Ana Vrsalović Presečki

Modelling as a tool of enzyme reaction engineering for enzyme reactor development

Strategy of the development of model for enzyme reactor at laboratory scale with respect to the modelling of kinetics is presented. The recent literature on the mathematic modelling on enzyme reaction rate is emphasized.

Impact of admission anemia, C-reactive protein and mean platelet volume on short term mortality in patients with acute ST-elevation myocardial infarction treated with primary angioplasty.

OBJECTIVES To investigate admission anemia, C-reactive protein (CRP) and mean platelet volume (MPV) together as prognostic markers in ST-elevation myocardial infarction (STEMI). DESIGN AND METHODS Baseline hemoglobin, CRP and MPV were determined in 543 patients with acute STEMI to whom primary angioplasty was performed and evaluated for short term mortality (30 days). RESULTS After multivariate analysis anemia (odds ratio 2.69, 95% confidence interval 1.24-5.86) and CRP (odds ratio 3.40, 95% confidence interval 1.13-10.22) remained significant independent predictors of short-term mortality. Addition of anemia and CRP to PAMI risk score improved prediction of short-term outcome; area under ROC curve rose from 0.76 to 0.87 (p<0.001). CONCLUSION Better ability to determine 30-day mortality was obtained when anemia and CRP were incorporated into the PAMI risk score.

Modulating role of alcohol and acetaldehyde on neutrophil and monocyte functions in vitro

Moderate alcohol intake lowers coronary heart disease risk. Because polymorphonuclear neutrophils (PMN) and monocytes (Mo) play a role in atherosclerotic plaque destabilization we investigated in vitro effects of clinically relevant concentrations of ethanol (0.05, 0.125, 0.25, and 0.5%) and its metabolite acetaldehyde (0.0625, 0.125, and 0.5 mM) on human PMN and Mo phagocytic functions. PMN and Mo from healthy volunteers were separated and purified according standard methods and the following parameters were determined: phagocytic activity (percent of phagocytes with at least one ingested particle), ingestion index (number of ingested particles per 100 phagocytic cells), and intracellular killing (percent of dead ingested particles per 100 phagocytes) using acridine orange method and living yeast cells as targets. Reactive oxygen species (ROS) formation of ethanol-treated PMN and Mo was evaluated using 2,7-dichlorofluorescin method and results were expressed as percent of fluorescence-positive cells. Ethanol and acetaldehyde significantly reduced PMN phagocytic functions, with the exception of phagocytic activity, starting at 0.125% for ethanol and 0.0625 mM for acetaldehyde. Mo ingestion and microbicidity were decreased at ethanol concentrations of 0.5% without effect on Mo phagocytic activity. Acetaldehyde impaired Mo ingestion ability starting at 0.0625 mM and phagocytic activity at 0.5 mM while was without effect on Mo microbicidity. ROS production was significantly increased at ethanol concentrations 0.25 and 0.5% in PMN and at 0.5% in Mo. These results might partly explain the benefitial role of moderate use of alcohol on cardiovascular disease.

C-reactive protein, renal function, and cardiovascular outcome in patients with symptomatic peripheral artery disease and preserved left ventricular systolic function.

Aim To investigate the prognostic role of C-reactive protein (CRP) and renal function for the occurrence of major adverse cardiovascular events (MACE) in patients with symptomatic peripheral artery disease (PAD) and preserved left ventricular ejection fraction (LVEF). Methods The occurrence of MACE, defined as composite endpoint of acute myocardial infarction, urgent coronary revascularization, stroke, and death was assessed in 319 consecutive PAD patients admitted to the University Hospital between January 2010 and January 2014 (66.5% men, mean [±standard deviation] age 70 ± 10 years, mean ankle brachial index 0.58 ± 0.14) with normal LVEF (>50%). Multivariate Cox regression analysis adjusted for age, sex, traditional cardiovascular risk factors, anemia, polyvascular disease, critical limb ischemia (CLI), statin treatment, CRP (>5 mg/L), and impaired renal function (estimated glomerular filtration rate <60 mL/min) was applied to assess the independent predictors of MACE. Results During median follow-up period of 24 months (interquartile range, 16-34 months), 77 patients (24%) experienced MACE. Compared to patients without MACE, these patients were older, more likely to have CLI, polyvascular disease, anemia, elevated CRP, and impaired renal function. In multivariate regression analysis, age (HR 1.04, 95% CI 1.01-1.07), polyvascular disease (HR 1.95, 95% CI 1.23-3.09), elevated CRP (HR 1.89, 95% CI 1.18-3.02), and impaired renal function (HR 1.68, 95% C 1.01-2.78) remained independent predictors of MACE. Patients with both impaired renal function and high CRP values on admission were 3.59 times more likely to experience MACE than patients with normal CRP and preserved renal function. Conclusion Elevated admission CRP and renal impairment are independent predictors of MACE in symptomatic PAD patients with preserved LVEF.

Impact of diabetes on mortality in peripheral artery disease: a meta-analysis

There are accumulating studies showing the association between diabetes and all‐cause mortality in peripheral vascular disease. However, the results in these studies are conflicting regarding the impact of diabetes on outcome.

C-reactive protein, not cardiac troponin T, improves risk prediction in hypertensives with type A aortic dissection

Abstract Background: The aim of the study was to evaluate prognostic role of inflammatory biomarkers, cardiac troponin T (cTnT) and D-dimer in type A acute aortic dissection (AAD) and to examine whether they might help in risk stratification beyond values of International Registry of Acute Aortic Dissection (IRAD) score. Methods: Baseline biomarkers were determined in 54 consecutive predominantly hypertensive patients with type A AAD and evaluated for in-hospital mortality. Results: After multivariable adjustment, the independent predictors of outcome were age (OR = 1.09; 95% CI 1.02–1.18), treatment strategy (OR = 0.11; 95% CI 0.02–0.06) and C-reactive protein (CRP) either as binary (OR = 7.06; 95% CI 1.34–37.36) or continuous variable (OR = 1.10; 95% CI 1.01–1.21). cTnT did not independently influence mortality. Receiver- operating characteristic (ROC) curve analysis showed significant link between CRP and outcome (area under the ROC curve, AUC = 0.79; p < 0.01). Values of CRP > 9.8 mg/l had 83% sensitivity and 80% specificity for predicting in-hospital mortality. Addition of CRP to IRAD score improved prediction of short-term outcome, AUC increased from 0.74 to 0.89 (p = 0.004). Conclusion: Admission CRP has independent prognostic value in type A AAD and the addition of CRP to IRAD score improved discriminative capacity of in-hospital mortality irrespective of symptom duration and treatment strategy.

Coenzyme regeneration in hexanol oxidation catalyzed by alcohol dehydrogenase.

The enzymatic ways of coenzyme regeneration include the addition of a second enzyme to the system or the addition of the co-substrate. In the present study, both methods of enzymatic coenzyme (NAD+) regeneration were studied and compared in the reaction of hexanol oxidation catalyzed by alcohol dehydrogenase (ADH). As a source of ADH, commercial isolated enzyme and the whole baker’s yeast cells were used. First, coenzyme regeneration was employed in the reaction of acetaldehyde reduction catalyzed by the same enzyme that catalyzed the main reaction, and then NAD+ regeneration was applied in the reaction of pyruvate reduction catalyzed by l-lactate dehydrogenase (l-LDH). Hexanal was obtained as the product of hexanol oxidation catalyzed by isolated ADH while hexaonic acid was detected as a product of the same reaction catalyzed by baker’s yeast cells. All of the used biocatalysts were kinetically characterized. The mass reactions were described by the mathematical models. All models were validated in the batch reactor. One hundred percent hexanol conversion was obtained using permeabilized yeast cells using both methods of cofactor regeneration. By using isolated enzyme ADH, the higher conversion was achieved in a system with cofactor regeneration catalyzed by l-LDH.

Atrial fibrillation and risk of cardiovascular events and mortality in patients with symptomatic peripheral artery disease: A meta-analysis of prospective studies

Atrial fibrillation (AF) is associated with adverse outcomes in terms of survival and morbidity. Peripheral artery disease (PAD) and AF share several common risk factors and often coexist. Whether AF has a prognostic role in patients with PAD has not been extensively studied.

Different strategies for multi-enzyme cascade reaction for chiral vic-1,2-diol production

The stereoselective three-enzyme cascade for the one-pot synthesis of (1S,2S)-1-phenylpropane-1,2-diol ((1S,2S)-1-PPD) from inexpensive starting substrates, benzaldehyde and acetaldehyde, was explored. By coupling stereoselective carboligation catalyzed by benzoylformate decarboxylase (BFD), L-selective reduction of a carbonyl group with alcohol dehydrogenase from Lactobacillus brevis (ADHLb) as well as the coenzyme regeneration by formate dehydrogenase (FDH), enantiomerically pure diastereoselective 1,2-diol was produced. Two different multi-enzyme system approaches were applied: the sequential two-step one-pot and the simultaneous one-pot cascade. All enzymes were kinetically characterized. The impact of acetaldehyde on the BFD and ADHLb stability was investigated. To overcome the kinetic limitation of acetaldehyde in the carboligation reaction and to reduce its influence on the enzyme stability, experiments were performed in two different excesses of acetaldehyde (100 and 300%). Due to the ADHLb deactivation by acetaldehyde, the simultaneous one-pot cascade proved not to be the first choice for the investigated three-enzyme system. In the sequential cascade with 300% acetaldehyde excess a 100% yield of vic 1,2-diol was reached.

Stereoselective synthesis of (1S,2S)-1-phenylpropane-1,2-diol by cell-free extract of Lactobacillus brevis

Abstract In this study Lactobacillus brevis cells were cultivated and then disrupted using various cell disruption methods to obtain maximal nicotinamide adenine dinucleotide phosphate (NADP(H))-dependent alcohol dehydrogenase (ADH) activity in cell-free extract. Evolutionary operation (EVOP) technique was used to find the optimal cell disruption method. The released ADH in cell-free extract was then used for biotransformation of (S)-2-hydroxypropiophenone ((S)-2-HPP) to (1S, 2S)-1-phenylpropane-1,2-diol ((1S, 2S)-1-PPD). Due to high coenzyme cost, the possibility of NADPH regeneration was considered by examining two substrate-coupled regeneration systems, and for that isopropanol and (R)-1-phenylethanol were used. The enzyme was kinetically characterized, and kinetics of all reactions were determined. Based on kinetic results, mathematical models were developed and were validated in batch reactor. Both regenerating systems successfully shifted the reaction in the desired direction; without coenzyme regeneration, obtained substrate equilibrium conversion was 27.5%, while with coenzyme regeneration by isopropanol oxidation was 99.0% and by (R)-1-phenylethanol was 70.1%.