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Articles |
-tocopherol-supplemented LDL
Department of Medical Sciences, 2nd Faculty of Medicine, University of Torino, Via Solaroli 17, I-28100 Novara, Italy.
a Author for correspondence. Fax +321 620 421.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-tocopherol (T), were oxidized with Cu2+,
2,2'-azobis-(2-amidino propane) hydrochloride (AAPH), and
H2O2 plus horseradish peroxidase (HRP). The
oxidation kinetics were measured spectrophotometrically at 234 nm to
follow the formation of conjugated dienes and evaluated as resistance
to oxidation (lag phase, LP) and maximal oxidation rate (propagation
rate, PR). The duration of LP in nonsupplemented LDL was different with
the three prooxidant stimuli (LP, in min: 96 ± 19 for
Cu2+, 28.7 ± 6.7 for HRP, and 67.1 ± 11.2 for
AAPH). No correlation was found between the values obtained with
Cu2+ and AAPH or HRP, but a significant correlation was
found with AAPH and HRP (r = 0.798, P
<0.002). In vitro T supplementation prolonged the LP and decreased
the PR with all the stimuli. The extent of increase in LP was highly
correlated (r = 0.872, P <0.001 for
Cu2+ and HRP; r = 0.603, P
<0.03 for Cu2+ and AAPH; r = 0.749,
P <0.005 for AAPH and HRP). Although the evaluation of
ex vivo LDL oxidation is dependent on the prooxidant
stimulus, the three prooxidant conditions used detect equally well the
efficiency of T supplementation in preventing LDL oxidation.
Key Words: indexing terms: free radicals • lipid peroxidation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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)(4)(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
-tocopherol (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
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)(4)(5)(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, T supplementation invariably
results in the prolongation of the LP and in the protection of LDL
lipids from peroxidation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T
supplementation and lipoprotein fractionation.
t supplementation
Three milliliters of EDTA-plasma were incubated at 37 °C in a
thermostated water bath with continuous, gentle stirring, with or
without 1 mmol/L 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.
lipoprotein isolation
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 (Econo-Pac 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 = 0.1 µmol/L).
Filtered LDL was immediately used for oxidation experiments.
lipoprotein oxidation
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: Cu2+, as CuSO4, 2.5
µmol/L final concentration at 30 °C; 2,2'-azobis-(2-amidino
propane) hydrochloride (AAPH), 1 mmol/L (freshly prepared) at 37 °C;
H2O2, 0.2 mmol/L, and horseradish peroxidase
(HRP), 5000 U/L at 37 °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 µmol dienes/min
per mg LDL), which is the maximal rate of LDL oxidation detected in the
kinetic curve.
t determination
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.
statistical analysis
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 ± SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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t-supplemented ldl
, 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 Cu2+ 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).
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The preincubation of plasma with T, a condition that promoted a
threefold increase in 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 T
supplementation was even increased when less dramatic oxidizing
conditions were used (e.g., 1.25 µmol/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 T supplementation
detected.
correlation between in vitro oxidation parameters with different
initiating conditions in nonsupplemented and t-supplemented ldl
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 T-supplemented LDL, for which LPs
were generally longer and PRs were generally lower as compared with
nonsupplemented LDL.
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When the same comparison was performed exclusively in native,
nonsupplemented LDL, no correlation was detected between the oxidation
kinetics obtained with Cu2+ and HRP plus hydrogen
peroxide or Cu2+ 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 Cu2+- 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).
effects of t concentration on oxidation triggered by
different initiating conditions in supplemented ldl
Conflicting results have been obtained concerning the relevance of
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 T content and
the resistance of LDL to oxidation triggered by Cu2+, AAPH,
and HRP plus hydrogen peroxide was detected.
The increase in 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 T concentration and the prolongation of the
LP in single LDL samples was observed with Cu2+
(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 T supplementation were highly correlated
independently from the experimental conditions used (Table 3
).
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An interesting and useful parameter introduced by Esterbauer et al.
(3) to describe the antioxidant activity of 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 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 T concentration in supplemented LDL (µmol/g LDL mass).
The efficiency of supplemented T in preventing LDL oxidation was
comparable with all three prooxidant conditions used, as suggested by
the highly significant correlations detected (Fig. 2
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. Cu2+ must bind to discrete sites of apo B100, forming apo B100-Cu2+ complexes that are subsequently reduced to the highly prooxidant apo B100-Cu+ complexes able to generate the alkoxyl radical LO·. It is assumed that Cu2+ is repeatedly reduced to Cu+ 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)(20)(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 Cu2+ 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 Cu2+, AAPH, and HRP/H2O2, 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 T located within
the LDL particle in preventing lipid peroxidation. Conflicting results
have been obtained indicating that T may act as antioxidant
(10), prooxidant (18)(22), or
neither (24). The results obtained in this study
demonstrate that an increased T content within the LDL molecule is
invariably associated with an increased resistance of LDL to oxidation
triggered by Cu2+, 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)(26)(27)(28)(29). A recent study has,
however, reported that addition of 1–3 µmol/L 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 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 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
Cu2+ and HRP/H2O2, 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 Cu2+-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 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.
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Acknowledgments |
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Footnotes |
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T, -tocopherol; AAPH, 2,2'-azobis-(2-amidinopropane) hydrochloride; HRP, horseradish peroxidase; and PR, propagation rate. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tocopherol supplementation on LDL oxidation: a dose–response study. Arterioscler Thromb Vasc Biol 1995;15:190-198.
-tocopherol on the vitamin E content of human low density lipoproteins and its oxidation resistance. J Lipid Res 1991;32:1325-1332.
[Abstract]
-tocopherol on the oxidative modification of low density lipoprotein. J Lipid Res 1992;33:899-906.
[Abstract]
-tocopherol and racemic -tocopherol in humans: effects on lipid levels and lipoprotein susceptibility to oxidation. Arterioscler Thromb 1993;13:601-608.
The following articles in journals at HighWire Press have cited this article:
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  M. A. Atkin, A. Gasper, R. Ullegaddi, and H. J. Powers Oxidative Susceptibility of Unfractionated Serum or Plasma: Response to Antioxidants in Vitro and to Antioxidant Supplementation Clin. Chem., November 1, 2005; 51(11): 2138 - 2144. [Abstract] [Full Text] [PDF]
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 A. Palomaki, K. Malminiemi, O. Malminiemi, and T. Solakivi Effects of Lovastatin Therapy on Susceptibility of LDL to Oxidation During {alpha}-Tocopherol Supplementation Arterioscler. Thromb. Vasc. Biol., June 1, 1999; 19(6): 1541 - 1548. [Abstract] [Full Text] [PDF]
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