All-Cause Mortality and Trimethylamine N-Oxide Levels in Patients with Cardiovascular Disease.

INTRODUCTION: Trimethylamine N-oxide (TMAO) is an organic compound with a well-established involvement in the pathogenesis of cardiovascular disease (CVD). However, data on the links between TMAO levels and cardiovascular mortality in Polish patients are lacking. OBJECTIVES: We aimed to assess the relationship between serum TMAO levels and 5-year mortality in Polish patients with CVD. PATIENTS AND METHODS: We retrospectively assessed serum TMAO levels in 1,036 consecutive patients (median age, 62 years; men, 61%) hospitalized between 2013 and 2015. Correlations between TMAO levels and 5-year mortality as well as anthropometric and biochemical parameters were assessed for the whole population and the subgroups of patients with acute coronary syndrome, stable coronary syndrome (SCS), chronic heart failure (HF), and atrial fibrillation (AF). RESULTS: In the univariate analysis, increased TMAO levels predicted 5-year mortality without clinically significant power (hazard ratio [HR], 1.01; 95% CI: 1.006-1.018; p < 0.0001). However, even this weak effect was lost in the multivariate analysis after adjustment for age, sex, comorbidities, and laboratory parameters. In the whole study group, TMAO levels in the fourth quartile of concentration (>6.01 µM) predicted 5-year mortality only in the univariate analysis (HR: 1.55; 95% CI: 1.34-1.79; p < 0.0001). In subgroup univariate analysis, TMAO levels predicted 5-year mortality in patients with SCS, chronic HF, and AF. CONCLUSIONS: Despite the promising results of previous studies, our study shows that the level of TMAO has at most moderate value in predicting all-cause mortality. TMAO levels depend on other clinical variables, which limits the use of TMAO as an independent predictor of mortality in these patients.

In Vitro Investigation of Binding Interactions between Albumin-Gliclazide Model and Typical Hypotensive Drugs.

Type 2 diabetes management usually requires polytherapy, which increases the risk of drug-to-drug interactions. Among the multiple diabetes comorbidities, hypertension is the most prevalent. This study aimed to investigate the binding interactions between the model protein, bovine albumin, and the hypoglycemic agent gliclazide (GLICL) in the presence of typical hypotensive drugs: quinapril hydrochloride (QUI), valsartan (VAL), furosemide (FUR), amlodipine besylate (AML), and atenolol (ATN). Spectroscopic techniques (fluorescence quenching, circular dichroism) and thermodynamic experiments were employed. The binding of the gliclazide to the albumin molecule was affected by the presence of an additional drug ligand, which was reflected by the reduced binding constant of the BSA-DRUG-GLICL system. This may indicate a possible GLICL displacement and its enhanced pharmacological effect, as manifested in clinical practice. The analysis of the thermodynamic parameters indicated the spontaneity of the reaction and emphasized the role of hydrogen bonding and van der Waals forces in these interactions. The secondary structure of the BSA remained almost unaffected.

How Does Glycation Affect Binding Parameters of the Albumin-Gliclazide System in the Presence of Drugs Commonly Used in Diabetes? In Vitro Spectroscopic Study.

In this research, the selected drugs commonly used in diabetes and its comorbidities (gliclazide, cilazapril, atorvastatin, and acetylsalicylic acid) were studied for their interactions with bovine serum albumin-native and glycated. Two different spectroscopic methods, fluorescence quenching and circular dichroism, were utilized to elucidate the binding interactions of the investigational drugs. The glycation process was induced in BSA by glucose and was confirmed by the presence of advanced glycosylation end products (AGEs). The interaction between albumin and gliclazide, with the presence of another drug, was confirmed by calculation of association constants (0.11-1.07 × 104 M-1). The nature of changes in the secondary structure of a protein depends on the drug used and the degree of glycation. Therefore, these interactions may have an influence on pharmacokinetic parameters.

Parameters of Oxidative and Inflammatory Status in a Three-Month Observation of Patients with Acute Myocardial Infarction Undergoing Coronary Angioplasty-A Preliminary Study.

Background and Objectives: Patients with acute myocardial infarction (MI) are usually treated with percutaneous transluminal coronary angioplasty (PTCA), which is burdened with a risk of postoperative complications, often accompanied by biochemical disturbances. The aim of our study was to evaluate a set of selected parameters of oxidative and inflammatory status, which could be useful in the management of post-procedural care in MI patients after PTCA. Materials and Methods: In this preliminary study, ischemia modified albumin (IMA), advanced oxidation protein products (AOPP), thiol groups (SH), total antioxidant status (TAS), insulin growth factor-1 (IGF-1), presepsin (PSP), and trimethylamine N-oxide (TMAO) were chosen as candidate biomarkers, and were determined in patients with MI who underwent PTCA at two time points: During cardiac episodes (at admission to the hospital, T0) and 3 months later (T3). Results: Most of the examined parameters were significantly different between patients and control subjects (except for IMA and TAS), but only hsCRP changed significantly during the time of observation (T0 vs. T3). Discriminant analysis created a model composed of AOPP, hsCRP, PSP, and TMAO, which differentiated male subjects into a group with MI and a control (without cardiovascular diseases). Conclusion: This set of parameters seems useful in evaluating inflammatory and oxidative status in MI patients after PTCA.

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.

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

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.

[Glycated albumin as a marker of glycemia in diabetes and its vascular complications]. / Glikowana albumina jako wskaznik glikemii w cukrzycy i jej naczyniowych powiklaniach.

Effective glycemic control is very important to prevent the onset and the progression of chronic complications in diabetic patients. It is known that glycation of various proteins is increased in diabetic patients compared with non-diabetics. Among these glycated proteins, glycated hemoglobin (HbA1c) is commonly used as a gold standard index of glycemic control in the clinical setting. However, it can be unreliable in conditions affecting the lifespan of erythrocytes (120 days) as well as in the clinical state in which glycemic control alleviates or deteriorates in a short period. By overcoming the shortcomings of HbA1c, glycated albumin (GA) has gained interest as a useful index for an intermediate glycation period (2 weeks) and pathogenic protein. After giving a brief overview of the key role of HbA1c as a long-term glycemic marker, this review focuses on (a) glycation of human albumin and its main properties, (b) methods of GA determination, (c) the recent clinical status of GA as a glycemic index in diabetic patients and its association with vascular complications. Finally, conditions with a possible inaccurate GA level are also mentioned.

In vitro studies of hemoglobin's affinity for the Vitamin B9 and control of its stability character.

Vitamin B9, known as folic acid, and hemoglobin play an important biological role in the human body. This study was designed to investigate the nature of the complex through multispectroscopic methods at physiological conditions due to the lack of research on the binding interactions between folic acid and hemoglobin. Structural analysis showed that the interactions between the molecules are mainly hydrophobic with binding constant of 0.73 × 104 L/mol at 37 °C. The secondary structure of the protein was stable after the addition of folic acid with a 20-fold excess of ligand per mol protein. The stability effect of folic acid on hemoglobin was examined as a function of release of iron ions and determination of the level of phenanthroline-Fe2+ complex. The protective function of folic acid was observed at a concentration of 6.12 nmol/L, and the release of iron ions was lower than in the control probe.

The impact of dietary nitrates and acrylamide intake on systemic redox status in healthy young adults.

OBJECTIVES: The nitrogen-containing xenobiotics, such as nitrates and acrylamide may potentially influence systemic redox status and contribute to the generation of oxidative stress (OS) in the human body, but there is still a lack of studies that would evaluate the various parameters assessing the oxidative-antioxidant balance. The aim of this study was to evaluate the exposure to nitrates and acrylamide derived from daily diet and to analyze the impact of these nitrate-containing xenobiotics on the parameters of systemic redox status in healthy young adults. MATERIAL AND METHODS: To assess nitrate and acrylamide intake in the study population, a semi-quantitative food frequency questionnaire was used. Systemic redox status was evaluated by measurement of a panel of biochemical parameters: enzymatic (glutathione S-transferase, glutathione reductase, glutathione peroxidase [GPx]) and non-enzymatic (uric acid, bilirubin and albumin), thiol/disulphide homeostasis parameters (total thiol, native thiol, and disulfide) and oxidative/ antioxidant balance indicators (total antioxidant status, total oxidant status, OS index). RESULTS: The average consumption of nitrates and acrylamide in the study population was 1.24 mg/kg b.w./day and 0.23 µg/kg b.w./day, respectively, which is within the normal value range. Of 12 measured parameters, significant differences were revealed for disulfide and total thiol levels, which were increased in the subgroup with the highest daily intake of nitrates compared to the subgroup with the lowest intake; for GPx, which was highest in the subgroup of the lowest daily intake of acrylamide; and for native thiols in the subgroup with the highest daily intake. CONCLUSIONS: The intake of nitrogen-containing xenobiotics within the range considered as normal does not markedly influence redox state parameters in healthy young adults. Some significant changes were revealed only for thiol/disulphide homeostasis parameters, which may be the first line of antioxidant defense, as well as for GPx activity. Compensative mechanisms in healthy young people are efficient enough to neutralize OS induced by slightly increased exposure to nitrogen-containing xenobiotics delivered with food. Int J Occup Med Environ Health. 2023;36(6):773-87.

Solid dispersion in pharmaceutical technology. Part II. The methods of analysis of solid dispersions and examples of their application.

In the first part of the article solid dispersions were classified the properties and methods of their preparation were described. This section presents methods of analysis of solid dispersions i.e.: thermoanalytical methods, XRPD, FTIR, microscopic methods, dissolution studies and examples of drug forms where solid dispersions were used.

Solid dispersions in pharmaceutical technology. Part I. Classification and methods to obtain solid dispersions.

There are many methods to increase solubility of a substance. These include, inter alia, preparation of solid dispersions, i.e. eutectic mixtures, solid solutions, glassy solutions and suspensions. When compared to the individual constituents prior to dispersion formation solid dispersion components are better soluble in water. Therefore, solid solutions became one of the most promising ways to modify solubility, ensuring improved bioavailability and consequently therapeutic efficacy of a substance. In this part of the publication solid dispersions were classified and described in regard to their properties and preparation methods, i.e. melting method, melt evaporation and melt extrusion methods, lyophilisation technique, melt agglomeration process as well as SCF technology and electrospinning.

Citrulline and long-term mortality in patients with cardiovascular disease.

BACKGROUND: Cardiovascular disease (CVD) is associated with intestinal barrier dysfunction and increased intestinal permeability. Increased intestinal permeability to gut microbial metabolites may accelerate the progression of CVD. Plasma citrulline levels are a marker of functional enterocyte mass, and reduced citrulline levels indicate intestinal epithelial damage. Citrulline was reported as a useful prognostic marker in critically ill patients. However, data are lacking on the association of citrulline with long-term mortality in patients with CVD and with the levels of trimethylamine N-oxide (TMAO), a microbiota-derived metabolite which has been implicated in the pathogenesis of CVD. OBJECTIVES: To assess the effect of citrulline levels, a marker of intestinal barrier disruption, on long-term mortality in patients with CVD. Moreover, the relationship between the concentrations of 2 biomarkers - citrulline and TMAO - was assessed. MATERIAL AND METHODS: Serum citrulline levels were retrospectively assessed in 1036 consecutive patients with CVD (median age: 62 years; 61% men) hospitalized between 2013 and 2015. Associations of citrulline levels with 5-year mortality rates as well as anthropometric and biochemical parameters were evaluated for the entire study group and in subgroups of patients with acute coronary syndrome (ACS), chronic coronary syndrome, chronic heart failure (chronic HF), and atrial fibrillation (AF). Correlations between serum citrulline and TMAO levels were assessed. RESULTS: The median citrulline level in the study population was 22.5 µM (interquartile range (IQR): 17.8-27.9). Citrulline levels were not associated with 5-year mortality in patients with CVD (hazard ratio (HR) = 0.99; 95% confidence interval (95% CI): 0.97-1.00; p = 0.49). Median citrulline levels differed significantly between deceased patients and survivors at 5 years in patients with ACS (p = 0.025). There were no significant correlations between citrulline and TMAO levels (Kendall's tau = 0.027). CONCLUSIONS: Decreasing citrulline levels do not predict long-term mortality of hospitalized patients with CVD. Moreover, they are not associated with the serum levels of TMAO in these patients.

Platelet Reactivity and Response to Aspirin and Clopidogrel in Patients with Platelet Count Disorders.

BACKGROUND: Platelet reactivity and response to antiplatelet drugs, acetylsalicylic acid (ASA) and clopidogrel, in patients with thrombocytopenia and thrombocythemia can have a potentially important effect on the outcome. The effectiveness and safety of antiplatelet drugs in such patients has not been well examined. Measuring the effect of ASA and clopidogrel on platelets could help guide the therapy. Nevertheless, platelet response to antiplatelet drugs is not routinely measured in platelet count disorders and relevant evidence is scarce. AIMS: The study aimed to measure platelet reactivity and response to ASA and clopidogrel in patients with platelet count disorders. MATERIALS AND METHODS: This was a cross-sectional study of consecutive patients hospitalized in cardiology and hematology departments in the years 2018-2019. The study included patients with thrombocytopenia (PLT < 150 G/L) and thrombocythemia (PLT > 450 G/L) on ASA or dual antiplatelet therapy (DAPT; ASA plus clopidogrel). Controls included patients on antiplatelet drugs with normal platelet count. Platelet reactivity was measured in whole blood (Multiplate aggregometer, Roche, Switzerland) using arachidonic acid (AA), adenosine-5'-diphosphate (ADP), and thrombin receptor agonist peptide-6 (TRAP) as agonists. Platelet aggregation was expressed in arbitrary units (AU). AA-induced aggregation was used as a measure of response to ASA with a cut-off above 30 AU showing high on-treatment platelet reactivity to ASA (HTPR-A). ADP-induced aggregation measured response to clopidogrel with a cut-off above 48 AU for high on-treatment platelet reactivity to clopidogrel (HTPR-C). TRAP-induced aggregation measured baseline platelet reactivity not affected by oral antiplatelet drugs. RESULTS: The study included 174 patients. There were 64 patients with thrombocytopenia, 30 patients with chronic thrombocythemia, and 80 controls. All patients were on 75 mg of ASA and 32% of them additionally on 75 mg of clopidogrel due to a history of recent coronary artery angioplasty. AA- and ADP-induced aggregation was comparable between thrombocytopenic patients and controls (median (IQR) 19 (7-28) vs. 23 (15-38) for AA AU and 32 (16-44) vs. 50 (32-71) for ADP AU, respectively), while it was significantly higher in thrombocythemic patients (median (IQR) 80 (79-118) for AA AU and 124 (89-139) for ADP AU). TRAP-induced aggregation showed significantly lowest aggregation in thrombocytopenic (median (IQR) 41 (34-60) for TRAP AU) and highest in thrombocythemic patients (median (IQR) 137 (120-180) for TRAP AU). HTPR-A was frequent in thrombocythemic patients in comparison with thrombocytopenic patients and controls (60% vs. 4% vs. 15%, respectively; p < 0.0002). HTPR-C was highly common in thrombocythemic patients and least common in thrombocytopenic ones in comparison with controls (80% vs. 8% vs. 40%, respectively; p < 0.001). CONCLUSION: Chronic thrombocytopenia does not significantly affect platelet reactivity and response to ASA and clopidogrel in comparison with controls. Thrombocytosis significantly increases platelet reactivity and attenuates response to both ASA and clopidogrel.

Platelet polyphosphate level is elevated in patients with chronic primary thrombocytopenia: A preliminary study.

BACKGROUND: Platelets are key players in hemostasis. These blood cells contain different types of granules. Recently, there has been a growing interest in the role of inorganic polyphosphate (polyP) structures stored in dense granules of platelets and secreted during platelet activation. OBJECTIVES: To measure platelet polyP levels in patients with thrombocytopenia and thrombocythemia, and to examine the relationship of this indicator with platelet aggregation. MATERIAL AND METHODS: The study included 36 patients with hematological disorders (26 with primary chronic thrombocytopenia and 10 with essential thrombocythemia (ET)) and 40 healthy subjects. Platelet reactivity was measured using whole blood impedance aggregometry. The polyP levels were isolated from lysed platelets, which were obtained from citrated platelet-rich plasma. The procedure included inactivating endogenous phosphatases, removing phosphate units derived from DNA and proteins, and finally hydrolyzing them into monophosphate units. A colorimetric assay using malachite green and ammonium molybdate was performed in order to quantify polyP levels. RESULTS: The polyP concentrations were significantly higher in the patients with thrombocytopenia than in the patients with thrombocythemia or the controls. The polyP level was not correlated with the level of aggregation. CONCLUSIONS: The higher polyP levels observed in the patients with low platelet counts may indicate the existence of a compensatory mechanism that prevents excessive bleeding in such patients. Our study provides evidence of an essential role of polyP in platelet function and the coagulation process.

[Ischemia modified albumin--specific marker in cardiological diagnostics?]. / Albumina modyfikowana niedokrwieniem--specyficzny marker w diagnostyce kardiologicznej?

Ischemia modified albumin (IMA) is a new biological marker for early identification of chest pain and ruling out myocardial infarction among patients with acute syndromes submitting to emergency department. Recently IMA has been investigated in the light of other cardiac markers (cTnT, CK-MBmas, NT-proBNP) in various states of ischemia (acute coronary syndromes, after percutaneous coronary intervention, in coronary vasospasm). Ischemia modified albumin levels were elevated in these states what suggests myocardial ischemia. However decrease in IMA concentration after exercise-induced skeletal muscle ischemia still remains unclear. Increased IMA concentration in patients with acute ischemic stroke and exposed to trauma limits its ability for detection myocardial ischemia. Specificity of IMA measurement is limited also in patients with peripheral vascular disease, systemic sclerosis, diabetes, end stage renal disease, pulmonary embolism and other pathological states with accompanying oxidative stress.

The effect of glycation on bovine serum albumin conformation and ligand binding properties with regard to gliclazide.

Albumin, the major serum protein, plays a variety of functions, including binding and transporting endogenous and exogenous ligands. Its molecular structure is sensitive to different environmental modifiers, among which glucose is one of the most significant. In vivo albumin glycation occurs under physiological conditions, but it is increased in diabetes. Since bovine serum albumin (BSA) may serve as a model protein in in vitro experiments, we aimed to investigate the impact of glucose-mediated BSA glycation on the binding capacity towards gliclazide, as well as the ability of this drug to prevent glycation of the BSA molecule. To reflect normo- and hyperglycemia, the conditions of the glycation process were established. Structural changes of albumin after interaction with gliclazide (0-14µM) were determined using fluorescence quenching and circular dichroism spectroscopy. Moreover, thermodynamic parameters as well as energy transfer parameters were determined. Calculated Stern-Volmer quenching constants, as well as binding constants for the BSA-gliclazide complex, were lower for the glycated form of albumin than for the unmodified protein. The largest, over 2-fold, decrease in values of binding parameters was observed for the sample with 30mM of glucose, reflecting the poorly controlled diabetic state, which indicates that the degree of glycation had a critical influence on binding with gliclazide. In contrast to significant changes in the tertiary structure of BSA upon binding with gliclazide, only slight changes in the secondary structure were observed, which was reflected by about a 3% decrease of the α-helix content of glycated BSA (regardless of glucose concentration) in comparison to unmodified BSA. The presence of gliclazide during glycation did not affect its progress. The results of this study indicate that glycation significantly changed the binding ability of BSA towards gliclazide and the scale of these changes depended on glucose concentration. It may have a direct impact on the free drug fraction and its pharmacokinetic behavior, including the risk of hypoglycemic episodes or unexpected interactions with other ligands. The use of BSA in examining binding effects upon glycation seems to be good model for preliminary research and may be used to identify a potential drug response in a diabetic state.

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 enzyme's 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.

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.

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.