A Microassay to Assess the Oxidative Resistance of Low-Density Lipoproteins

¹ Medicine,
² Pediatrics, and
³ Clinical Chemistry, Medical University of Debrecen, Debrecen H-4012, Hungary;
⁴ Department of Medicine, University of Minnesota, Minneapolis, MN 55455;

Oxidative modification of LDL is implicated in the pathogenesis of atherosclerosis (1)(2). Susceptibility of LDL to oxidative modification is suggested to be an independent risk factor for coronary atherosclerosis, and recent epidemiological studies revealed protective effects of antioxidants on development and progression of atherosclerosis (3)(4)(5).

We have previously demonstrated that hemin readily intercalates into LDL particles and rapidly oxidizes LDL in vitro (6). Hemin-catalyzed oxidation of LDL can be accelerated by activated inflammatory cells, small amounts of hydrogen peroxide, or preformed lipid hydroperoxides within the LDL. That such hemin-induced oxidative modification of LDL may be involved in atherogenesis is supported by the finding that hemin-sensitive genes in endothelium (7)(8) are up-regulated in atherosclerotic lesions (9).

The aim of the present study was to establish a clinical laboratory microassay, based on the time kinetics of hemin-catalyzed lipid peroxidation of LDL, for assessing LDL resistance to oxidative modification, and to determine the optimal conditions and reproducibility of the assay.

Plasma LDL was isolated from 1 g/L Na₂EDTA-anticoagulated venous blood after a 2000g centrifugation for 20 min at 4 °C, and the density of plasma was adjusted to 1210 g/L with KBr. After a two-layer gradient was made in a 5.1-mL Quick-Seal polyallomer tube (Beckman Instruments) by layering normal saline containing 100 mg/L of Na₂EDTA on 1.5 mL of density-adjusted plasma, a single spin gradient ultracentrifugation at 228 000g at 4 °C for 90 min (VTi 65.2 rotor, Beckman Instruments) was performed to isolate LDL. The LDL fraction proved pure in agarose gel electrophoresis, running homogeneously with ß-lipoprotein. The LDL protein concentration, which is proportional to LDL molarity, was determined by the bicinchoninic acid (BCA) protein assay (Pierce). During preparative procedures and storage, the samples were kept at 4 °C in room air and protected from shaking and light.

Hemin-catalyzed lipid peroxidation of LDL was monitored spectrophotometrically at 405 nm in a reaction mixture containing LDL (200 mg/L protein), hemin (4 µmol/L), hydrogen peroxide (75 µmol/L), and HEPES buffer (10 mmol/L, pH 7.4), in a final volume of 200 µL, in triplicate. In the hemin-hydrogen peroxide-mediated LDL modification system, hemin degradation was shown to occur inversely with conjugated diene formation (6)(10); thus hemin degradation may function as a probe of lipid peroxidation process. The reaction was monitored in an Automated Microplate Reader Model EL340 (Bio-Tek Instruments) in a 96-well flat bottom tissue culture plate at 37 °C. To run the reader and to analyze serial measurements taken every minute for 4 h, we applied KC3 software (Bio-Tek Instruments). The oxidative resistance of LDL was characterized by T at maximum velocity (V_max) in seconds, the time period until the maximal velocity of hemin disappearance in the propagation phase. The V_max of hemin degradation, as defined by the maximum change in absorbance of hemin at 405 nm, was calculated using absorbance values detected every minute, with four absorbance values used to calculate the slope. Data are given as means ± SD. The linearity of the relationship between T at V_max and lag time was assessed by use of correlation coefficients.

Fig. 1A demonstrates the kinetics of lipid peroxidation of an LDL sample catalyzed by hemin. The lipid peroxidation process and hemin degradation were monitored spectrophotometrically at 405 nm, using a kinetic microplate reader. The maximal velocity of the propagation phase was -6.4848 milliabsorbance units/min, and T at V_max was 4590 s. Because T at V_max has a strong linear relationship (r = 0.957) with the lag time, the length of initiation phase, the T at V_max also characterizes the oxidative resistance of LDL (Fig. 1B ). The lag time was 84% of T at V_max.

View larger version (16K): [in this window] [in a new window]   Figure 1. Kinetic curve of lipid peroxidation in an LDL sample (A) and the relationship of T at Vmax with lag time (B). Lipid peroxidation was generated by 4 µmol/L hemin and 75 µmol/L hydrogen peroxide in an LDL suspension of 200 mg/L protein concentration in 200 µL final volume. The reaction was monitored spectrophotometrically by an automated microplate reader at 405 nm.

We analyzed how the concentrations of hemin and hydrogen peroxide influence T at V_max. Between 3.0 and 5.0 µmol/L hemin, T at V_max had minimum values; therefore, 4.0 µmol/L of hemin was optimal for assays generating the characteristic, shortest T at V_max. Increases in the concentration of hydrogen peroxide decreased T at V_max. From a technical point of view, i.e., length of kinetic spectrophotometric measurement, 75 µmol/L was the optimal hydrogen peroxide concentration. The presence or absence of Na₂EDTA in the reaction mixture does not affect the reaction kinetic curve of LDL lipid peroxidation catalyzed by hemin; therefore, dialysis of samples is not required for running the assay.

We tested the effect of storage of isolated LDL on the values of T at V_max. Five LDL samples were stored in 150 mmol/L NaCl solution containing 100 mg/L of Na₂EDTA at 4 °C in room air, and T at V_max was determined on 5 consecutive days. The decreases in T at V_max were 2.6% ± 0.9% and 9.4% ± 5.7% at 24 and 48 h after LDL isolation, respectively. There were more substantial changes at later time points, even in the presence of Na₂EDTA. We emphasize that the interassay CVs were 10–20% for these LDL samples. The decrease in T at V_max during storage of LDL samples is the consequence of spontaneous LDL lipid peroxidation and not the failure of our method. The spontaneous lipid peroxidation of LDL was reflected in endogenous lipid hydroperoxide formation (not shown). It is advisable to process the samples within 24 h after blood drawing. Because isolation of LDL from plasma (n = 10) stored at -70 °C for 4 weeks also decreased T at V_max by 8.7% ± 12.4%, we advise not to use frozen plasma for the assay.

The intraassay CVs of T at V_max were 1.6–2.7% (n = 20, Table 1 ). When the T at V_max values of 54 LDL samples from healthy subjects were analyzed, a broad spectrum of LDL resistance to oxidative stress was observed (Table 1 ). The extreme values were 1275 s and 8495 s.

View this table: [in this window] [in a new window]   Table 1. Intraassay precision of T at Vmax of LDL samples (n = 20) and T at Vmax of LDL samples (n = 54) from healthy subjects.

Because -tocopherol increases LDL resistance to oxidation, we assessed the effect of oral vitamin E supplementation on T at V_max in 11 healthy volunteers taking 800 IU of DL--tocopherol acetate per day for 2 weeks. The T at V_max increased 1.2- to 2.8-fold (mean, 1.8-fold) and the -tocopherol content of LDL increased 1.5- to 4.1-fold (mean, 2.4-fold). Significant correlation between the increase in T at V_max and the increase in LDL -tocopherol content was found during supplementation (r = 0.609).

This novel assay is suitable for testing large numbers of LDL samples on an automated microplate reader. The advantages of our method over existing measurements (2)(11) are the ability to follow the kinetics of LDL lipid peroxidation at a visible wavelength and the use of Na₂EDTA during isolation and analysis of LDL. Because the readily and exactly measurable T at V_max has a strong linear relationship with the lag time, the T at V_max also characterizes the oxidative resistance of LDL.

Acknowledgments

This work was supported in part by US-Hungarian joint fund 349/93-B, ETT 116,136/96, OTKA T 21.023, and MEC-1/96. We thank Alice G. Dobolyi for technical assistance.

Footnotes

and * address correspondence to this author at: Department of Pediatrics, Medical University of Debrecen, Nagyerdei krt. 98. Pf. 19., Debrecen H-4012, Hungary

fax 36-52-413 653, e-mail balla{at}ibel.dote.hu

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