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Suitability of chemical in vitro models to investigate LDL oxidation: study with different initiating conditions in native and [alpha]-tocopherol-supplemented LDL.

The oxidation of LDL plays a fundamental role in the formation and progression of early atherosclerotic lesions [1]. Oxidized lipoproteins are more atherogenic than their parent forms and the demonstration that LDL oxidation does actually occur in vivo [2] has promoted clinical investigations to ascertain the role played by this process in the progression of atherosclerosis in humans.

Oxidative modifications in LDL result from the imbalance between the prooxidant challenges occurring in vivo and the antioxidant defenses making LDL resistant to oxidation [3-5]. The molecular processes initiating the peroxidation of LDL lipids in vivo are poorly understood. Nevertheless, a variety of methodologies have been developed to evaluate in vitro the relative contribution exerted by various intrinsic factors in modulating the susceptibility of the LDL particle to undergo oxidative modifications [5]. Lipoproteins are isolated from plasma by density gradient ultracentrifugation and subsequently challenged with prooxidant stimuli. The kinetics of LDL oxidation is then measured as the formation of conjugated dienes [6], oxysterols [7], and various aldehydic products (thiobarbituric acid-reactive substances) [8] by fluorescence development [9] and by changes of the electrophoretic mobility of apolipoprotein (apo) B100 [10]. (1) Various prooxidant stimuli are generally used, including copper, iron or chelated iron [11], free radical generators [12], ionizing radiations [13], lipoxygenase(s) [14], and peroxidase(s) plus hydrogen peroxide or organic hydroperoxides [15]. Irrespective of the difference of the initiating stimulus, the extensive peroxidation of LDL lipids begins after an initial lag phase (LP), the length of which is taken as an index of the resistance of LDL to oxidation. Major determinants of the duration of the LP are the concentration and the efficiency of the different antioxidants in LDL [3]. Various in vitro and in vivo supplementation studies have demonstrated that the increase in [alpha]-tocopherol ([alpha]T) concentration within the LDL particle results in an augmented resistance of the lipoproteins to ex vivo oxidation, as demonstrated by the prolongation of the LP [10, 16].

The availability of these different methodologies and their indiscriminate use and abuse, however, have generated some confusion concerning (a) the comparison of the results obtained in clinical studies and (b) the evaluation of the efficiency of various antioxidant supplementations. Furthermore, the consistency of the results obtained with different methodologies in samples from the same subjects or groups of patients has never been validated.

The present study was designed to compare the effects of three different prooxidant stimuli on the oxidation kinetics of LDL isolated from human volunteers and used as nonsupplemented or supplemented with [alpha]T. The stimuli were specifically chosen for their ability to promote LDL oxidation via completely different mechanisms and were used at the same concentrations and in the same experimental conditions described in literature [3-6].

The results obtained indicate that the evaluation of the susceptibility of isolated LDL to oxidation heavily depends on the nature of the initiating stimulus. Nevertheless, [alpha]T supplementation invariably results in the prolongation of the LP and in the protection of LDL lipids from peroxidation.

Materials and Methods


Twelve unselected healthy volunteers were used. Twenty milliliters of venous blood were taken after an overnight fast and drawn in polypropylene tubes containing EDTA (1 g/L blood). Plasma was separated by low-speed centrifugation and immediately processed for [alpha]T supplementation and lipoprotein fractionation.

[alpha]T supplementation

Three milliliters of EDTA-plasma were incubated at 37[degrees]C in a thermostated water bath with continuous, gentle stirring, with or without 1 mmol/L [alpha]T dissolved in absolute ethanol (final ethanol concentration <10 g/L) for 3 h. Ethanol (at the same final concentration) was included in control, nonsupplemented samples.


The LDL fraction was isolated from whole plasma by ultracentrifugation through a KBr discontinuous gradient and collected as the fraction floating at a density of 1.019-1.063 kg/L [6]. EDTA was then removed by rapid filtration through disposable desalting columns (EconoPac 10 DG, Bio-Rad) and LDL was resuspended in oxygen-saturated PBS (10 mmol/L phosphate, pH 7.2) at a concentration of 0.25 g/L (50 mg/L LDL protein 5 0.1 [micro]mol/L). Filtered LDL was immediately used for oxidation experiments.


LDL oxidation was continuously monitored spectrophotometrically at 234 nm in a Beckman DU-650 spectrophotometer equipped with a six-position thermostated cuvette holder, to follow the formation of conjugated dienes. Three different experimental conditions were used to induce LDL oxidation: [Cu.sup.2+], as CuS[O.sub.4], 2.5 [micro]mol/L final concentration at 30[degrees]C; 2,2'-azobis-(2-amidino propane) hydrochloride (AAPH), 1 mmol/L (freshly prepared) at 37[degrees]C; [H.sub.2][O.sub.2], 0.2 mmol/L, and horseradish peroxidase (HRP), 5000 U/L at 37[degrees]C. These conditions are essentially identical to those previously described (see ref. 5 for a review). In selected conditions, dose-dependency experiments were performed with all the oxidizing stimuli. The oxidation curve obtained was characterized by two parameters: the LP (expressed in minutes), i.e., the interval between the addition of the prooxidant and the beginning of the extensive oxidation, which was measured on the basis of the intercept between the baseline and the tangent to the rapid oxidation phase; and the propagation rate (PR) (expressed in [micro]mol dienes/min per mg LDL), which is the maximal rate of LDL oxidation detected in the kinetic curve.

[alpha]T determination

[alpha]T in LDL was determined as described [17]. Briefly, LDL was precipitated with ethanol and subsequently extracted with hexane. The hexane phase was then evaporated and the residue was dissolved in methanol and separated by HPLC.


All data were analyzed with Student's t-test and linear regression analysis with the CSS:Statistica program for personal computers. Results are expressed as mean [+ or -] SD.



A general comparison of the major parameters describing the oxidation profile of LDL samples obtained from the same patients and challenged with three prooxidant conditions was made. As summarized in Table 1, the LP and the PR differed depending on the oxidizing stimulus. The use of HRP in combination with hydrogen peroxide invariably promoted a more rapid and accelerated formation of conjugated dienes as compared with [Cu.sup.2+] and AAPH when the three oxidants were at the concentrations indicated in the Table. However, a decrease in HRP or hydrogen peroxide concentrations led to the progressive prolongation of the LP and decrease of the PR (not shown).

The preincubation of plasma with [alpha]T, a condition that promoted a threefold increase in [alpha]T concentration within the LDL (Table 1), increased the resistance to the oxidation induced by the three stimuli. The duration of the LP increased by about two- to threefold, whereas the PR decreased by ~23-57%. The protective effect of [alpha]T supplementation was even increased when less dramatic oxidizing conditions were used (e.g., 1.25 [micro]mol/L copper, 0.5 mmol/L AAPH, 2500 U/L HRP, or 0.1 mmol/L hydrogen peroxide). In none of the experimental conditions, however, was a prooxidant effect of [alpha]T supplementation detected.


Highly significant correlations were found between the LPs measured in different supplemented and nonsupplemented LDL samples challenged with the three prooxidant conditions (Table 2). Similar, although less significant, correlations were found comparing the PRs (Table 2). However, this kind of analysis was biased by the relative contribution of [alpha]T-supplemented LDL, for which LPs were generally longer and PRs were generally lower as compared with nonsupplemented LDL.

When the same comparison was performed exclusively in native, nonsupplemented LDL, no correlation was detected between the oxidation kinetics obtained with [Cu.sup.2+] and HRP plus hydrogen peroxide or [Cu.sup.2+] and AAPH (Table 2). A statistically significant correlation was still detectable, however, between the length of the LPs obtained with AAPH and HRP plus hydrogen peroxide (Table 2). It is noteworthy that the presence or the absence of these correlations did not rely on the experimental conditions. For example, the lengths of the LPs in [Cu.sup.2+]and HRP-induced LDL oxidation did not correlate even when HRP concentration was lowered to 2500 U/L (r = 0.122, not significant) or to 1000 U/L (r = 0.098, not significant).


Conflicting results have been obtained concerning the relevance of [alpha]T as a major determinant of the resistance of native LDL to oxidation as measured by the duration of the LP [10, 18]. In nonsupplemented LDL samples investigated in this study, no correlation between the [alpha]T content and the resistance of LDL to oxidation triggered by [Cu.sup.2+], AAPH, and HRP plus hydrogen peroxide was detected.

The increase in [alpha]T concentration in LDL obtained by in vitro supplementation of plasma invariably resulted in an increased resistance of LDL to the oxidation induced by the three prooxidant stimuli (Table 1). Moreover, a statistically significant correlation between the increase in [alpha]T concentration and the prolongation of the LP in single LDL samples was observed with [Cu.sup.2+] (r = 0.581, P <0.047), AAPH (r = 0.595, P <0.041), and HRP plus hydrogen peroxide (r = 0.605, P <0.037) (Fig. 1). Furthermore, in contrast to nonsupplemented LDL, the relative increases of the LP due to [alpha]T supplementation were highly correlated independently from the experimental conditions used (Table 3).

An interesting and useful parameter introduced by Esterbauer et al. [3] to describe the antioxidant activity of [alpha]T in supplemented LDL is the so-called "vitamin E efficiency." This parameter can be easily calculated from the slope of the correlation between the length of the LP and the concentration of [alpha]T in differentially loaded LDL. In the present study, a similar approach was used by measuring the increase of the LP (in minutes) per unit of increase in [alpha]T concentration in supplemented LDL ([micro]mol/g LDL mass). The efficiency of supplemented [alpha]T in preventing LDL oxidation was comparable with all three prooxidant conditions used, as suggested by the highly significant correlations detected (Fig. 2).



The results reported in this study demonstrate that the detection of interindividual variability in the resistance of isolated LDL to in vitro oxidative modification is heavily dependent on the conditions used to initiate the peroxidative process. Moreover, the artificial enhancement of antioxidant defenses obtained by increasing [alpha]T concentration within the LDL particles invariably makes these lipoproteins more resistant to peroxidative modifications, irrespective of the prooxidant initiator.

The three prooxidant conditions used in this study are believed to initiate the process of lipid peroxidation in LDL via different mechanisms. [Cu.sup.2+] must bind to discrete sites of apo B100, forming apo B100-[Cu.sup.2+] complexes that are subsequently reduced to the highly prooxidant apo B100-[Cu.sup.+] complexes able to generate the alkoxyl radical [LO.sup.*]. It is assumed that [Cu.sup.2+] is repeatedly reduced to [Cu.sup.+] by a variety of reducing equivalents; the copper-binding sites would therefore be centers for repeated site-specific, free radical production [4]. Conversely, AAPH is able to form radicals by thermal decomposition in the aqueous phase surrounding the LDL particle, thus producing a more or less random attack on the surface of the LDL [4]. Several evidences have been obtained in the last few years indicating that iron-containing proteins, such as peroxidases, may oxidize LDL in the absence of added metals [15, 19-21], and it has been postulated that this type of oxidation may provide a model for in vivo oxidation [15]. However, Santhanam and Parthasarathy have recently demonstrated the absolute requirement for apo B100 in peroxidase-induced in vitro LDL oxidation [22]. These findings indicate that the initiation of lipid peroxidation in LDL induced by [Cu.sup.2+] and by peroxidases is relatively complex and requires the active participation of some still unknown molecular residues in apo B100.

The beginning of extensive peroxidation of polyunsaturated fatty acids in LDL, measured by the duration of the LP in the oxidation kinetics, is believed to result from the imbalance between the strength of the initiating stimuli and the efficiency of the intrinsic antioxidant defenses [4], although a more complex relation has been postulated [23]. The data reported in this study demonstrate that in the same LDL samples obtained from the same donors having the same antioxidant content, the susceptibility to oxidation is strictly dependent on the prooxidant stimulus. This conclusion is heavily biased by the impossibility of doing comparable dose-response experiments with [Cu.sup.2+], AAPH, and HRP/[H.sub.2][O.sub.2], as it is practically impossible to quantify the radical flux generated by the three conditions. However, the lack of correlation between the length of the LPs measured for each LDL sample in the three different experimental conditions supports the conclusion.


An increasing number of clinical and epidemiological studies carried out to evaluate the possible association between the progression of atherosclerosis and enhanced LDL oxidation relies on the demonstration that lipoproteins isolated from selected patients exhibit a reduced LP during in vitro oxidation initiated by a variety of prooxidant stimuli (see ref. 5 for a review). However, because the exact mechanism responsible for triggering in vivo LDL oxidation has not been elucidated in detail (and thus there is no evidence that any of the models developed in vitro exactly mimic in vivo conditions), any firm conclusion concerning an augmented susceptibility of LDL to oxidation is, at least, questionable.

A still open question concerns the real efficacy of [alpha]T located within the LDL particle in preventing lipid peroxidation. Conflicting results have been obtained indicating that [alpha]T may act as antioxidant [10], prooxidant [18, 22], or neither [24]. The results obtained in this study demonstrate that an increased [alpha]T content within the LDL molecule is invariably associated with an increased resistance of LDL to oxidation triggered by [Cu.sup.2+], AAPH, and HRP plus hydrogen peroxide. These findings are in perfect agreement with those obtained in a large series of studies performed with in vivo supplementation [20, 25-29]. A recent study has, however, reported that addition of 1-3 [micro]mol/L [alpha]T to the incubation medium had a paradoxical action, potentiating LDL oxidation induced by a variety of peroxidases [22]. However, the experimental conditions used were quite different from those used in the present study and very far from physiological conditions, since [alpha]T was added to the incubation medium instead of included in the LDL particle. Experiments carried out in our (M. Seccia and G. Bellomo, unpublished results) as well as in other laboratories [24] have, in fact, provided convincing evidence that the exact location of [alpha]T outside or within different chemical compartments of the LDL molecule may greatly affect the antioxidant properties.

Few conclusive issues can be drawn from the results obtained in this study. It appears rather clear that the data concerning the susceptibility of isolated LDL to in vitro oxidation and obtained in various clinical studies performed with different initiating conditions are not comparable. In addition, the interindividual variability in the duration of the LPs measured with selected initiators, such as [Cu.sup.2+] and HRP/[H.sub.2][O.sub.2], which require specific domains in apo B100, may simply reflect alterations in the complex initiating machinery rather than real differences in the LDL susceptibility to oxidation. The concomitant use of the three methodologies here described, however, may have advantageous perspectives. We have recently demonstrated the existence in human sera of circulating antibodies selectively recognizing HRP-modified LDL and not [Cu.sup.2+]-modified LDL and vice versa [30]. This finding would reflect a selective operation of a single mechanism of LDL oxidation over others and a selected propensity of LDL to be modified by a specific stimulus over others. Further research is needed to better clarify the exact mechanism of in vivo initiation of lipid peroxidation in LDL and to develop a reliable assay for its in vitro evaluation. However, the demonstration that with all the initiating conditions used a protective activity of [alpha]T supplementation could be detected points out that the choice of the methodological approach used is not critical when the effects of antioxidant regimens in longitudinal and intervention studies are investigated.

This work was supported by grants from Ministero dell'Universita e della Ricerca Scientifica e Tecnologica (MURST) and from the University of Torino. The excellent technical assistance of Maria Grazia Moretti is gratefully acknowledged.

Received November 11, 1996; revised and accepted April 10, 1997.


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Milfred Seccia, Emanuele Albano, and Giorgio Bellomo *

Department of Medical Sciences, 2nd Faculty of Medicine, University of Torino, Via Solaroli 17, I-28100 Novara, Italy.

* Author for correspondence. Fax 1321 620 421.

(1) Nonstandard abbreviations: apo, apolipoprotein; LP, lag phase; [alpha]T, [alpha]-tocopherol; AAPH, 2,2'-azobis-(2-amidinopropane) hydrochloride; HRP, horseradish peroxidase; and PR, propagation rate.
Table 1. In vitro oxidation parameters of native LDL and
[alpha]T-supplemented LDL.

Parameters Native LDL LDL

LP, min
 Copper 96.0 [+ or -] 19.4 167.3 [+ or -] 32.2 (b)
 HRP 28.7 [+ or -] 6.70 94.1 [+ or -] 31.6 (b)
 AAPH 67.1 [+ or -] 11.2 115.0 [+ or -] 22.5 (b)
PR, [micro]mol
 Copper 3.86 [+ or -] 0.42 2.65 [+ or -] 0.72 (a)
 HRP 7.73 [+ or -] 1.60 3.37 [+ or -] 1.30 (b)
 AAPH 3.80 [+ or -] 1.69 2.87 [+ or -] 1.41
[alpha]T, nmol/mg 3.01 [+ or -] 1.32 10.14 [+ or -] 3.02 (b)

Parameters Difference

LP, min
 Copper 71.3 [+ or -] 26.1
 HRP 65.3 [+ or -] 28.1
 AAPH 48.5 [+ or -] 17.08
PR, [micro]mol
 Copper -1.21 [+ or -] 0.61
 HRP -4.35 [+ or -] 1.02
 AAPH -0.93 [+ or -] 0.55
[alpha]T, nmol/mg 7.12 [+ or -] 2.59

Significantly different from native LDL ((a) P <0.05,
(b) P <0.01).

Table 2. Correlation between resistance to in vitro oxidation and
maximal oxidation rate of nonsupplemented and [alpha]T-supplemented
LDL challenged with different initiating conditions.

 Native, Nonsupplemented
 nonsupplemented Supplemented + supplemented
 LDL (n = 12) LDL (n = 12) LDL (n = 24)

Parameters r P r P r P
LP, min
 [Cu.sup.2+]/AAPH -0.005 NS 0.542 NS 0.804 0.001
 [Cu.sup.2+]/HRP -0.184 NS 0.644 0.023 0.840 0.001
 HRP/AAPH 0.769 0.002 0.785 0.002 0.923 0.001
PR, nmol dienes/min
per mg LDL
 [Cu.sup.2+]/AAPH 0.414 NS 0.587 0.044 0.473 0.019
 [Cu.sup.2+]/HRP -0.176 NS 0.317 NS 0.651 0.006
 HRP/AAPH 0.262 NS 0.671 0.016 0.533 0.007

NS, not significant (P >0.05).

Table 3. Correlation between increased resistance to in
vitro oxidation and decreased maximal oxidation rate in
[alpha]T-supplemented LDL challenged with different
initiating conditions.

 Supplemented LDL

Parameters r P

Difference in LP, min
 [Cu.sup.2+]/AAPH 0.603 0.037
 [Cu.sup.2+]/HRP 0.873 0.002
 HRP/AAPH 0.749 0.005
Difference in PR, nmol dienes/min
 per mg LDL
 [Cu.sup.2+]/AAPH -0.340 NS
 [Cu.sup.2+]/HRP 0.206 NS
 HRP/AAPH -0.030 NS

NS, not significant (P >0.05).
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Title Annotation:Lipids and Lipoproteins
Author:Seccia, Milfred; Albano, Emanuele; Bellomo, Giorgio
Publication:Clinical Chemistry
Date:Aug 1, 1997
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