Ethyl pyruvate inhibits oxidation of LDL in vitro and attenuates oxLDL toxicity in EA.hy926 cells

<div><p>Background</p><p>Ethyl pyruvate (EP) exerts anti-inflammatory and anti-oxidative properties. The aim of our study was to investigate whether EP is capable of inhibiting the oxidation of LDL, a crucial step in atherogenesis. Additionally, we examined whether EP attenuates the cytotoxic effects of highly oxidized LDL in the human vascular endothelial cell line EA.hy926.</p><p>Methods</p><p>Native LDL (nLDL) was oxidized using Cu²⁺ ions in the presence of increasing amounts of EP. The degree of LDL oxidation was quantified by measuring lipid hydroperoxide (LPO) and malondialdehyde (MDA) concentrations, relative electrophoretic mobilities (REMs), and oxidation-specific immune epitopes. The cytotoxicity of these oxLDLs on EA.hy926 cells was assessed by measuring cell viability and superoxide levels. Furthermore, the cytotoxicity of highly oxidized LDL on EA.hy926 cells under increasing concentrations of EP in the media was assessed including measurements of high energy phosphates (ATP).</p><p>Results</p><p>Oxidation of nLDL using Cu²⁺ ions was remarkably inhibited by EP in a concentration-dependent manner, reflected by decreased levels of LPO, MDA, REM, oxidation-specific epitopes, and diminished cytotoxicity of the obtained oxLDLs in EA.hy926 cells. Furthermore, the cytotoxicity of highly oxidized LDL on EA.hy926 cells was remarkably attenuated by EP added to the media in a concentration-dependent manner reflected by a decrease in superoxide and an increase in viability and ATP levels.</p><p>Conclusions</p><p>EP has the potential for an anti-atherosclerotic drug by attenuating both, the oxidation of LDL and the cytotoxic effect of (already formed) oxLDL in EA.hy926 cells. Chronic administration of EP might be beneficial to impede the development of atherosclerotic lesions.</p></div

Anticoagulant action of low, physiologic, and high albumin levels in whole blood

<div><p>Albumin is the most abundant plasma protein. Critical illness is often associated with altered, predominately decreased, serum albumin levels. This hypoalbuminaemia is usually corrected by administration of exogenous albumin. This study aimed to track the concentration-dependent influence of albumin on blood coagulation in vitro. Whole blood (WB) samples from 25 volunteers were prepared to contain low (19.3 ± 7.7 g/L), physiological (45.2 ± 7.8 g/L), and high (67.5 ± 18.1 g/L) levels of albumin. Haemostatic profiling was performed using a platelet function analyzer (PFA) 200, impedance aggregometry, a Cone and Platelet analyzer (CPA), calibrated automated thrombogram, and thrombelastometry (TEM). Platelet aggregation-associated ATP release was assessed via HPLC analysis. In the low albumin group, when compared to the physiological albumin group, we found: i) shortened PFA 200-derived closure times indicating increased primary haemostasis; ii) increased impedance aggregometry-derived amplitudes, slopes, ATP release, as well as CPA-derived average size indicating improved platelet aggregation; iii) increased TEM-derived maximum clot firmness and alpha angles indicating enhanced clot formation. TEM measurements indicated impaired clot formation in the high albumin group compared with the physiological albumin group. Thus, albumin exerted significant anticoagulant action. Therefore, low albumin levels, often present in cancer or critically ill patients, might contribute to the frequently occurring venous thromboembolism.</p></div

Effects of low, physiologic, and high albumin concentrations on primary haemostasis.

<p>Effects of low, physiologic, and high albumin concentrations on primary haemostasis.</p

Effects of low, physiologic, and high albumin concentrations on platelet aggregation.

<p>Effects of low, physiologic, and high albumin concentrations on platelet aggregation.</p

Effects of low, physiologic, and high albumin concentrations on thrombin generation assessed by means of CAT and by F1+2 plasma levels.

<p>Effects of low, physiologic, and high albumin concentrations on thrombin generation assessed by means of CAT and by F1+2 plasma levels.</p

Cytotoxic effects of highly oxidized LDL on EA.hy926 cells in the presence of increasing medium concentrations of EP.

<p>nLDL (1.5 mg/mL) was oxidized by addition of 10 μmol/L CuCl₂ in the absence of EP in order to obtain the highly cytotoxic form of oxLDL. EP added to the culture medium concentration-dependently attenuated the cytotoxic effect of this oxLDL on EA.hy926 cells, reflected in increased viability <b>(A)</b> and decreased levels of superoxide radical anion formation <b>(B)</b>. Data represent mean ± SD (n = 6 for metabolic activity, n = 4 for superoxide levels), * p < 0.05, *** p < 0.001.</p

Effects of low, physiologic, and high albumin concentrations on platelet adhesion/aggregation assessed by means of CPA.

<p>Effects of low, physiologic, and high albumin concentrations on platelet adhesion/aggregation assessed by means of CPA.</p

ATP levels and EC in EA.hy926 cells treated with highly or mildly oxidized LDL.

<p>EP concentration-dependently attenuated the cytotoxic effect of Cu²⁺-oxidized LDL demonstrated by the increase of ATP <b>(A)</b> and EC <b>(B)</b> in EA.hy926 cells. Data represent mean ± SD (n = 6); ** p < 0.05, *** p < 0.001.</p

Effect of increasing amounts of EP on nLDL oxidation induced by Cu²⁺ ions in vitro.

<p>nLDL (1.5 mg/mL) was preincubated in the absence or presence of EP (100 and 500 μg/mL) and then oxidized by addition of 10 μmol/L CuCl₂ for up to 8 h. <b>(A)</b> EP significantly decreased LPO formation (p < 0.05), <b>(B)</b> MDA formation (p < 0.05), and <b>(D)</b> the amount of oxidation-specific epitopes (p < 0.05). <b>(C)</b> REM concentration-dependently decreased when LDL was oxidized in the presence of increasing amounts of EP (p < 0.005). Data represent mean ± SD from three separate experiments.</p

ATP levels and EC in EA.hy926 cells treated with highly oxidized LDL in the presence of increasing medium concentrations of EP.

<p>EP concentration-dependently attenuated the cytotoxic effects of highly Cu²⁺-oxidized LDL demonstrated by the increase of ATP levels <b>(A)</b> and EC <b>(B)</b> in EA.hy926 cells. Data represent mean ± SD (n = 6), ** p < 0.05, *** p < 0.001.</p