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© 2003 The American Society for Nutritional Sciences J. Nutr. 133:3047-3051, October 2003

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

3To whom correspondence should be addressed. E-mail: slynch{at}midwestern.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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 µmol/L) in the absence (control) or presence of vitamin C (20–200 µmol/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 µmol/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 µmol/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% for native HDL, P < 0.05). The addition of vitamin C (50–200 µmol/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.

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

HDL cholesterol is an independent risk factor for atherosclerotic cardiovascular disease (1,2), one of the major causes of mortality in the United States (3). Results from epidemiologic 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 "reverse cholesterol transport" as the mechanism likely responsible 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 properties. These include preservation of vascular endothelial function, inhibition of platelet activation, anticoagulant and profibrinolytic activities, and protection of LDL from oxidation (5). HDL, like LDL, are susceptible to lipid oxidation with consequent loss of some cardioprotective properties (6). However, although several studies have demonstrated that vitamin 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 protected lipids in human HDL from oxidation. We also investigated 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Materials.

Vacutainer blood collection systems and tubes (1.43 x 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 x g for 300 min in a Beckman VTi50 rotor cooled to 7°C. The identity of the isolated HDL and LDL 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 chromatography (Supelco Sephadex G-25M PD-10 columns), and the purified fractions filter-sterilized (Gelman 0.2 µm Acrodisc filter). The protein 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 µmol/L) in the absence (control) or presence of vitamin C (20–200 µmol/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 assessment of LDL oxidation (18,19).



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  		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.

 


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  		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) independent experiments; {alpha}, ß, {gamma}, and {delta}: different from control, P < 0.05 for incubations containing vitamin C at concentrations of 20, 50, 100 and 200 µmol/L, respectively; {epsilon}, different from control, P < 0.05 for vitamin C >= 50 µmol/L (C).

 


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  		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.

 
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 incubated at 37°C with Cu2+ (0.5 µmol/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:

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 during co-incubation of LDL with HDL and Cu2+ for 3 h. For HDL oxidized by preincubation with Cu2+, the reaction was terminated by the addition of diethylenetriaminepentaacetic acid (DTPA,3 20 µmol/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 reported as means ± SEM for pooled data from three independent experiments; within each experiment, single analyses of each measured variable were performed. Statistical analysis was performed after testing for variance homogeneity (Levene’s test) by either two-way (Figs. 1and 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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Vitamin C was rapidly consumed during incubation with HDL and Cu2+ (Fig. 1). Even for the highest added concentration (i.e., 200 µmol/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 oxidation as evidenced by a decline in cholesteryl linoleate content 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 remained 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 µmol/L) significantly retarded oxidation of lipids in HDL. At the lowest concentration (i.e., 20 µmol/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. Similarly, in contrast to control incubations in which lipid oxidation 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 µmol/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 µmol/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 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 µmol/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 µmol/L; HDL-50 to HDL-200, respectively) decreased lipid oxidation in HDL compared with the vitamin C–free control (<=4.1 ± 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 µmol/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 antioxidant 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 concentrations 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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
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 exposed to prooxidant Cu2+, HDL underwent rapid oxidation in control incubations lacking vitamin C. Immediately upon exposure to Cu2+, levels of HDL-associated cholesteryl linoleate, a substrate for lipid oxidation in lipoproteins (21), 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 consistent with those of numerous other investigators who reported 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 µmol/L) to our standard incubation system 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 µmol/L), initiation of HDL lipid oxidation was delayed such that complete oxidation was not observed until after at least 2 h had elapsed after incubation with Cu2+. Indeed, at vitamin C concentrations within the range normally found in human blood [i.e., 30–150 µmol/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 concentrations, vitamin C affords significant protection against Cu2+-mediated lipid oxidation in HDL. This observation is consistent with the known antioxidant capabilities of vitamin C in human blood (11).

Prior studies investigating the effect of antioxidant nutrients on HDL oxidation focused mainly on vitamin E, the principal lipid-soluble antioxidant present in HDL (31). Laureaux and co-workers (7) reported that in vitro enrichment of isolated HDL with {alpha}-tocopherol stabilized lipid hydroperoxides 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 volunteers’ blood from 55.6 ± 4.0 to 111.9 ± 7.4 µmol/L, none of this vitamin C would have been present during the ex vivo oxidation of the isolated HDL. In contrast, in our experimental system, vitamin C was added directly to HDL in the ex vivo prooxidant experimental system mimicking the in vivo situation 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 preventing lipid oxidation in HDL exposed to Cu2+. Thus, in control incubations lacking vitamin C, levels of HDL-associated vitamin 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 disappearance 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 vitamin C from oxidation, it is clear from our results that other 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 preincubated 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, therefore, 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 preservation 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 promote 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 vitamin 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 hypochlorous 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 hypochlorous acid (46), it is not known how it will affect oxidation of HDL by myeloperoxidase-generated oxidants.

In summary, our results demonstrate that physiologic concentrations of vitamin C inhibit Cu2+-mediated lipid oxidation 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 investigation in our laboratory.


   ACKNOWLEDGMENTS
 
The authors thank Allan Campione and Michael Moore for their expert technical assistance.


   FOOTNOTES
 
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.)]. Back

2 Supported by Midwestern University’s Office of Research and Sponsored Programs. Back

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 µmol/L, respectively); PON1, paraoxonase-1. Back

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


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