The Chemiluminescence Assay of Lipid Peroxidation Products in Human Blood Plasma Lipoproteins

Abstract

A chemiluminescence (CL) flash kinetics on the addition of Fe2+ ions into oxidized low density lipoprotein (LDL) suspension has been studied. LDL oxidation was carried out at 37 degrees C without and in the presence of 5 or 50 microM of Cu2+. It has been found that under certain experimental conditions (the addition of excess iron ions, more than 1 mM) the amplitude of CL flash depended almost linearly (1) on the concentration of oxidized LDL and (2) on the extent of LDL oxidation measured as diene conjugates (DC) and 2-thiobarbituric acid-reactive substance (TBARS) accumulation. The corresponding correlation coefficients were: for TBARS -0.94 and for DC -0.97, in the case of LDL autooxidation; 0.72 and 0.98, in the case of copper-induced LDL oxidation. A sensitivity of the CL method was shown to be significantly enhanced (by more than two orders) in the presence of CL sensitizer--2,3,5,6-1H,4H-tetrahydro-9-(2'-benzoimidazolyl)-qui nolizin-(9,9a,1- gh)coumarin.

Light Emission Resulting from Hydroxylamine‐Induced Singlet Oxygen Formation of Oxidizing LDL Particles

“…In order to understand the particular role of NH,OH in Cu2+-induced LPO of lipid membranes, we compared our observations with the extensive studies on lipid peroxidederived chemiluminescence of Vladimirov and his coworkers (10,30,31). As already mentioned in the introduction, they developed a reaction scheme of Cu2+-oxidized LDL, where the cupric ion first initiates the peroxidation of lipids starting with the decomposition of adventitious lipid hydroperoxides that are always present in LDL at low concentrations: oxidation was started with lipoxygenase (27) light emission was detected when NH,OH was added (Fig.…”

“…A similar observation was recently made by Driomina el al. (10). In that case light emission was found when ferrous iron was added to LDL particles that had been preoxidized by Cu2+ ions.…”

Chemiluminescence study of the contents of the products of Cu2+-induced lipid peroxidation in different fractions of human blood lipoproteins

“…The LP were dialyzed for 18 h against 500 volumes of the standard buffer (10 mM Tris-HC1, 100 mM NaC1, pH 7.4). Oxidation was carried out for 4 h at 37~ in a dry thermostat in the presence of varied concentrations of Cu 2 § The amplitude of chemiluminescence bursts was measured in an LKB-1251 chemiluminometer as described elsewhere [3] after the addition of Fe 2+ (1 mM) to LP samples containing 0.08 mg protein/ ml in the standard buffer. Oxidation was carried out for 4 h at 37~ in a dry thermostat in the presence of varied concentrations of Cu 2 § The amplitude of chemiluminescence bursts was measured in an LKB-1251 chemiluminometer as described elsewhere [3] after the addition of Fe 2+ (1 mM) to LP samples containing 0.08 mg protein/ ml in the standard buffer.…”

“…Spectrophotometric determination of diene conjugates formed upon free-radical oxidation of polyunsaturated fatty acids is a method commonly used for analysis of the hydroperoxide content [12]. Recently, we showed that the reaction of lipid peroxides in LDL with Fe 2+ is accompanied by a chemiluminescence burst, the intensity of which under certain conditions depends on the peroxide content in human plasma LDL [3]. However, this method is not widely applied in clinical laboratories, since it requires complicated equipment.…”

The cytoprotective effect of nitrite is based on the formation of dinitrosyl iron complexes

“…The accumulation of TBARS, a lipid peroxidation product, induced by iron in mitochondria is well established [29]. Mitochondrial suspensions were treated as described above and samples were drawn at different time points during reoxygenation.…”