Maria Knapik-Kordecka

Ischemia-modified albumin level in type 2 diabetes mellitus - Preliminary report.

Aim: The main goal of the present study was the evaluation of ischemia-modified albumin (IMA) in patients with type 2 diabetes mellitus and estimation of its connection with vascular complications, glycemic control, hypertension, dyslipidemia and obesity. Methods: In 76 diabetic patients and 25 control subjects, a plasma level of IMA by manually performed, spectrophotometric Co(II)-albumin binding assay was determined. Other parameters such as glucose, fructosamine, HbA1c, total cholesterol and its fractions (HDL, LDL), triglicerydes were estimated by routine methods. Results: Diabetic patients had significantly higher level of IMA in comparison with control subjects. There were not significant differences between groups with various states of vascular complications although the lowest concentration of IMA was observed in patients with microangiopathy. Patients with poor glycemic control had higher IMA level in comparison with these with good glycemic control. Significant correlation was observed between IMA and HbA1c. Among the risk factors, only blood pressure and LDL showed a weak relationship with IMA level. Conclusions: Our results revealed, for the first time, higher level of IMA in diabetic patients which confirms that it may be of non-cardiac origin. We can suggest that the albumin molecule in plasma of diabetic patients is modified in the chronic hypoxia conditions provoked mainly by hyperglycemia and oxidative stress in diabetes.

Concentration of leukocyte elastase in plasma and polymorphonuclear neutrophil extracts in type 2 diabetes.

Abstract The concentration of leukocyte elastase/α1-proteinase inhibitor complexes in plasma and polymorphonuclear neutrophil extracts, and plasma trypsin inhibitory capacity were determined in 88 patients with type 2 diabetes and 47 control subjects. Higher values of these variables were found in patients as compared to controls (p < 0.001). The concentration of elastase was higher in obese patients than in lean ones (p < 0.05 for plasma, p < 0.01 for polymorphonuclear leukocytes). Only leukocyte elastase levels were significantly higher in the group with both micro- and macroangiopathy in comparison to the group with microangiopathy (p < 0.01) or macroangiopathy (p < 0.05) alone. Poor short-term glycaemic control was associated with higher elastase concentration in plasma and neutrophils (p < 0.05). The present study demonstrates that measurements of plasma polymorphonuclear neutrophil elastase level can be considered as a marker of development of diabetic angiopathy.

Plasma glycooxidation protein products in type 2 diabetic patients with nephropathy

In diabetes mellitus, hyperglycaemia accelerates non‐enzymatic glycation and oxidative stress leading to damage of macromolecules, among others proteins. This manifests in the increased levels of advanced glycation end products (AGE) and advanced oxidation protein products (AOPP).

Markers of oxidative protein damage in plasma and urine of type 2 diabetic patients

Abstract This study aims to measure selected markers of oxidative protein damage (OPD) in patients with type 2 diabetes mellitus (T2DM) in order to estimate their utility as indicators of oxidative stress (OS) and to search for possible associations between them. The concentrations of advanced oxidation protein products (AOPP) and total sulphydryl (TSH) and reactive carbonyl (RCO) groups are measured in the plasma (P) and urine (U) of 60 patients and 22 controls using spectrophotometric methods. Significantly higher plasma concentrations of AOPP (P<0.001), RCO groups (P<0.01) and their P/U indexes (P<0.001) as well as urinary levels of the RCO and TSH groups (P<0.001) were observed in the diabetic patients compared with the controls. In contrast, the plasma levels and P/U index of the TSH groups were significantly lower (P<0.001). A progressive increase in AOPP (plasma, urine and P/U index) in the course of albuminuria was noted, but significant differences among the subgroups of patients (with normoalbuminuria, microalbuminuria and macroalbuminuria) were found only in plasma. Plasma levels of all the measured parameters of OPD showed significant changes in T2DM patients compared with the control group. The largest increase was observed for AOPP. As the urinary AOPP concentration was not significantly different to that of the controls, it cannot be recommended as a marker of oxidative stress for monitoring the development of diabetic nephropathy. The P/U indexes did not provide any more information than the plasma concentrations of the studied markers.

Proteins from the 18 glycosyl hydrolase family are associated with kidney dysfunction in patients with diabetes type 2

Abstract Objectives: To investigate chitotriosidase (CHIT1) activity and chitinase-3-like protein 1 (YKL-40) concentration in plasma of type 2 diabetic patients and evaluate their relationship with kidney dysfunction. Materials and methods: 94 diabetic subjects and 33 controls were enrolled in the study. Plasma CHIT1 activity and YKL-40 concentration were measured along with routine laboratory parameters. Results: Levels of CHIT1 and YKL-40 in plasma of type 2 diabetic patients increased progressively with the degree of albuminuria. CHIT1 discriminated normoalbuminuric subjects from those with abnormal albuminuria better than YKL-40. Conclusions: CHIT1represent a supportive biomarker connected with development of diabetic vascular complications, especially kidney dysfunction.

Neutrophils as a Source of Chitinases and Chitinase-Like Proteins in Type 2 Diabetes

Purpose The pathophysiological role of human chitinases and chitinase-like proteins (CLPs) is not fully understood. We aimed to determine the levels of neutrophil-derived chitotriosidase (CHIT1), acidic mammalian chitinase (AMCase) and chitinase 3-like protein 1 (YKL-40) in patients with type 2 diabetes (T2D) and verify their association with metabolic and clinical conditions of these patients. Methods Neutrophils were obtained from the whole blood by gradient density centrifugation from 94 T2D patients and 40 control subjects. The activities of CHIT1 and AMCase as well as leukocyte elastase (LE) were measured fluorometrically and concentration of YKL-40 immunoenzymatically. Also, routine laboratory parameters in serum/plasma were determined by standard methods. Results The levels of all three examined proteins were about 2-times higher in diabetic patients in comparison to control subjects. They were significantly correlated with the activity of LE and increased progressively across tertiles of LE activity. Moreover, the activities of CHIT1 and AMCase were significantly correlated with each other. Metabolic compensation of diabetes did not influence the levels of these proteins. In the subgroup of patients with inflammatory evidence only YKL-40 concentration was significantly higher compared to those without inflammation. The highest levels of all three proteins were observed in patients with macroangiopathies. Insulin therapy was associated with lower levels of examined proteins. Conclusions We revealed that neutrophils may be an important source of the increased levels of chitinases and CLPs in T2D, and these proteins may participate in inflammatory mechanisms in the course of the disease and consequent development of diabetic angiopathies.

Changes in glycosylation of human blood plasma chitotriosidase in patients with type 2 diabetes

Human blood plasma chitotriosidase (CHIT1) is a glycoprotein with chitinolytic activity with not fully elucidated biological function. Its increased level is observed in type 2 diabetes mellitus (T2DM) and is associated with development of diabetic complications. The CHIT1 glycosylation profile and degree is still poorly studied and never investigated in T2DM. Therefore the aim of the present study was to examine the association between glycosylation profile and degree and diabetes with accompanying nephropathy. In blood plasma of 28 patients with T2DM and 11 healthy subjects the CHIT1 concentration and specific activity were examined. The profile and degree of CHIT1 glycosylation were determined by lectin-ELISA using lectins specific to O-glycans (Jacalin, MPL, VVL) and sialo-specific SNA and MAA. We revealed that both concentration and specific activity of CHIT1 significantly increased in T2DM, especially in nephropathy with elevated albuminuria. The relative reactivities with lectins, except Jacalin, decreased progressively with T2DM occurrence and albuminuria progression. The most significant differences were observed between control vs. albuminuric group (Micro and Macro). It is also possible that the observed differences in immunoblotting pattern in molecular masses of CHIT1 bands between T2DM patients and healthy subjects may be caused by the differences in degree of CHIT1 glycosylation. The analysis of CHIT1 glycosylation status and the determination of CHIT1 concentration together with its enzymatic activity in blood plasma might constitute additional valuable diagnosis tools for the evaluation the T2DM patients with accompanying nephropathy. Extension of the lectin panel specific to O-glycans occurs useful for the further research using microarray formats, which are expected to accelerate “lectin-based glycan profiling” of glycoproteins.

Increased chitotriosidase activity in plasma of patients with type 2 diabetes

Introduction Chitotriosidase (CHIT1) is a chitinolytic enzyme involved mainly in the immune and inflammatory response. It shows increased activity in many pathologies, including in newly diagnosed type 2 diabetes (T2D). This study aimed to investigate this enzymes activity in plasma of patients with ongoing T2D and indicate factors related to the increased activity of this enzyme. Material and methods Ninety-one patients and 46 control subjects without abnormalities in carbohydrate metabolism and inflammatory states were enrolled in the study. Plasma CHIT1 activity was measured by a spectrofluorometric method. Routine laboratory parameters such as blood glucose, total cholesterol and HDL fraction, triglyceride, glycated hemoglobin, white blood cell count and C-reactive protein were measured by standard methods. Results We found that the chitotriosidase activity was significantly higher (p < 0.001) in type 2 diabetic patients and positively associated with parameters of glycemic control (levels of glucose and glycated hemoglobin) and blood pressure. Plasma glucose level and systolic blood pressure were independent determinants of increased CHIT1 activity in T2D patients, even after adjustment for disease duration, body mass index, parameters of inflammation and lipid metabolism. We also found that increased CHIT1 activity was associated with occurrence of diabetic angiopathies. Conclusions This investigation indicates a possible role of chitotriosidase in the course of T2D, especially in relation to development of diabetic angiopathies.

Activities of Neutrophil Membrane-bound Proteases in Type 2 Diabetic Patients

BACKGROUND AND AIMS Hyperglycemia and oxidative stress in type 2 diabetes (T2DM) provoke neutrophil overstimulation and the release and/or translocation of proteases from granules to the cell surface. Although the expression of neutrophil membrane-bound elastase (MLE) is well documented, the presence of the membrane-bound form of cathepsin B (MCB) is unknown. The aim of our study was to evaluate the neutrophil MLE and MCB activities in T2DM patients and their associations with the metabolic and clinical parameters of the disease. METHODS Neutrophils were obtained from 47 T2DM patients and 20 control subjects. The activities of MLE and MCB and the intracellular activities of the examined proteases (ILE and ICB, respectively) were measured using fluorometric substrates. Additionally, the percentage equivalents of the activities, namely, MLEtot/ILEtot and MCBtot/ICBtot, were calculated. The susceptibility to inhibitors of both forms of the studied proteases was also determined. RESULTS A significant increase in the activities of MLE, MCB, ILE, and ICB was found in neutrophils from T2DM patients compared with the control group. The percentage equivalent (contribution of the total membrane-bound activities to the total intracellular activities) was also higher. A partial resistance of the membrane-bound forms toward their inhibitors was revealed. Higher activities of both the membrane-bound and the intracellular proteases were also observed in patients with poor glycemic and metabolic control. The differences between subgroups with different therapeutic schemes were also revealed. CONCLUSIONS The pathophysiological implications of the neutrophil membrane-bound forms of leukocyte elastase and cathepsin B are of great importance in the development of T2DM and its complications.