Slavica Spasic

LDL and HDL subclasses in acute ischemic stroke: Prediction of risk and short-term mortality

OBJECTIVE Small, dense low-density lipoprotein (sdLDL) and small-sized high-density lipoprotein (HDL) particles are established risk factors for ischemic heart disease. However, their clinical significance for acute ischemic stroke (AIS) is uncertain. This study evaluates associations of LDL and HDL particle sizes and subclasses with AIS risk and short-term mortality after AIS. METHODS Two hundred AIS patients hospitalised for first-in-a-lifetime stroke and 162 apparently healthy controls were included in the study. LDL and HDL particles were separated by gradient gel electrophoresis and serum lipid parameters were measured by standard laboratory methods. Baseline characteristics of LDL and HDL particles were evaluated for the prediction of AIS and short-term mortality after AIS. RESULTS AIS patients had significantly more LDL III and IVb, but less LDL I and II particles. They also had significantly smaller HDL size, more HDL 3a, 3b and 3c and less HDL 2b subclasses. The relative content of both sdLDL and small-sized HDL particles was significantly increased in patients (P<0.001 and P<0.001, respectively). In addition, sdLDL was significantly higher in AIS fatalities (n=25) compared with survivors (n=175, P<0.05). Increased sdLDL was a significant predictor of AIS (OR=4.31; P<0.001) and in-hospital mortality after AIS (OR=5.50; P<0.05). The observed relationships persisted after adjustment for conventional risk factors. CONCLUSIONS AIS is associated with adverse distributions of LDL and HDL subclasses. In addition, short-term mortality after AIS is associated with increased sdLDL particles. Our results indicate that sdLDL is an independent predictor of both AIS onset and consecutive short-term mortality.

Pulmonary function, oxidative stress and inflammatory markers in severe COPD exacerbation.

BACKGROUND Oxidative stress and inflammation play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). OBJECTIVE Pulmonary function, oxidative stress parameters and inflammatory markers were measured in 74 patients with severe COPD exacerbation and 41 healthy subjects. In patients all parameters were assessed at two time points: Firstly, one day after admission and secondly, after 7 10 days when they were clinically stable enough to be discharged. Patients were divided in two groups according the presence of ischemic heart disease (IHD): IHD positive (IHD+) patients and IHD negative (IHD-) patients. METHODS AND RESULTS During hospitalisation O2•-, malondialdehyde (MDA), advanced oxidation protein products (AOPP) and total oxidant status (TOS) increased and were higher at discharge compared with admission and the control group. Superoxide dismutase (SOD) activity was significantly lower in COPD patients at both time points compared with the control group. Total antioxidant status (TAS) was significantly lower and the prooxidant-antioxidant balance (PAB) was higher at both time points in COPD patients compared with the control group. High sensitive C-reactive protein (hsCRP) and also the neutrophil count were significantly higher at admission compared with discharge. Paraoxonase 1 (PON1) enzymatic activities in COPD patients did not differ compared with the control group. IHD+ COPD patients had significantly lower PON1 activity but higher PAB levels and hsCRP concentrations, compared with IHD COPD patients. CONCLUSION The oxidant/antioxidant imbalance was significantly pronounced in patients with COPD exacerbation for at least 24 hours following their admission and when they were clinically stable enough to be discharged. Increased oxidative stress, elevated systemic inflammation and decreased antioxidant defence were common in end-stage disease and particularly COPD patients with ischemic heart disease.

Evaluation of different formulas for LDL-C calculation

BackgroundFriedewalds formula for the estimation of LDL-C concentration is the most often used formula in clinical practice. A recent formula by Anandaraja and colleagues for LDL-C estimation still needs to be evaluated before it is extensively applied in diagnosis. In the present study we validated existing formulas and derived a more accurate formula to determine LDL-C in a Serbian population.MethodsOur study included 2053 patients with TG ≤ 4.52 mmol/L. In an initial group of 1010 patients, Friedewalds and Anandarajas formulas were compared to a direct homogenous method for LDL-C determination. The obtained results allowed us to modify Friedewalds formula and apply it in a second group of patients.ResultsThe mean LDL-C concentrations were 3.9 ± 1.09 mmol/L, 3.63 ± 1.06 mmol/L and 3.72 ± 1.04 mmol/L measured by a direct homogenous assay (D-LDL-C), calculated by Friedewalds formula (F-LDL-C) and calculated by Anandarajas formula (A-LDL-C), respectively in the 1010 patients. The Students paired t-test showed that D-LDL-C values were significantly higher than F-LDL-C and A-LDL-C values (p < 0.001). The Passing-Bablok regression analysis indicated good correlation between calculated and measured LDL-Cs (r > 0.89). Using lipoprotein values from the initial group we modified Friedewalds formula by replacing the term 2.2 with 3. The new modified formula for LDL-C estimation (S-LDL-C) showed no statistically significant difference compared to D-LDL-C. The absolute bias between these two methods was -0.06 ± 0.37 mmol/L with a high correlation coefficient (r = 0.96).ConclusionsOur modified formula for LDL-C estimation appears to be more accurate than both Friedewalds and Anandarajas formulas when applied to a Serbian population.

High serum uric acid and low-grade inflammation are associated with smaller LDL and HDL particles

Elevated serum uric acid (UA) is associated with higher risk for cardiovascular disease (CVD). Smaller, denser low density lipoprotein (LDL) and high-density lipoprotein (HDL) particles are the potential risk factors for CVD, while the role and diagnostic value of inflammatory markers are firmly established. This current cross-sectional study investigates interrelationships between UA, high sensitivity C-reactive protein (hsCRP) and fibrinogen concentrations with LDL and HDL sizes in healthy middle-aged subjects. The outcomes-of-interest were smaller, denser LDL and HDL particles (LDL size <or=25.5nm and HDL size <or=8.8nm). Serum UA, hsCRP and plasma fibrinogen concentrations were measured by standard laboratory methods in a sample of 194 healthy volunteers (112 men and 82 women). LDL and HDL particle sizes were determined by gradient gel electrophoresis. The subjects in the highest UA tertile had significantly smaller LDL and HDL particle sizes (P<0.05 and P<0.01, respectively) and higher concentrations of fibrinogen and hsCRP (P<0.05 and P<0.01, respectively). Elevated UA (>or=318micromol/L) was a significant predictor of smaller, denser LDL and HDL particles (OR=3.09; P<0.01; n=19 and OR=4.40; P<0.001; n=23, respectively). The observed relationship with smaller HDL size persisted after adjustment for conventional cardiovascular risk factors. UA strongly correlated with both markers of inflammation. In addition, the higher hsCRP level correlated with smaller LDL size (P<0.05), while fibrinogen concentration was inversely related to HDL size (P<0.05). Multiple regression analysis revealed that HDL size and inflammatory markers remained independent determinants of UA concentration. In conclusion, higher serum UA and low-grade inflammation are closely linked to alterations in lipoprotein metabolism which may represent an early sign of atherosclerosis in asymptomatic subjects.

Association of oxidative stress and PON1 with LDL and HDL particle size in middle‐aged subjects

Background  Alterations in plasma lipoprotein subclass distributions affect atherosclerosis risk. Smaller, denser low‐density lipoprotein (LDL) particles (sdLDL) are more susceptible to oxidation. In contrast, most of the protective effects of high‐density lipoproteins (HDL) are attributable to larger particles. This study investigates the connection between LDL and HDL particle heterogeneity and oxidative stress, antioxidative defence (AOD) and paraoxonase (PON1) status in a healthy middle‐aged Serbian population.

Circulating sTWEAK improves the prediction of coronary artery disease.

OBJECTIVES Decreased concentrations of circulating soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) were recently reported to be associated with atherosclerosis, but there are still no data concerning its predictive performance. DESIGN AND METHODS The current cross-sectional study investigates the potential of the novel atherosclerotic biomarker for the prediction of coronary artery disease (CAD). Serum sTWEAK was measured by ELISA in 76 CAD patients and 82 CAD-free subjects. RESULTS Serum sTWEAK concentrations were significantly lower in the patients (534.5+/-110.9 microg/L) than in the controls (688.1+/-150.0 microg/L, p<0.001), even after adjusting for different confounders (p<0.001). The areas under ROC curves (AUC)s calculated for logistic regression models that included different known risk factors were significantly increased when sTWEAK was added to the corresponding model (p=0.011-0.035). CONCLUSIONS The measurement of serum sTWEAK concentrations improves the prediction of CAD based on existing biomarkers.

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.

Cytotoxic effects of the Viscum album L. extract on Ehrlich tumour cells in vivo.

To date most pharmacological studies on mistletoe (Viscum album L.) have focused on the therapeutic properties of its polar extracts. This study examined the non‐polar constituents of Viscum album and their biological activities. Supercritical CO2 extraction coupled with gas chromatography/mass spectrometry (GC/MS) was used to selectively extract and identify compounds in Viscum album leaves. Several non‐polar classes of compounds were identified in the extract. In addition, a volatile fraction was identified that contained several novel terpene molecules. The hypothesis was tested that the Viscum album extract exhibits cytotoxic properties in Ehrlich carcinoma (EAC) cells in vivo due to the induction of oxidative stress. A significant reduction in the incidence of cancer was observed in all groups that received the Viscum album extract compared with the EAC control group. The largest decrease was observed in mice pretreated with the Viscum album extract, although significantly reduced numbers of EAC cells were also observed in animals with developed carcinoma. The activities of antioxidative enzymes in the EAC cells suggested the absence of oxidative stress. However, changes in the antioxidative enzymes activities observed after administration of the Viscum album extract might be due to the induction of oxidative stress in the EAC cells. Copyright

Paraoxonase-1 (PON1) activity, but not PON1Q192R phenotype, is a predictor of coronary artery disease in a middle-aged Serbian population

Abstract Background: Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme that protects lipoproteins from oxidative modifications. Polymorphisms in the gene, including PON1Q192R, have been studied. However, inconsistencies regarding the above-mentioned polymorphism obscure its association with vascular disease. Methods: Using a two-substrate (paraoxon/diazoxon) activity method, we investigated the frequencies of PON1Q192R phenotypes in 261 middle-aged subjects: 156 patients with angiographically assessed coronary heart disease (CHD) and 105 CHD-free subjects as the control group. The PON1192 phenotype was predicted from examination of the two-dimensional plot of hydrolysis rates of diazoxon vs. paraoxon and by using the antimode of the histogram of the ratio of diazoxonase/paraoxonase activity. Results: The PON1Q192R phenotype frequencies in 113 patients with occlusion >50% (coronary artery disease-positive, CAD+ group) vs. control population were as follows: QQ (0.552 vs. 0.510), QR (0.382 vs. 0.408) and RR (0.066 vs. 0.082); χ2=0.414, p=0.813. We found lower paraoxonase (POase) and diazoxonase (DZOase) activities in the CAD+ patients when compared to the control population. According to logistic regression analysis, POase activity was a better predictor of coronary disease onset compared with DZOase activity measurements and PON1Q192R phenotyping. Conclusions: We conclude that enzyme activity (within a particular phenotypic group) is more important than phenotype alone in predicting susceptibility to coronary artery disease. Clin Chem Lab Med 2006;44:1206–13.

Capillary electrophoresis method optimized with a factorial design for the determination of glutathione and amino acid status using human capillary blood

Plasma aromatic and sulfur containing amino acids are good indicators of protein anabolism/catabolism, while blood reduced and oxidized glutathione reflect oxidative status in an organism. Using a full factorial design for screening important variables (pH, concentration, temperature) we developed a capillary zone electrophoresis method permitting their measurements in the single run, without any derivatization procedures. The best separations were obtained within less than 30 min employing a 10 mmol/l phosphate buffer, pH 2.8, 18 degrees C, 15 kV voltage. Fairly good precision with a linear relationship between peak area and concentrations (r=0.995-0.999) were obtained. The method was used to analyze human capillary blood.