabetes) and individuals who stayed normoglycaemic during the same condition: AcuteInflammation (59 cases vs 49 controls) and AcuteInflammation Replication (52 cases vs 14 controls) populations. N-glycome was also studied in individuals from FinRisk (37 incident cases of type 2 diabetes collected at baseline vs 37 controls), Orkney Complex Disease Study (ORCADES; 94 individuals with HbA1c > 6.5% [47.5 mmol/mol] vs 658 controls) and Southall and Brent Revisited (SABRE) cohort studies (307 individuals with HbA1c > 6.5% [47.5 mmol/mol] vs 307 controls). RESULTS: Individuals with increased risk for diabetes type 2 development (AcuteInflammation and AcuteInflammation Replication populations), incident cases of type 2 diabetes collected at baseline (FinRisk population) and individuals with elevated HbA1c (ORCADES and SABRE populations) all presented with increased branching, galactosylation and sialylation of plasma protein N-glycans and these changes were of similar magnitude. CONCLUSIONS/INTERPRETATION: Increased complexity of plasma N-glycan structures is associated with higher risk of developing type 2 diabetes and poorer regulation of blood glucose levels. Although further research is needed, this finding could offer a potential new approach for improvement in prevention of diabetes and its complications.

Changes in IgG and total plasma protein glycomes in acute systemic inflammation.

Recovery after cardiac surgery is a complex process that has to compensate for both individual variability and extensive tissue damage in the context of systemic inflammation. Protein glycosylation is essential in many steps of the inflammatory cascade, but due to technological limitations the role of individual variation in glycosylation in systemic inflammation has not been addressed until now. We analysed composition of the total plasma and IgG N-glycomes in 107 patients undergoing cardiac surgery. In nearly all individuals plasma N-glycome underwent the same pattern of changes in the first 72 h, revealing a general mechanism of glycosylation changes. To the contrary, changes in the IgG glycome were very individualized. Bi-clustering analysis revealed the existence of four distinct patterns of changes. One of them, characterized by a rapid increase in galactosylated glycoforms, was associated with nearly double mortality risk measured by EuroSCORE II. Our results indicate that individual variation in IgG glycosylation changes during acute systemic inflammation associates with increased mortality risk and indicates new avenues for the development of personalized diagnostic and therapeutic approach.

Hyperglycemia in sepsis is a risk factor for development of type II diabetes.

BACKGROUND: Hyperglycemia is frequent in sepsis, even in patients without diabetes or impaired glucose metabolism. It is a consequence of inflammatory response and stress, so its occurrence is related to severity of illness. However, not all severely ill develop hyperglycemia and some do even in mild disease. We hypothesized the existence of latent disturbance of glucose metabolism that contributes to development of hyperglycemia and that those patients might have increased risk for diabetes. METHODS: Patients admitted with sepsis and no history of impaired glucose metabolism were included and divided in the hyperglycemia group (glucose >or=7.8 mmol/L) and normoglycemia group. Severity of sepsis was assessed. Surviving patients without diabetes at discharge were followed-up for 5 years to investigate risk for development of diabetes. RESULTS: Hyperglycemia was related to severity of sepsis. Follow-up was finished for 55 patients with hyperglycemia, of which 8 (15.7%) developed diabetes, and 118 patients with normoglycemia, of which 5 (4.2%) developed diabetes (P = .002). Relative risk for developing type 2 diabetes was 4.29 (95% CI, 1.35-13.64). CONCLUSION: Patients with hyperglycemia in sepsis who are not diagnosed with diabetes before or during the hospitalization should be considered a population at increased risk for developing diabetes.

Hyperglycaemia in critical illness is a risk factor for later development of type II diabetes mellitus.

Hyperglycaemia caused by stress and inflammation is common during critical illness. We hypothesised that a latent glucose metabolism disturbance contributes to development of hyperglycaemia and that those patients have increased risk for diabetes. We included patients with sepsis, acute coronary syndrome and acute heart failure with no history of impaired glucose metabolism and divided them in the hyperglycaemia group (glucose ≥ 7.8 mmol/l) and normoglycaemia group. Patients were followed for 5 years. Follow-up was completed for 115 patients in the normoglycaemia group, of which 4 (3.5%) developed type 2 diabetes. In the hyperglycaemia group 51 patients finished follow-up and 8 (15.7%) developed type 2 diabetes. Relative risk in 5-year period for patients with hyperglycaemia was 4.51 for development of type 2 diabetes. Patients with hyperglycaemia during critical illness who are not diagnosed with diabetes before or during the hospitalisation should be considered a population at increased risk for developing diabetes.