L. Selmeci

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 γGT: 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 γGT: 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 γGT, 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.

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.

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°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.

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

Abstract 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 21.3years (range 18–33). Over a period of 18months 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.

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

Abstract 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.

Intrapericardial infusion of endothelin-1 induces ventricular arrhythmias in dogs.

OBJECTIVES Recently, extremely high levels of endothelin-1 (ET-1) were detected in the pericardial fluid of patients with heart disease; however, the pathophysiological importance of this finding is not known. The present study was designed to characterize ET-1 levels in canine pericardial fluid and to investigate the effects of local high concentrations of exogenous ET-1 in vivo. METHODS In anesthetized, open-chest dogs ET-1 (Groups 1 and 2: 11 and 33 pmol.kg-1.min-1; n = 6 and 6, respectively) or physiological saline (Group 3, n = 5) were infused into the closed pericardial sac for 40 min. In serial pericardial fluid and aortic blood plasma samples, ET-1 levels were measured by radioimmunoassay, and analysed by high-performance liquid chromatography (HPLC). Systemic arterial blood pressure, heart rate, cardiac output (CO), standard ECG and right ventricular endocardial monophasic action potentials (MAPs) were recorded. RESULTS Basal pericardial fluid ET-1 levels were significantly higher than respective plasma levels (342 +/- 210 vs. 8.0 +/- 5.2 pmol.l-1, n = 14, P < 0.001. In HPLC analysis pericardial fluid ET-1 was indistinguishable from ET-1(1-21). Infusion of exogenous ET-1 into the pericardial space induced ventricular arrhythmias in all instances, which were associated with 9.7-fold increase in pericardial fluid ET-1 levels. Ventricular tachycardias developed in 9 of 12 animals. The arrhythmogenic effect of ET-1 was more apparent in dogs with the larger dose. Before the onset of arrhythmias, intrapericardial infusion of ET-1 increased QT time (Group 1: 207 +/- 18 to 230 +/- 23 ms, P < 0.01; Group 2: 220 +/- 12 to 277 +/- 17 ms, P < 0.01) and MAP duration at 90% repolarization (at 300 ms cycle length) (Group 1: 192 +/- 9 to 216 +/- 9 ms, P < 0.01; Group 2: 205 +/- 9 to 255 +/- 9 ms, P < 0.001). Hemodynamic variables did not change significantly prior to the onset of ventricular tachyarrhythmias. In Group 3, arrhythmias were not observed and all electrophysiological and hemodynamic parameters remained unchanged. CONCLUSIONS Administration of exogenous ET-1 into the pericardial space induces ventricular arrhythmias associated with prolongation of QT time and MAP duration. Whether pericardial fluid ET-1 under pathophysiological conditions can ever reach sufficiently high levels to induce ventricular arrhythmias remains to be elucidated.

Investigating the dual nature of endothelin-1: ischemia or direct arrhythmogenic effect?

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide, which may also elicit severe ventricular arrhythmias. The aims of our study were to compare the effects of total left anterior descending coronary artery (LAD) occlusion to intracoronary (ic.) ET-1 administration and to investigate the pathomechanism of ET-1 induced arrhythmias in 3 groups of anesthetized, open-chest mongrel dogs. In group A (n=10) a total LAD occlusion was carried out for 30 min, followed by a 60 min reperfusion period. In groups B and C ET-1 was administered into LAD for 30 min at a rate of 30 pmol/min (n=6) and 60 pmol/min (n=8). Epi- and endocardial monophasic action potential (MAP) recordings were performed to detect electrophysiologic changes and ischemia Blood samples for lactate measurements were collected from the coronary sinus (CS) and from the femoral artery. Infrared imaging was applied to follow epimyocardial heat emission changes. At the end of the ET-1 infusion period coronary blood flow (CBF) was reduced significantly in groups B and C (deltaCBF30MIN B: 21+/-2%, p<0.05; C: 35+/-2%, p<0.05), paralleled by a significant epimyocardial temperature decrease in group C (deltaT30MIN: -0.65+/-0.29 degrees C, p<0.05). Two dogs died of ventricular fibrillation (VF) in the reperfusion period in group A. Ventricular premature contractions and non-sustained ventricular tachycardic episodes appeared in group B, whereas six dogs died of VF in group C. Significant CS lactate level elevation indicating ischemia was observed only in group A from the 30th min occlusion throughout the reperfusion period (control vs. 30 min: 1.3+/-0.29 vs. 2.2+/-0.37 mmol/l, p<0.05). Epi- and endocardial MAP durations (MAPD90) and left ventricular epicardial (LV(EPI)) upstroke velocity decreased significantly in group A in the occlusion period. ET-1 infusion significantly increased LV(EPI) MAPD90 in group B and both MAPD90-s in group C. In conclusion, ischemic MAP and CS lactate changes were observed only in group A. Although ET-1 reduced CBF significantly in groups B and C, neither MAP nor lactate indicated ischemic alterations. ET-1 induced major ventricular arrhythmias appeared before signs of myocardial ischemia developed, though reduced CBF presumably contributed to sustaining the arrhythmias.

PRESENCE OF IMMUNOREACTIVE ENDOTHELIN-1 AND ATRIAL NATRIURETIC PEPTIDE IN HUMAN PERICARDIAL FLUID

This study was undertaken to characterize endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) concentrations in human pericardial fluid, blood plasma, right atrial appendage and papillary muscle by use of specific radioimmunoassays. In patients undergoing cardiac surgery (n=16) pericardial fluid mean immunoreactive (ir-) ET-1 and ir-ANP levels were 36-fold and 4-fold higher than corresponding plasma levels, respectively. In high performance liquid chromatography (HPLC) pericardial fluid ir-ET-1 was indistinguishable from human ET-1[1-21] and the majority of pericardial fluid ir-ANP coeluted with human ANP[99-126]. Atrial tissue ir-ET-1 and ir-ANP concentrations were 17-fold and 870-fold higher than in ventricular tissue. Our present study demonstrated for the first time the presence of ir-ET-1 in the pericardial fluid in humans. Human pericardial fluid contained far the highest concentrations of ET-1 among all biological fluids tested thus far. The functions of pericardial fluid ET-1 and ANP on cardiac performance and coronary vascular tone require further investigations.

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.

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

Abstract 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. Twentyfive 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 crossclamping, 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. Clin Chem Lab Med 2003; 41(10):13201322