Assay of oxidized fibrinogen reactivity (OFR) as a biomarker of oxidative stress in human plasma: the role of lysine analogs.

BACKGROUND: There is accumulating evidence that fibrinogen is also a biomarker of oxidative stress in human plasma. Results of in vitro studies demonstrated that fibrinogen can bind to apolipoprotein(a) [apo(a)] component of lipoprotein(a) [Lp(a)] through both lysine-sensitive and lysine-insensitive mechanisms. The goal of the present study was to investigate oxidized fibrinogen reactivity (OFR) as a biomarker of oxidative stress in human plasma in the presence and absence of lysine analogs. METHODS: Citrate anticoagulated peripheral venous blood samples were collected from 65 (36 M/29 F) consecutive patients with various peripheral vascular diseases. After centrifugation, the plasma was used promptly. Plasma OFR was determined in duplicate using a recently described kinetic photometric assay (358 nm, 37 degrees C) in the presence and in the absence of lysine analogs. RESULTS: The inclusion of tranexemic acid (TRA) or epsilon-aminocaproic acid in the incubation medium resulted in a rapid increase in OFR in a dose-dependent manner. The peak effect was observed at a final concentration of 200 mmol/L TRA. OFR was significantly higher in patient plasma assayed in the presence of TRA compared with no TRA (163.1 +/- 73.5 vs. 63.4 +/- 20.7 U/L; p < 0.0001). Bound OFR was also significantly higher than free OFR (99.7 +/- 56.3 vs. 63.4 +/- 20.7; p < 0.001). CONCLUSIONS: On the basis of the present results it appears that oxidized fibrinogen resides in plasma in two compartments: free and bound to apo(a) of Lp(a). The relatively simple and cost-effective kinetic approach applied in this study makes routine determination of OFR available as a biomarker of oxidative stress, separately in both compartments.

Low c1-inhibitor levels predict early restenosis after eversion carotid endarterectomy.

OBJECTIVE: Homozygotes for the normal (A) allele of mannose-binding lectin (MBL2) gene have higher risks to develop an early restenosis after eversion carotid endarterectomy (CEA). Activation of the lectin pathway is regulated by C1-inhibitor (C1-INH). The objective of the present study was to determine the predictive value of C1-INH in restenosis after CEA. METHODS AND RESULTS: C1-INH and MBL-associated serine protease-2 (MASP-2) were determined in samples serially taken from 64 patients with CEA, who were followed-up with carotid duplex scan (CDS) examinations for 14 months. MBL2 genotypes were also determined. Patients with >50% restenosis had lower C1-INH levels at 6 weeks (P=0.0052) and at 4 days (P=0.0277) postsurgery. C1-INH levels at 6 weeks correlated inversely with the CDS values at 14 months (r=-0.3415, P=0.0058), but only in MBL2 A/A homozygotes (r=-0.5044, P=0.0015). Patients with low C1-INH levels (C1-INH <115%) had higher CDS values already at 7 months postsurgery. Patients with MBL2 A/A and low C1-INH levels at 6 weeks postsurgery had 13.97 (95% CI:1.95 to 100.21, P=0.0087) times higher risk to develop an early restenosis. Differences in the MASP-2 concentration were not associated with restenosis. CONCLUSIONS: Determining C1-INH levels at 6 weeks postsurgery-together with genotyping of MBL2-might be a useful marker in the identification of patients with high risk for early carotid restenosis.

Early rise in serum VEGF and PDGF levels predisposes patients with a normal MBL2 genotype to restenosis after eversion endarterectomy.

BACKGROUND AND PURPOSE: Recently we found that the incidence of restenosis after carotid endarterectomy was significantly higher in patients homozygous for the normal genotype of mannose-binding lectin (MBL2) than in with patients with MBL2 variant genotypes. Several growth factors are also known to contribute to restenosis. Therefore, we investigated whether early postoperative changes in serum vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) concentrations and MBL2 genotypes interact in the development of restenosis. METHODS: Eighty-two patients who underwent carotid eversion endarterectomy and were followed up by carotid duplex scan sonography for 14 months were studied. Growth factors were measured preoperatively and 4 days after surgery. RESULTS: Pronounced significant increases in both VEGF and PDGF predicted restenosis but only in patients who were homozygous for the normal MBL2 genotype. In this group, the adjusted odds ratios of restenosis at 14 months in patients with high versus low early VEGF and PDGF increases were 27.73 (2.42 to 317.26) and 9.23 (1.45 to 58.70), respectively. CONCLUSIONS: These findings indicate that the development of restenosis depends on both complement activation regulated by the MBL2 gene and pathologic processes leading to enhanced production of VEGF and PDGF during the very early postoperative period.

Elevated complement C3 is associated with early restenosis after eversion carotid endarterectomy.

Early restenosis following carotid endarterectomy (CEA) is an inflammatory process leading to myointimal hyperplasia of smooth muscle cells. The risk for restenosis is increased in homozygous carriers of the normal (A) allele of mannose-binding lectin (MBL2) gene. Our objective was to study the associations of C3 and as control three non-complement acute-phase reactants (APRs) (C-reactive protein, haptoglobin and alpha2HSglycoprotein) with early restenosis following CEA. We also considered, whether MBL2 genotype relates to C3 levels and to the risk of restenosis. Concentrations of the APRs were determined by radial immunodiffusion or immunoturbidimetric methods in 64 patients who underwent eversion CEA and were followed up with carotid duplex scan (CDS) examinations for at least one year. MBL2 genotypes were determined by a PCR-SSP method. C3 levels increased during the follow-up and correlated with the percentage of restenosis detected by CDS at 14 months postsurgery, in MBL2 A/A allele carriers. Patients with high C3 levels had nearly five-fold higher odds for the presence of significant restenosis (>50% reduction in diameter) even after adjusting for MBL2 genotype, age and gender. By contrast, no such associations were detected between the non-complement APRs and early restenosis. C3 is associated with and might have a direct role in the development of an early restenosis following CEA, which is partially related to an intact MBL lectin pathway, thus determining C3 levels might have clinical importance. On the other hand, our results indicate that the regulation of C3 differs from non-complement APRs.

Human blood plasma advanced oxidation protein products (AOPP) correlates with fibrinogen levels.

In 1996 a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPP) was detected in the plasma of chronic uremic patients. The aim of the present studies was to find out that which plasma fraction(s) is responsible for AOPP reactivity. Thermal treatment of pooled samples of human citrate-plasma or EDTA-plasma at 50 degrees C resulted in a rapid and parallel loss of fibrinogen concentration and AOPP reactivity. On the basis of time course and t1/2 values following thermal treatment, AOPP was indistinguishable from fibrinogen. There was a statistically significant (p < 0.0001) correlation between levels of blood plasma fibrinogen and AOPP in patients (n = 61) with various peripheral vascular or cardiovascular diseases. There was also a significant (p < 0.0001) relationship between plasma levels of fibrinogen and molar AOPP/fibrinogen ratio indicating that higher fibrinogen concentrations were associated with more oxidatively transformed groups on the molecule. Results of the present studies suggest that post-translationally modified fibrinogen is a key molecule responsible for human plasma AOPP reactivity. It remains to be elucidated what is the pathophysiological significance of the post-translationally modified fibrinogen in the inflammation-associated events of atherosclerosis, in platelet aggregation, and as a cardiovascular risk biomarker.

High rate of early restenosis after carotid eversion endarterectomy in homozygous carriers of the normal mannose-binding lectin genotype.

BACKGROUND AND PURPOSE: Mannose-binding lectin (MBL) is thought to influence the pathophysiology of cardiovascular disease by decreasing the risk of advanced atherosclerosis and by contributing to enhanced ischemia reperfusion injury. Thus, we investigated the role of MBL in restenosis after eversion endarterectomy in patients with severe carotid atherosclerosis. METHODS: In a prospective study, 123 patients who underwent carotid endarterectomy were followed-up by carotid duplex scan (CDS) sonography for 14 months. In a retrospective study, we examined 17 patients and 29 patients, respectively, who had or had not at least 50% restenosis 29 months after carotid eversion endarterectomy. MBL genotypes were analyzed by a polymerase chain reaction-based method, and MBL serum concentrations were measured. RESULTS: In the prospective study in the patients homozygous for the normal MBL genotype, CDS values were significantly higher after 14 months of follow-up compared with the values measured 6 weeks after surgery (P<0.001). In contrast, only a slight increase was registered in patients carrying MBL variant alleles. The differences were much more pronounced in female than in male patients. Similar differences were observed when patients with high and low MBL serum concentrations were compared. In the retrospective study, a significant increase in the frequency of MBL variant genotypes was observed in patients not experiencing restenosis compared with the patients with restenosis (P=0.007). CONCLUSIONS: These results indicate that reoccurrence of stenosis after carotid endarterectomy is partially genetically determined and imply that MBL contributes significantly to the pathophysiology of this condition.

Relationship between eradication of Helicobacter pylori and gastric mucosal superoxide dismutase activity.

BACKGROUND: Helicobacter pylori (HP) is the main pathogenic factor in the development of gastritis and gastric cancer. Superoxide-dismutase (SOD) is a key enzyme of mucosal antioxidant protection. In the presence of HP there is a significant increase of SOD activity in the antrum. Changes in gastric mucosal SOD activity were detected in response to eradication treatment of HP infection. PATIENTS AND METHODS: Biopsies were taken from 13 patients upon gastroscopy performed prior to and 88.3 +/- 12.6 days after treatment. The activity of SOD was determined by spectrophotometry. RESULTS: The activity of SOD in the gastric mucosa decreased significantly following the successful eradication, whereas in the corpus activity did not change significantly. CONCLUSION: In the presence of HP there is an oxidative stress in the gastric mucosa triggered by the bacterium. It may represent the final common path of HP carcinogenesis. Successful eradication treatment prevents the production of reactive oxygen metabolites.

Superoxide-dismutase activity of the gastric mucosa in patients with Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori (HP) is a pathogenic factor in the development of different alterations in the gastric mucosa. Superoxide-dismutase (SOD) is a key enzyme of mucosal antioxidant protection. We have detected changes in the activity of mucosal SOD in different diseases caused by HP. MATERIALS AND METHODS: Biopsies were taken upon gastroscopy (n = 131). Activity of SOD was measured by photometry, referred to the amount of protein in the sample. RESULTS: SOD activity of the antrum of HP-positive patients was significantly higher than that of HP-negative ones. There was a significant increase in erosive gastritis. CONCLUSION: In the presence of HP there was a significant increase of the SOD activity in the antrum but not in the corpus. In chronic antral gastritis there was a positive relationship between the SOD activity and both the severity and activity of inflammation. We presume that HP-associated gastritis is predominant in the antrum.

[C-reactive protein levels determined by an ultrasensitive method in a healthy Hungarian population]. / Az ultraszenzitív módszerrel meghatározott C-reaktív fehérjekoncentráció megoszlása az egészséges magyar populációban.

INTRODUCTION: C-reactive protein (CRP) is a well-known acute phase protein. The concentration of CRP in serum is increased in response to inflammatory stimuli. Increased levels serve to identify organic disease, to monitor disease activity and to assist differential diagnosis. AIM: The aim of the authors' cross-sectional study was to determine CRP distribution of the healthy Hungarian population. METHOD: 207 (79 male, 128 female; mean age: 4 +/- 68 years) healthy blood donors were enrolled for the study. The following parameters were registered: sex, age, body mass index, smoking habits, diabetes mellitus and blood pressure. Serum samples were assayed for total serum cholesterol, triglyceride, erythrocyte sedimentation rate, hemoglobin and for white blood cell count. CRP was measured by ultrasensitive, particle enhanced immunoturbidimetric assay. RESULTS: CRP levels were less than 5 mg/L in 81% of the blood donors. Mean level of CRP in the study population was 3.57 mg/L (SD +/- 5.33); the distribution was comparable to the data of already published studies. Comparing laboratory parameters and the risk status stratified according to CRP levels (less or more than 5 mg/L) significant differences were found in BMI (p = 0.0015), in total serum cholesterol (p = 0.0136), in triglyceride (p < 0.0001), in erythrocyte sedimentation rate (p < 0.0001), in white blood cell (p = 0.0007) and granulocyte count (p = 0.0014). Significant correlation was found between age and the concentration CRP (r = 0.22; p = 0.0011). CONCLUSION: The CRP measurement by ultrasensitive method is suitable for cardiovascular risk estimation in apparently healthy men and women. Risk prediction adapted for the Hungarian situation may be stimulated by these data.

Advanced oxidation protein products (AOPP): novel uremic toxins, or components of the non-enzymatic antioxidant system of the plasma proteome?

In 1996, a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPP), was detected in the plasma of chronic uremic patients. It was suggested that AOPP measure highly oxidized proteins, especially albumin. Recent data in turn appear to indicate that oxidized fibrinogen is the key molecule responsible for the AOPP reaction in the human plasma. Since fibrinogen is an acute-phase reactant, it is evident that during each episode of inflammatory response, the antioxidant capacity of the plasma is enhanced. In this context, fibrinogen can be regarded as a component of the antioxidant system of the plasma proteome. It was also demonstrated that oxidized fibrinogen is bound to apolipoprotein(a) of lipoprotein(a) via lysine binding sites. Thus, apo(a) could compete with plasminogen (and/or tissue plasminogen activator) for its binding sites of fibrin(ogen), causing inhibition of fibrinolysis, and thereby promote atherosclerosis and cardiovascular disease.

Higher serum DPP-4 enzyme activity and decreased lymphocyte CD26 expression in type 1 diabetes.

Dipeptidyl peptidase-4 (DPP-4) is involved in the metabolism of peptide hormones, T-cell activation and proliferation. In type 1 diabetes mellitus (T1DM) ß-cell destruction involves a number of dysregulated T-cells. Our aim was to assess the serum DPP-4 activity and the lymphocyte membrane bound CD26 expression in patients with type 1 diabetes and healthy controls. Ninety-eight (T1DM: 48, F/M = 20/28, mean age: 34.4y; control: 50, F/M = 39/11 mean age: 32,4y) individuals were included. Fasting serum DPP-4 enzymatic activity, plasma glucose (FPG), CD26 expression on CD3+, CD4+ and CD8+ lymphocytes, HbA1c and body mass index (BMI) were assessed. ICA and GAD antibodies were assessed in the T1DM group. DPP-4 enzymatic activity was determined by kinetic enzyme assay, ICA and GAD were assessed by ELISA. Determination of the CD26 expression on CD3+, CD4+ and CD8+ lymphocytes was performed by flow-cytometric analysis. We found higher serum DPP-4 activity (Mean: T1DM: 30.069 U/L, control: 22.62 U/L, p < 0.0001) and decreased CD26 lymphocyte expression on all lymphocyte subpopulations in T1DM. Fasting serum DPP-4 activity was independent from the ICA or GAD status of patients with T1DM. Here we first present that the serum DPP-4 activity is increased and the lymphocyte membrane bound CD26 expression is decreased in type 1 diabetes. Decreased lymphocyte membrane bound CD26 expression in T1DM might be a novel part of the T-lymphocyte regulatory dysfunction observed in type 1 diabetes mellitus. These results might provide some basis for the clinical implication of DPP-4 inhibition in patients with T1DM.

Serum dipeptidyl peptidase-4 activity in insulin resistant patients with non-alcoholic fatty liver disease: a novel liver disease biomarker.

BACKGROUND: In a cross-sectional study we studied the fasting serum DPP-4 enzymatic activity (sDPP-4) and the insulin resistance index (HOMA2-IR) in gliptin naïve patients with type 2 diabetes and in non-alcoholic fatty liver disease (NAFLD) and in healthy controls (CNTRL). METHODS AND FINDINGS: sDPP-4 was measured by kinetic assay in 39 NAFLD (F/M:19/20, mean age: 47.42 yrs) and 82 type 2 diabetes (F/M:48/34, 62.8 yrs) patients and 26 (F/M:14/12, 35.3 yrs) controls. Definition of T2D group as patients with type 2 diabetes but without clinically obvious liver disease created non-overlapping study groups. Diagnosis of NAFLD was based on ultrasonography and the exclusion of other etiololgy. Patients in T2D and NAFLD groups were similarly obese. 75 g CH OGTT in 39 NAFLD patients: 24-NGT, 4-IGT or IFG ("prediabetes"), 11-type 2 diabetes. HOMA2-IR: CNTRL: 1.44; T2D-group: 2.62 (p = 0.046 vs CNTRL, parametric tests); NAFLD(NGTonly): 3.23 (p = 0.0013 vs CNTRL); NAFLD(IFG/IGT/type 2 diabetes): 3.82 (p<0.001 vs CNTRL, p = 0.049 vs 2TD group). sDPP-4 activity was higher in NAFLD both with NGT (mean:33.08U/L) and abnormal glucose metabolism (30.38U/L) than in CNTRL (25.89U/L, p<0.001 and p = 0.013) or in T2D groups (23.97U/L, p<0.001 and p = 0.004). Correlations in NAFLD among sDPP-4 and ALT: r = 0.4637,p = 0.0038 and gammaGT: r = 0.4991,p = 0.0017 and HOMA2-IR: r = 0.5295,p = 0.0026 and among HOMA2-IR and ALT: r = 0.4340,p = 0.0147 and gammaGT: r = 0.4128,p = 0.0210. CONCLUSIONS: The fasting serum DPP-4 activity was not increased in T2D provided that patients with liver disease were intentionally excluded. The high serum DPP-4 activities in NAFLD were correlated with liver tests but not with the fasting plasma glucose or HbA1C supporting that the excess is of hepatic origin and it might contribute to the speedup of metabolic deterioration. The correlation among gammaGT, ALT and serum DPP-4 activity and also between serum DPP-4 activity and HOMA2-IR in NAFLD strongly suggests that serum DPP-4 activity should be considered as a novel liver disease biomarker.

Advanced oxidation protein products (AOPP) for monitoring oxidative stress in critically ill patients: a simple, fast and inexpensive automated technique.

Oxidative stress is known to be involved in many human pathological processes. Although there are numerous methods available for the assessment of oxidative stress, most of them are still not easily applicable in a routine clinical laboratory due to the complex methodology and/or lack of automation. In research into human oxidative stress, the simplification and automation of techniques represent a key issue from a laboratory point of view at present. In 1996 a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPP), was detected in the plasma of chronic uremic patients. Here we describe in detail an automated version of the originally published microplate-based technique that we adapted for a Cobas Mira Plus clinical chemistry analyzer. AOPP reference values were measured in plasma samples from 266 apparently healthy volunteers (university students; 81 male and 185 female subjects) with a mean age of 21.3 years (range 18-33). Over a period of 18 months we determined AOPP concentrations in more than 300 patients in our department. Our experiences appear to demonstrate that this technique is especially suitable for monitoring oxidative stress in critically ill patients (sepsis, reperfusion injury, heart failure) even at daily intervals, since AOPP exhibited rapid responses in both directions. We believe that the well-established relationship between AOPP response and induced damage makes this simple, fast and inexpensive automated technique applicable in daily routine laboratory practice for assessing and monitoring oxidative stress in critically ill or other patients.

Antioxidant capacity of the human pericardial fluid: does gender have a role?

The aim of the present study was to assess the antioxidant capacity in the serum and pericardial fluid of patients undergoing heart surgery for coronary heart disease (CHD) or valvular heart disease (VHD) and to find out whether there are gender-related differences in the antioxidant defense. This study involved 85 patients (35 VHD and 50 CHD) undergoing elective heart surgery. Blood samples from the peripheral vein and from the pericardial fluid were taken intraoperatively. Variables determined in the serum and pericardial fluid were: total protein, albumin, uric acid and antioxidant capacity. In the total patient population the antioxidant capacity in the pericardial fluid was lower than in the serum but still relatively high as determined by two independent techniques. No major differences were seen in serum or pericardial fluid antioxidant capacity between the two patient groups. In the overall patient population uric acid (p<0.05), albumin (p<0.01) and total protein concentrations (p<0.01) were, however, significantly greater in the pericardial fluid of male than of female patients. The pericardial fluid may contribute to the local antioxidant defense of the myocardium. It appears that male gender confers advantage in this respect. It remains to be elucidated whether this finding has any implication for the higher risk for women of perioperative complications and of cardiovascular mortality after coronary bypass grafting or coronary angioplasty.

Impairment of blood-brain barrier integrity during carotid surgery as assessed by serum S-100B protein concentrations.

During carotid endarterectomy (CEA) the internal carotid artery is cross-clamped for a period of several minutes. This maneuver may cause cerebral hypoperfusion and/or impairment of the blood-brain barrier (BBB) even in cases where clinical signs are absent. The aim of the present study was to examine whether such alterations could be detected by monitoring the cerebral marker S-100B protein concentrations during and after CEA in the serum. Twenty-five consecutive patients (17 M/8 F, mean age: 64.2 years, range 47-79 years) undergoing elective CEA at our department were studied. All of these patients were without perioperative neurological deficit. Intraoperative samples were collected from internal jugular and peripheral venous blood: 1) before carotid cross-clamping; 2) immediately before declamping; 3) after clamp release. Postoperative samples were taken from peripheral blood at 6 and 24 h, respectively. S-100B was assayed in sera using an immunoluminometric technique. During carotid cross-clamping, S-100B protein concentrations in the ipsilateral jugular serum significantly (p < 0.02) increased to pre-clamp values. After declamping, however, S-100B returned to the baseline level. No differences were seen between the responses of hypertensive and normotensive patients. There was no correlation between carotid occlusion time and S-100B protein concentrations. In the peripheral venous serum no significant changes in S-100B concentrations were detected during or after CEA. We presume that the elevation of S-100B protein concentration during CEA in patients with no neurological deficits indicates the transient opening of the BBB elicited by carotid cross-clamping.