See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/5606345

Vitamin C Inhibits Lipid Oxidation in Human HDL1

Article  in  Journal of Nutrition · October 2003

DOI: 10.1093/jn/133.10.3047 · Source: PubMed

CITATIONS READS

61 118

3 authors, including:

Sean Lynch
Midwestern University
52 PUBLICATIONS   3,251 CITATIONS   

SEE PROFILE

All content following this page was uploaded by Sean Lynch on 11 March 2014.

The user has requested enhancement of the downloaded file.

 Biochemical and Molecular Actions of Nutrients

Vitamin C Inhibits Lipid Oxidation in Human HDL1,2

Robert J. Hillstrom, Angela K. Yacapin-Ammons* and Sean M. Lynch3
Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers
Grove, IL 60515 and *Department of Biology, North Central College, Naperville, IL 60540

ABSTRACT HDL are susceptible to oxidation, which affects their cardioprotective properties. Although several
studies have reported inhibition of HDL oxidation by vitamin E, none has determined the potential protective effect
of vitamin C, another important blood antioxidant. We investigated whether vitamin C protects HDL from oxidation
by incubating HDL (0.2 g of protein/L) at 37°C with cupric (Cu2⫹) ions (10 ␮mol/L) in the absence (control) or
presence of vitamin C (20 –200 ␮mol/L). In the absence of vitamin C, lipid oxidation in HDL began immediately and
proceeded rapidly. Cholesteryl linoleate declined to a minimum, whereas lipid oxidation products (lipid dienes and
TBARS) increased to near-maximal levels within 1 h. Vitamin C (50 –200 ␮mol/L) retarded initiation of lipid oxidation
for at least 4 h under the same conditions. The ability of vitamin C to preserve the cardioprotective antioxidant
function of HDL was also assessed. HDL (0.5 g of protein/L) preincubated with Cu2⫹ (10 ␮mol/L) for 2 h in the
absence of vitamin C lost antioxidant activity (45.4 ⫾ 6.2% inhibition of LDL oxidation compared with 93.2 ⫾ 3.6%

Downloaded from jn.nutrition.org by guest on June 9, 2013

for native HDL, P ⬍ 0.05). The addition of vitamin C (50 –200 ␮mol/L) during preincubation of HDL with Cu2⫹,
however, resulted in no significant loss of HDL antioxidant activity (77.3 ⫾ 0.3 to 89.8 ⫾ 5.4% inhibition of LDL
oxidation, P ⬎ 0.05 compared with native HDL). Our results demonstrate that vitamin C inhibits lipid oxidation in
HDL and preserves the antioxidant activity associated with this lipoprotein fraction. J. Nutr. 133: 3047–3051, 2003.

KEY WORDS: ● ascorbic acid ● antioxidants ● lipoproteins ● oxidant stress ● cardiovascular diseases

HDL cholesterol is an independent risk factor for athero-
sclerotic cardiovascular disease (1,2), one of the major causes
of mortality in the United States (3). Results from epidemio-
logic studies have convincing demonstrated that low blood
levels of HDL cholesterol are associated with increased risk for
heart disease and every 10 g/L increase in HDL cholesterol is
associated with a 2–3% decrease in disease incidence (1,2).
Although attention has focused mainly on the ability of HDL
to participate in the removal of cholesterol from sites of
atherosclerotic lesion development via a process termed “re-
verse cholesterol transport” as the mechanism likely responsi-
ble for the observed inverse relationship between blood HDL
cholesterol levels and incidence of heart disease (4), HDL also
exhibit a number of other potentially cardioprotective prop-
erties. These include preservation of vascular endothelial func-
tion, inhibition of platelet activation, anticoagulant and pro-
fibrinolytic activities, and protection of LDL from oxidation
(5). HDL, like LDL, are susceptible to lipid oxidation with
consequent loss of some cardioprotective properties (6). How-
ever, although several studies have demonstrated that vitamin
1
Presented in part at Experimental Biology 02, April 2002, New Orleans, LA
[Lynch S. M., Hillstrom R. J., Yoon, P. S., Campione, A. L. & Moore, M. K. (2002)
Vitamin C protects human high-density lipoprotein (HDL) from oxidation. FASEB
J. 16: A1107 (abs.)] and at Oxygen 2002, November 2002, San Antonio, TX
[Yacapin-Ammons, A. K., Hillstrom R. J. & Lynch S. M. (2002) Vitamin C and
HDL antioxidant activity. Free Radic. Biol. Med. 33: S387–S388 (abs.)].
2
Supported by Midwestern University’s Office of Research and Sponsored
Programs.
3
To whom correspondence should be addressed.
E-mail: slynch@midwestern.edu.
E can protect HDL from lipid oxidation with preservation of
cardioprotective properties (7–10), the ability of vitamin C,
another important blood antioxidant (11), to protect HDL
from oxidation has not been investigated. The aim of our
study, therefore, was to determine whether vitamin C pro-
tected lipids in human HDL from oxidation. We also investi-
gated whether the putative protective effect of vitamin C
against lipid oxidation could, in turn, preserve the ability of
HDL to protect LDL from oxidation, an important early event
in atherosclerotic cardiovascular disease (12).
MATERIALS AND METHODS
Materials. Vacutainer blood collection systems and tubes (1.43
⫻ 104 USP units sodium heparin/L) were purchased from Becton
Dickinson (Franklin Lakes, NJ), Acrodisc LC13 filters were from
Gelman Sciences (Ann Arbor, MI), Sephadex G-25M PD-10 gel-
filtration columns and analytical HPLC columns were from Supelco
(Bellefonte, PA), and Lipo gels for lipoprotein electrophoresis were
from Beckman Instruments (Fullerton, CA). Solvents for HPLC
analyses were purchased from Fisher Scientific (Fair Lawn, NJ).
Ion-pairing reagent (dodecyltriethylammonium phosphate; Q12)
used for vitamin C analysis was purchased from Regis Technologies,
(Morton Grove, IL). All other chemicals were purchased from Sigma
(St. Louis, MO).
Lipoprotein isolation. Blood was collected by venipuncture from
a healthy, normolipidemic male volunteer after an overnight fast, and
used immediately for isolation of lipoproteins by single vertical spin
density gradient ultracentrifugation (13). Density-adjusted plasma
(1.21 kg/L; 0.012 L) was layered under NaCl (1.006 kg/L; 0.028 L)
and centrifuged at 206,000 ⫻ g for 300 min in a Beckman VTi50
rotor cooled to 7°C. The identity of the isolated HDL and LDL

0022-3166/03 $3.00 © 2003 American Society for Nutritional Sciences.

Manuscript received 8 April 2003. Initial review completed 22 May 2003. Revision accepted 5 July 2003.

3047
3048 HILLSTROM ET AL.
FIGURE 1 Vitamin C levels during incubation of human HDL with
Cu2⫹ in the absence (control) or presence of vitamin C. Values are
means ⫾ SEM for pooled data from two independent experiments;
*different from control, P ⬍ 0.05.
fractions, and their lack of contamination with other lipoproteins,
was confirmed by agarose gel electrophoresis (Lynch, S. M., data not
shown). Low-molecular-weight contaminants (including KBr) were
removed from the lipoprotein fractions by size-exclusion chromatog-
raphy (Supelco Sephadex G-25M PD-10 columns), and the purified
fractions filter-sterilized (Gelman 0.2 ␮m Acrodisc filter). The pro-
tein content of the purified lipoprotein fractions was estimated by a
modification (14) of the Lowry procedure (15) using bovine serum
albumin as the standard. The collection of blood from a human
subject for isolation of lipoproteins was approved by the Institutional
Review Board of Midwestern University.
Quantitation of vitamin C. The vitamin C content of samples
was determined by ion-pairing HPLC (16,17) as described previously
(18).
Oxidation of HDL. HDL [either 0.2 g of protein/L (Figs. 1, 2) or
0.5 g of protein/L (Fig. 3A)] were incubated at 37°C with Cu2⫹ (10
FIGURE 2 Oxidation of human
HDL by Cu2⫹ in the absence (control)
or presence of vitamin C monitored
by measuring its content of vitamin E
(A), cholesteryl linoleate (B), lipid
dienes (C) and TBARS (D). Values are
means ⫾ SEM for pooled data from
either two (A) or three (B–D) indepen-
dent experiments; ␣, ␤, ␥, and ␦: dif-
ferent from control, P ⬍ 0.05 for incu-
bations containing vitamin C at
concentrations of 20, 50, 100 and 200
␮mol/L, respectively; ⑀, different from
control, P ⬍ 0.05 for vitamin C ⱖ 50
␮mol/L (C).
␮mol/L) in the absence (control) or presence of vitamin C (20 –200
␮mol/L). Lipid oxidation in HDL was assessed by quantitation of
lipoprotein-associated vitamin E, cholesteryl linoleate, lipid dienes
and TBARS (4) using the methods described previously for assess-
ment of LDL oxidation (18,19).
Inhibitory effect of HDL on LDL oxidation. The physiologic
antioxidant activity of HDL was assessed by the method of Raveh and
co-workers (20). HDL (either native or oxidized by preincubation
with Cu2⫹ for 2 h in the absence or presence of vitamin C; 0.06 g of
protein/L), LDL (0.17 g of protein/L), and their mixtures were incu-
bated at 37°C with Cu2⫹ (0.5 ␮mol/L), and lipid oxidation assessed
as the increase in absorbance at 245 nm during 3 h of incubation. The
inhibition of lipid oxidation by HDL under these conditions was
calculated according to the formula:
Inhibition of LDL oxidation 共%兲
共AHDL ⫹ ALDL) ⫺ AHDL⫹LDL
⫽ ⫻ 100
(AHDL ⫹ ALDL)
where (AHDL ⫹ ALDL) is the sum of the absorbances observed for
HDL and LDL incubated separately with Cu2⫹ for 3 h [corresponds
to the level of oxidation predicted for a (theoretical) HDL devoid of
antioxidant activity] and AHDL⫹LDL is the absorbance observed dur-
ing co-incubation of LDL with HDL and Cu2⫹ for 3 h. For HDL
Downloaded from jn.nutrition.org by guest on June 9, 2013
oxidized by preincubation with Cu2⫹, the reaction was terminated by
the addition of diethylenetriaminepentaacetic acid (DTPA,4 20
␮mol/L), and the HDL purified by size-exclusion chromatography
(Supelco Sephadex G-25M PD-10 column) before assessment of its
antioxidant activity in this experimental system.
Statistical analysis. Unless otherwise indicated, results are re-
ported as means ⫾ SEM for pooled data from three independent
experiments; within each experiment, single analyses of each mea-
4
Abbreviations used: DTPA, diethylenetriaminepentaacetic acid; HDL-0, HDL
oxidized with Cu2⫹ in the absence of vitamin C; HDL-20 to HDL-200, HDL
oxidized with Cu2⫹ in the presence of vitamin C (20 –200 ␮mol/L, respectively);
PON1, paraoxonase-1.
 VITAMIN C INHIBITS LIPID OXIDATION IN HDL
FIGURE 3 Antioxidant activity of human HDL (B) after oxidation
by Cu2⫹ in the absence (HDL-0) or presence (HDL-20 to HDL-200) of
vitamin C (A). Values are means ⫾ SEM for pooled data from three
independent experiments; *different from control [(A) HDL-O; (B) native
(n)HDL], P ⬍ 0.05.
sured variable were performed. Statistical analysis was performed after
testing for variance homogeneity (Levene’s test) by either two-way
(Figs. 1 and 2) or one-way (Fig. 3) ANOVA with a Bonferroni
post-test to compare means from experimental incubations with the
appropriate control. All statistical analyses were performed using
GraphPad Prism Version 3.00 for Windows (GraphPad Software, San
Diego, CA); differences were considered significant when P ⬍ 0.05.
RESULTS
Vitamin C was rapidly consumed during incubation with
HDL and Cu2⫹ (Fig. 1). Even for the highest added concen-
tration (i.e., 200 ␮mol/L), complete consumption occurred
within 2 h. However, despite its rapid consumption, vitamin C
still conferred significant protection from Cu2⫹-mediated lipid
oxidation in HDL (Fig 2). In the absence of vitamin C,
exposure of HDL to prooxidant Cu2⫹ caused rapid lipid oxi-
dation as evidenced by a decline in cholesteryl linoleate con-
tent from 170.4 ⫾ 19.8 to a near-minimal level of 14.6 ⫾ 14.6
nmol/mg of HDL protein within 1 h of incubation with Cu2⫹;
Fig. 2B). During this same time, a rapid increase in the levels
of two markers of lipid oxidation (lipid dienes and TBARS)
from nondetectable to near-maximal levels of 106.5 ⫾ 6.8 and
23.6 ⫾ 1.9 nmol/mg of HDL protein, respectively, was also
observed (Fig. 2C,D). Interestingly, during this period of rapid
lipoprotein oxidation, the vitamin E content of HDL re-
3049
mained relatively constant (declined from 5.9 ⫾ 0.5 only to
5.4 ⫾ 0.5 nmol/mg of HDL protein during 1 h of incubation
with Cu2⫹; Fig. 2A). In contrast to the rapid lipid oxidation
occurring in control incubations, the addition of vitamin C
(20 –200 ␮mol/L) significantly retarded oxidation of lipids in
HDL. At the lowest concentration (i.e., 20 ␮mol/L), vitamin
C retarded the decline in HDL-associated cholesteryl linoleate
such that after 1 h of incubation with Cu2⫹, the level had
declined to only 57.9 ⫾ 23.1 nmol/mg of HDL protein. Sim-
ilarly, in contrast to control incubations in which lipid oxida-
tion began immediately after exposure of HDL to Cu2⫹ and
increased to near-maximal levels within 1 h, inclusion of
vitamin C at a concentration of 20 ␮mol/L delayed formation
of both lipid dienes and TBARS for ⬃1 h with maximal levels
(134.0 ⫾ 10.6 and 25.3 ⫾ 11.7 nmol/mg of HDL protein,
respectively) being attained only after ⬃2 h of incubation.
Addition of higher concentrations of vitamin C (i.e., 50 –200
␮mol/L) further delayed initiation of lipid oxidation in HDL
for at least 4 h (Fig. 2 C,D). Although the HDL content of
vitamin E and cholesteryl linoleate declined (P ⬍ 0.05) over
time (Fig. 2 A,B), vitamin C had no effect on these variables
(P ⬎ 0.05). However, there was an inhibitory effect (P
⬍ 0.05) of vitamin C on the accumulation of lipid dienes and
Downloaded from jn.nutrition.org by guest on June 9, 2013
TBARS in HDL under the same circumstances (Fig. 2C,D).
To assess the physiologic relevance of the ability of vitamin
C to inhibit lipid oxidation in HDL, the ability of HDL to
protect LDL from oxidation was measured. Consistent with
our previous results (Fig. 2D), incubation of HDL (0.5 mg of
HDL protein/mL) with Cu2⫹ (10 ␮mol/L) for 2 h in the
absence of vitamin C (HDL-0) resulted in extensive lipid
oxidation (33.9 ⫾ 6.8 nmol TBARS/mg of HDL protein; Fig.
3A). Inclusion of vitamin C (50 –200 ␮mol/L; HDL-50 to
HDL-200, respectively) decreased lipid oxidation in HDL
compared with the vitamin C–free control (ⱕ4.1 ⫾ 3.1 nmol
TBARS/mg HDL protein; Fig 3A). Lipid oxidation tended to
be decreased (P ⫽ 0.22) when vitamin C was added at a
concentration of 20 ␮mol/L (HDL-20; 15.0 ⫾ 6.4 nmol
TBARS/mg HDL protein). Vitamin C was nondetectable in
all preincubations after 2 h of exposure to Cu2⫹ (Hillstrom et
al., unpublished data). Native HDL were highly effective at
protecting LDL from Cu2⫹-mediated oxidation (93.2 ⫾ 3.6%
inhibition of LDL oxidation compared with that predicted for
a HDL devoid of antioxidant activity; Fig. 3B). This antioxi-
dant effect was clearly lost for HDL-0 with only 45.4 ⫾ 6.2%
inhibition of LDL oxidation (Fig. 3B; P ⬍ 0.05 compared with
native HDL). Antioxidant activity for HDL-20 also decreased
(71.1 ⫾ 4.0% inhibition of LDL oxidation; Fig. 3B; P ⬍ 0.05
vs. native HDL). For the other HDL preparations (i.e., HDL-
50, HDL-100 and HDL-200), the extent of inhibition of LDL
oxidation increased progressively with increasing concentra-
tions of vitamin C in the preincubation until it was essentially
the same as that observed for native HDL (inhibition of LDL
oxidation ⫽ 89.8 ⫾ 5.4 and 88.4 ⫾ 4.4% for HDL-100 and
HDL-200, respectively; Fig. 3B; P ⬎ 0.05 vs. native HDL).
DISCUSSION
Our results demonstrate an important new role for vitamin
C in preventing lipid oxidation in HDL and preserving the
cardioprotective ability of this lipoprotein fraction to inhibit
atherogenic modification (i.e., oxidation) of LDL. In our ex
vivo experimental system, in which isolated HDL were ex-
posed to prooxidant Cu2⫹, HDL underwent rapid oxidation in
control incubations lacking vitamin C. Immediately upon
exposure to Cu2⫹, levels of HDL-associated cholesteryl li-
noleate, a substrate for lipid oxidation in lipoproteins (21),
3050 HILLSTROM ET AL.
began to decline, whereas markers of lipid oxidation (lipid
dienes and TBARS) increased concomitantly. Within 1 h of
incubation with Cu2⫹, HDL-associated cholesteryl linoleate
declined to a near-minimal level, whereas lipid dienes and
TBARS attained near-maximal levels. These results are con-
sistent with those of numerous other investigators who re-
ported that lipids in HDL are highly susceptible to oxidation
during incubation with Cu2⫹ (7,8,20,22–29). The addition of
vitamin C (20 –200 ␮mol/L) to our standard incubation sys-
tem containing HDL and Cu2⫹ inhibited lipid oxidation in
HDL. Thus, although lipid oxidation was essentially complete
within 1 h of incubation with Cu2⫹ in the absence of vitamin
C, in the presence of vitamin C (20 –200 ␮mol/L), initiation
of HDL lipid oxidation was delayed such that complete oxi-
dation was not observed until after at least 2 h had elapsed
after incubation with Cu2⫹. Indeed, at vitamin C concentra-
tions within the range normally found in human blood [i.e.,
30 –150 ␮mol/L; (30)], there was no increase above baseline of
the levels of either of the two markers of lipid oxidation used
in our study for at least 4 h. Thus, at physiologic concentra-
tions, vitamin C affords significant protection against Cu2⫹-
mediated lipid oxidation in HDL. This observation is consis-
tent with the known antioxidant capabilities of vitamin C in
human blood (11).
Prior studies investigating the effect of antioxidant nutri-
ents on HDL oxidation focused mainly on vitamin E, the
principal lipid-soluble antioxidant present in HDL (31). Lau-
reaux and co-workers (7) reported that in vitro enrichment of
isolated HDL with ␣-tocopherol stabilized lipid hydroperox-
ides formed during Cu2⫹-mediated oxidation and retarded
their conversion to more reactive, and potentially deleterious
lipid aldehydes. Studies investigating the effects of in vivo
supplementation with vitamin E have consistently found that
HDL isolated from volunteers after consumption of a vitamin
E supplement are more resistant to Cu2⫹-mediated oxidation
assessed by monitoring the formation of either lipid dienes
(8,32), lipid peroxides (8) or reactive lipid aldehydes (9). Only
one study (25) previously investigated the possibility that
vitamin C might protect HDL from lipid oxidation. In that
study, supplementation of volunteers with a combination of
vitamins E and C for 10 d decreased the susceptibility of HDL
isolated from their blood to Cu2⫹-mediated ex vivo oxidation
assessed as the accumulation of TBARS. However, due to
manner in which this study was performed, it is likely that the
observed antioxidant effect resulted exclusively from increased
vitamin E content in the volunteers’ HDL (10.9 ⫾ 0.9
nmol/mg of HDL protein compared with 6.5 ⫾ 0.5 nmol/mg of
HDL protein before supplementation). Although vitamin C
supplementation doubled the vitamin C content of the vol-
unteers’ blood from 55.6 ⫾ 4.0 to 111.9 ⫾ 7.4 ␮mol/L, none
of this vitamin C would have been present during the ex vivo
oxidation of the isolated HDL. In contrast, in our experimen-
tal system, vitamin C was added directly to HDL in the ex vivo
prooxidant experimental system mimicking the in vivo situa-
tion in which HDL is presumably exposed to oxidant stress in
the presence of vitamin C. Interestingly, in our experimental
system, vitamin E seemed relatively unimportant in prevent-
ing lipid oxidation in HDL exposed to Cu2⫹. Thus, in control
incubations lacking vitamin C, levels of HDL-associated vita-
min E were still relatively high even after 1 h of incubation
with Cu2⫹ (when lipid oxidation was essentially complete),
and began to decline only after ⬎2 h of incubation. Although
the presence of vitamin C did appear to retard the disappear-
ance of vitamin E from HDL exposed to Cu2⫹ with at least
some vitamin E remaining even after 4 h of incubation, the
large variability in our vitamin E data makes it difficult to
interpret this observation.
Our study also investigated whether the ability of vitamin
C to protect HDL from lipid oxidation influenced physiologic
function of this lipoprotein fraction. In addition to a well-
documented role in reverse cholesterol transport, HDL have
recently been recognized to have several other important
cardioprotective properties including the ability to protect
LDL from oxidative modification (5). Consistent with the
recent observation of Jaouad and co-workers (10), we found
that oxidation of lipids in HDL lowered the ability to protect
LDL from oxidative (i.e., atherogenic) modification. However,
prevention of lipid oxidation by physiologic concentrations of
vitamin C attenuated this loss of HDL antioxidant activity in
our experimental system. This observation is similar to that of
Yoshikawa and co-workers (33) who reported a synergistic
protective effect of a combination of HDL and vitamin C,
compared with either HDL or vitamin C alone, against LDL
oxidation. However, although these investigators suggested
that the effectiveness of the combination of vitamin C and
HDL against LDL oxidation resulted from protection of vita-
min C from oxidation, it is clear from our results that other
Downloaded from jn.nutrition.org by guest on June 9, 2013
mechanisms are likely involved. In contrast to Yoshikawa and
co-workers (33) who assessed oxidation of LDL during its
co-incubation with HDL and Cu2⫹ in the absence or presence
of vitamin C, in our experimental system, HDL was preincu-
bated with Cu2⫹ in the absence or presence of vitamin C
before testing for ability to protect LDL from oxidation. Thus,
vitamin C (and any of its oxidation products resulting from
exposure to prooxidant Cu2⫹) was absent when we tested the
antioxidant function of (oxidized) HDL. Our results are, there-
fore, more consistent with the idea that vitamin C prevents
loss of the physiologic antioxidant activity associated with
HDL during oxidation, rather than HDL protecting vitamin C
from oxidation. The ability of HDL to protect LDL from
oxidation is linked to HDL-associated paraoxonase-1 (PON1)
activity, which catalyzes the breakdown of lipid peroxides in
LDL (34). Oxidation of HDL is associated with inactivation of
PON1 and loss of antioxidant activity (10). It is tempting to
speculate that the ability of vitamin C to prevent loss of HDL
antioxidant activity reported here may result from preserva-
tion of PON1 activity. Consistent with this speculation, it is
interesting to note that PON1 activity is highly correlated
with dietary consumption of vitamin C and vitamin E (35).
It is important to note that although our study investigated
only the ability of vitamin C to protect HDL from Cu2⫹-
mediated oxidation, a number of other prooxidants also pro-
mote lipid oxidation in HDL. These include peroxyl (26,31)
and hydroxyl (36,37) radicals, lipoxygenase (26) and cultured
endothelial cells (38) and macrophages (39). Whether vita-
min C will protect HDL from metal ion–independent lipid
oxidation remains to be determined. Interestingly, although
oxidation of HDL by Cu2⫹ and other prooxidants capable of
promoting extensive lipid oxidation results in the loss of
cardioprotective properties associated with this lipoprotein
(6,10), oxidation of the constituent apoproteins of HDL in the
absence of significant lipid oxidation, actually enhances their
ability to facilitate reverse cholesterol transport (6,40 – 42).
Such protein oxidation is mediated in vivo by phagocyte-
associated myeloperoxidase activity through the generation of
chemical species including the tyrosyl radical and hypochlo-
rous acid (43). Although vitamin C has been shown to protect
LDL from oxidation by activated human neutrophils (44,45),
a cell-free myeloperoxidase oxidizing system (44) and hypo-
chlorous acid (46), it is not known how it will affect oxidation
of HDL by myeloperoxidase-generated oxidants.
 VITAMIN C INHIBITS LIPID OXIDATION IN HDL
In summary, our results demonstrate that physiologic con-
centrations of vitamin C inhibit Cu2⫹-mediated lipid oxida-
tion in HDL and preserve the cardioprotective ability of this
lipoprotein fraction to prevent atherogenic modification of
LDL. Whether vitamin C will protect HDL from metal ion-
independent oxidation, and how such an antioxidant effect
might modulate other physiologic functions of HDL, such as
reverse cholesterol transport, is the subject of ongoing inves-
tigation in our laboratory.
ACKNOWLEDGMENTS
The authors thank Allan Campione and Michael Moore for their
expert technical assistance.
LITERATURE CITED
1. Maron, D. J. (2000) The epidemiology of low levels of high-density
lipoprotein cholesterol in patients with and without coronary artery disease.
Am. J. Cardiol. 86: 11–14.
2. Franceschini, G. (2001) Epidemiologic evidence for high-density li-
poprotein cholesterol as a risk factor for coronary artery disease. Am. J. Cardiol.
88: 9 –13.
3. Centers for Disease Control and Prevention (1999) Mortality pat-
terns—United States, 1997. Morb. Mortal. Wkly. Rep. 48: 664 – 668.
4. von Eckardstein, A., Nofer, J.-R. & Assmann, G. (2001) High density
lipoproteins and arteriosclerosis: role of cholesterol efflux and reverse cholesterol
transport. Arterioscler. Thromb. Vasc. Biol. 21: 13–27.
5. Nofer, J.-R., Kehrel, B., Fobker, M., Levkau, B., Assmann, G. & von
Eckardstein, A. (2002) HDL and arteriosclerosis: beyond reverse cholesterol
transport. Atherosclerosis 161: 1–16.
6. Francis, G. A. (2000) High density lipoprotein oxidation: in vitro sus-
ceptibility and potential in vivo consequences. Biochim. Biophys. Acta 1483:
217–235.
7. Laureaux, C., Therond, P., Bonnefont-Rousselot, D., Troupel, S. E., Leg-
rand, A. & Delattre, J. (1997) ␣-Tocopherol enrichment of high-density lipopro-
teins: stabilization of hydroperoxides produced during copper oxidation. Free
Radic. Biol. Med. 22: 185–194.
8. Arrol, S., Mackness, M. I. & Durrington, P. N. (2000) Vitamin E sup-
plementation increases the resistance of both LDL and HDL to oxidation and
increases cholesteryl ester transfer activity. Atherosclerosis 150: 129 –134.
9. Schnell, J. W., Anderson, R. A., Stegner, J. E., Schindler, S. P. & Wein-
berg, R. B. (2001) Effects of a high polyunsaturated fat diet and vitamin E
supplementation on high-density lipoprotein oxidation in humans. Atherosclerosis
159: 459 – 466.
10. Jaouad, L., Milochevitch, C. & Khalil, A. (2003) PON1 paraoxonase
activity is reduced during HDL oxidation and is an indicator of HDL antioxidant
capacity. Free Radic. Res. 37: 77– 83.
11. Carr, A. C., Zhu, B. Z. & Frei, B. (2000) Potential antiatherogenic
mechanisms of ascorbate (vitamin C) and ␣-tocopherol (vitamin E). Circ. Res. 87:
349 –354.
12. Steinberg, D. (1997) Low density lipoprotein oxidation and its patho-
biological significance. J. Biol. Chem. 272: 20963–20966.
13. Chung, B. H., Segrest, J. P., Ray, M. J., Brunzell, J. D., Hokanson, J. E.,
Krauss, R. M., Beaudrie, K. & Cone, J. T. (1986) Single vertical spin density
gradient ultracentrifugation. Methods Enzymol. 128: 181–209.
14. Peterson, G. L. (1977) A simplification of the protein assay method of
Lowry et al. which is more generally applicable. Anal. Biochem. 83: 346 –356.
15. Lowry, O., Rosebrough, N., Farr, A. & Randall, R. (1951) Protein
estimation with the Folin phenol reagent. J. Biol. Chem. 193: 265–275.
16. Barja de Quiroga, G., Lopez-Torres, M., Perez-Campo, R. & Rojas, C.
(1991) Simultaneous determination of two antioxidants, uric and ascorbic acid,
in animal tissue by high-performance liquid chromatography. Anal. Biochem. 199:
81– 85.
17. Motchnik, P. A., Frei, B. & Ames, B. N. (1994) Measurement of anti-
oxidants in human blood plasma. Methods Enzymol. 234: 269 –279.
18. Alul, R. H., Wood, M., Longo, J., Marcotte, A. L., Campione, A. L., Moore,
M. K. & Lynch, S. M. (2003) Vitamin C protects low-density lipoprotein from
homocysteine-mediated oxidation. Free Radic. Biol. Med. 34: 881– 891.
19. Lynch, S. M., Campione, A. L. & Moore, M. K. (2000) Plasma thiols
inhibit hemin-dependent oxidation of human low-density lipoprotein. Biochim.
Biophys. Acta 1485: 11–22.
20. Raveh, O., Pinchuk, I., Fainaru, M. & Lichtenberg, D. (2001) Kinetics of
lipid peroxidation in mixtures of HDL and LDL, mutual effects. Free Radic. Biol.
Med. 31: 1486 –1497.
21. Keaney, J. F., Jr. & Frei, B. (1994) Antioxidant protection of low-density
lipoprotein and its role in the prevention of atherosclerotic vascular disease. In:
Natural Antioxidants in Human Health and Disease. (Frei, B., ed.), pp. 303–351,
Academic Press, San Diego, CA.
View publication stats
3051
22. Nagano, Y., Arai, H. & Kita, T. (1991) High density lipoprotein loses its
effect to stimulate efflux of cholesterol from foam cells after oxidative modifica-
tion. Proc. Natl. Acad. Sci. U.S.A. 88: 6457– 6461.
23. Bradamante, S., Barenghi, L., Giudici, G. A. & Vergani, C. (1992) Free
radicals promote modifications in plasma high-density lipoprotein: nuclear mag-
netic resonance analysis. Free Radic. Biol. Med. 12: 193–203.
24. Kontush, A., Meyer, S., Finckh, B., Kohlschutter, A. & Beisiegel, U.
(1996) ␣-Tocopherol as a reductant for Cu(II) in human lipoproteins. Triggering
role in the initiation of lipoprotein oxidation. J. Biol. Chem. 271: 11106 –11112.
25. Rifici, V. A. & Khachadurian, A. K. (1996) Effects of dietary vitamin C
and E supplementation on the copper mediated oxidation of HDL and on HDL
mediated cholesterol efflux. Atherosclerosis 127: 19 –26.
26. Garner, B., Witting, P. K., Waldeck, A. R., Christison, J. K., Raftery, M. &
Stocker, R. (1998) Oxidation of high density lipoproteins. I. Formation of
methionine sulfoxide in apolipoproteins AI and AII is an early event that accom-
panies lipid peroxidation and can be enhanced by ␣-tocopherol. J. Biol. Chem.
273: 6080 – 6087.
27. Julier, K., Mackness, M. I., Dean, J. D. & Durrington, P. N. (1999)
Susceptibility of low- and high-density lipoproteins from diabetic subjects to in
vitro oxidative modification. Diabetes Med. 16: 415– 423.
28. Ivanov, V., Carr, A. C. & Frei, B. (2001) Red wine antioxidants bind to
human lipoproteins and protect them from metal ion-dependent and -indepen-
dent oxidation. J. Agric. Food Chem. 49: 4442– 4449.
29. Thomas, M. J., Chen, Q., Zabalawi, M., Anderson, R., Wilson, M., Wein-
berg, R., Sorci-Thomas, M. G. & Rudel, L. L. (2001) Is the oxidation of
high-density lipoprotein lipids different than the oxidation of low-density lipopro-
tein lipids? Biochemistry 40: 1719 –1724.
30. Stocker, R. & Frei, B. (1991) Endogenous antioxidant defences in
human blood plasma. In: Oxidative Stress: Oxidants and Antioxidants. (Sies, H.,
Downloaded from jn.nutrition.org by guest on June 9, 2013
ed.), pp. 213–243, Academic Press, San Diego, CA.
31. Bowry, V. W., Stanley, K. K. & Stocker, R. (1992) High density lipopro-
tein is the major carrier of lipid hydroperoxides in human blood plasma from
fasting donors. Proc. Natl. Acad. Sci. U.S.A. 89: 10316 –10320.
32. Suzukawa, M., Ishikawa, T., Yoshida, H. & Nakamura, H. (1995) Effect
of in-vivo supplementation with low-dose vitamin E on susceptibility of low-
density lipoprotein and high-density lipoprotein to oxidative modification. J. Am.
Coll. Nutr. 14: 46 –52.
33. Yoshikawa, M., Sakuma, N., Hibino, T., Tamai, N., Sasai, K., Yoshimata,
T., Jin-no, Y. & Kamiya, Y. (1998) Strong synergistic anti-peroxidative effects
of HDL3 and ascorbic acid against copper-catalyzed LDL peroxidation. Biochim.
Biophys. Acta 1406: 307–314.
34. Durrington, P. N., Mackness, B. & Mackness, M. I. (2001) Paraoxo-
nase and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 21: 473– 480.
35. Jarvik, G. P., Tsai, N. T., McKinstry, L. A., Wani, R., Brophy, V. H., Richter,
R. J., Schellenberg, G. D., Heagerty, P. J., Hatsukami, T. S. & Furlong, C. E.
(2002) Vitamin C and E intake is associated with increased paraoxonase activity.
Arterioscler. Thromb. Vasc. Biol. 22: 1329 –1333.
36. Bonnefont-Rousselot, D., Khalil, A., Delattre, J., Jore, D. & Gardes-Albert,
M. (1997) Oxidation of human high-density lipoproteins by .OH and 䡠OH/O2䡠⫺
free radicals. Radiat. Res. 147: 721–728.
37. Bonnefont-Rousselot, D., Khalil, A., Gardes-Albert, M. & Delattre, J.
(1997) Reciprocal protection of LDL and HDL oxidised by .OH free radicals in the
presence of oxygen. FEBS Lett. 403: 70 –74.
38. Parthasarathy, S., Barnett, J. & Fong, L. G. (1990) High-density li-
poprotein inhibits the oxidative modification of low-density lipoprotein. Biochim.
Biophys. Acta 1044: 275–283.
39. Rifici, V. A., Schneider, S. H. & Khachadurian, A. K. (2002) Lipoprotein
oxidation mediated by J774 murine macrophages is inhibited by individual red
wine polyphenols but not by ethanol. J. Nutr. 132: 2532–2537.
40. Francis, G. A., Mendez, A. J., Bierman, E. L. & Heinecke, J. W. (1993)
Oxidative tyrosylation of high density lipoprotein by peroxidase enhances cho-
lesterol removal from cultured fibroblasts and macrophage foam cells. Proc. Natl.
Acad. Sci. U.S.A. 90: 6631– 6635.
41. Francis, G. A., Oram, J. F., Heinecke, J. W. & Bierman, E. L. (1996)
Oxidative tyrosylation of HDL enhances the depletion of cellular cholesteryl esters
by a mechanism independent of passive sterol desorption. Biochemistry 35:
15188 –15197.
42. Wang, W. Q., Merriam, D. L., Moses, A. S. & Francis, G. A. (1998)
Enhanced cholesterol efflux by tyrosyl radical-oxidized high density lipoprotein is
mediated by apolipoprotein AI-AII heterodimers. J. Biol. Chem. 273: 17391–
17398.
43. Heinecke, J. W. (1997) Pathways for oxidation of low density lipopro-
tein by myeloperoxidase: tyrosyl radical, reactive aldehydes, hypochlorous acid
and molecular chlorine. Biofactors 6: 145–155.
44. Savenkova, M. L., Mueller, D. M. & Heinecke, J. W. (1994) Tyrosyl
radical generated by myeloperoxidase is a physiological catalyst for the initiation
of lipid peroxidation in low density lipoprotein. J. Biol. Chem. 269: 20394 –20400.
45. Carr, A. C. & Frei, B. (2002) Human neutrophils oxidize low-density
lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C.
Biol. Chem. 383: 627– 636.
46. Carr, A. C., Tijerina, T. & Frei, B. (2000) Vitamin C protects against and
reverses specific hypochlorous acid– and chloramine-dependent modifications of
low-density lipoprotein. Biochem. J. 346: 491– 499.