CURRENT
THERAPEUTIC
RESEARCH®
VOL. 58, NO. 5, MAY 1997
E F F E C T S OF VITAMIN C AND VITAMIN E ON PLASMA LEVELS
OF LIPID HYDROPEROXIDES AND THIOBARBITURIC ACID
REACTIVE SUBSTANCE IN HUMANS
NAGAHIKO SAKUMA, SEIJI IWATA, TAKESHI HIBINO, NOZOMU TAMAI,
KANNA SASAI, TAKAYUKI YOSHIMATA, YOSHINOBU KAMIYA,
MASANOBU KAWAGICHI, AND TAKAO FUJINAMI
The Third Department of Internal Medicine, Nagoya City University Medical School,
Nagoya, Japan
ABSTRACT
A s t u d y was c o n d u c t e d t o i n v e s t i g a t e t h e effects o f v i t a m i n C a n d
v i t a m i n E o n p l a s m a levels o f lipid h y d r o p e r o x i d e s (LPO) a n d thiob a r b i t u r i c acid r e a c t i v e s u b s t a n c e (TBARS) i n h u m a n v o l u n t e e r s to
i d e n t i f y t h e s t e p i n t h e lipid p e r o x i d a t i o n cascade a t which t h e s e
v i t a m i n s act. F o r t y s u b j e c t s (20 m e n a n d 20 w o m e n ) w e r e r a n d o m l y
a s s i g n e d to r e c e i v e e i t h e r v i t a m i n C o r v i t a m i n E. T w e n t y s u b j e c t s
r e c e i v e d 500 mg o f v i t a m i n C d a i l y f o r 4 weeks, a n d 20 s u b j e c t s
r e c e i v e d 300 mg o f v i t a m i n E daily f o r 4 weeks. Blood s a m p l e s w e r e
collected before and 4 weeks after vitamin treatment, in the morning, a f t e r t h e s u b j e c t s h a d f a s t e d for 12 h o u r s . P l a s m a levels o f LPO
a n d TBARS w e r e d e t e r m i n e d . P l a s m a l e v e l s o f lipids, a p o l i p o p r o t e i n s , v i t a m i n C, a n d v i t a m i n E w e r e also m e a s u r e d . V i t a m i n C sign i f i c a n t l y r e d u c e d p l a s m a levels o f LPO a n d TBARS. V i t a m i n E sign i f i c a n t l y i n c r e a s e d p l a s m a levels o f LPO a n d s i g n i f i c a n t l y r e d u c e d
p l a s m a levels o f TBARS. P l a s m a c o n c e n t r a t i o n s o f v i t a m i n C a n d
vitamin E significantly increased after 4 weeks of vitamin treatm e n t . T h e r e w e r e n o s i g n i f i c a n t c h a n g e s i n t h e p l a s m a levels o f
lipids e x c e p t LPO, TBARS, a n d a p o l i p o p r o t e i n s . F r o m t h e s e resuits, i t was c o n c l u d e d t h a t v i t a m i n C r e d u c e d LPO a n d TBARS levels, a n d v i t a m i n E i n c r e a s e d LPO levels a n d r e d u c e d TBARS levels.
Key words: v i t a m i n C, v i t a m i n E, lipid h y d r o p e r o x i d e s , t h i o b a r b i t u ric acid r e a c t i v e s u b s t a n c e .
INTRODUCTION
The plasma levels of the products of lipid peroxidation are increased in
patients with atherosclerotic disease. 1-6 Glavind et al 7 detected lipid peroxides in atheromatous h u m a n arteries but not in healthy arteries. These
findings suggest that an increased plasma concentration of lipid peroxides
Address correspondenceto: Nagahiko Sakuma, MD, PhD, Associate Professor of Internal Medicine,The
Third Department of Internal Medicine,Nagoya City UniversityMedical School,Mizuho-cho,Mizuho-ku,
Nagoya 467, Japan.
Received for publication on February 4, 1997. Printed in the U.S.A.
Reproduction in whole or part is not permitted.
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VITAMIN C AND VITAMIN E EFFECTS ON LIPID PEROXIDATION
may be a risk factor for atherosclerosis. Peroxidative modification of lowdensity lipoprotein (LDL) is also believed to contribute to the pathogenesis
of atherosclerosis in vivo. s Therefore, the study of the effects of antioxidants against lipid peroxidation is of interest. 9 Ascorbic acid (vitamin C),
an aqueous antioxidant, and alpha-tocopherol (vitamin E), a lipid-soluble
antioxidant, are radical-scavenging antioxidants.
Peroxidation of fatty-acyl groups proceeds via the formation of a diene
conjugate and results in the formation of a peroxy radical. The peroxy
radical then either forms a metastable hydroperoxide or breaks down to
release a wide variety of aldehydes, ketones, and hydroxylipids, l°'u Vitamin C and vitamin E may have a preventive (ie, primary) effect and]or a
chain-breaking (ie, secondary) effect on the peroxy radical, b u t these
potential effects have not been elucidated. Therefore, the present study
was designed to investigate the short-term (4 weeks) effects of vitamin
C and v i t a m i n E on p l a s m a concentrations of lipid hydroperoxides
(LPO) and thiobarbituric acid reactive substance (TBARS), which reflects
mainly an end peroxidative product such as malondialdehyde in healthy
volunteers. 12
SUBJECTS
AND METHODS
Forty volunteers (20 men and 20 women) ranging in age from 36 to 81
years (mean age, 61.7 + 9.0 years) were randomly assigned to the outpatient clinic of The Third Department of Internal Medicine, Nagoya City
University Hospital, Nagoya, Japan. They were healthy subjects who had
visited the hospital for their regular physical examinations. Informed consent for participation in the study was obtained from all subjects.
The subjects were randomly divided into two equal groups. There were
no differences between groups with regard to age, sex, or body mass index.
Subjects were instructed to maintain their same dietary and physical activity habits, smoking habits, and their normal alcohol intake. Also, subjects were instructed not to take nonstudy vitamin supplements. One
group (n = 20) was given 500 mg of vitamin C (ascorbic acid*) daily (250
mg in the morning and 250 mg in the evening) for 4 weeks. The other group
(n = 20) was given 300 mg of vitamin E (tocopherol acetatet) daffy (100 mg
in the morning, 100 mg at noon, and 100 mg in the evening) for 4 weeks.
Blood samples were collected into Vacutainer tubes (Terumo Corporation, Tokyo, Japan) containing ethylenediaminetetraacetic acid before
and 4 weeks after vitamin treatment, in the morning, after the subjects
had fasted for 12 hours. The plasma levels of LPO, the primary product of
* Trademark: Hicee ® granules (Takeda Chemical Industries, Ltd., Osaka, Japan).
t Trademark: Juvela ® (Eisai Co., Ltd., Tokyo, Japan).
318
N. S A K U M A ET AL.
lipid peroxide in the sample, were determined by the methylene-blue hemoglobin method of Ohishi et a113 using the determiner LPO kit (Kyowa
Medix, Tokyo, Japan). Cumene hydroperoxide was used as a standard.
This method is specific for measuring LPO.
For comparison, the plasma levels of TBARS were measured fluorometrically using the lipid peroxide test Wako kit (Wako Pure Chemical
Industries, Ltd., Osaka, Japan) according to the methods of Yagi. 12 This
method is not specific, since many lipid peroxides in plasma produce the
same red pigment as malondialdehyde when reacted with thiobarbituric
acid. Results of this assay, however, provide a convenient estimate of total
lipid peroxides and their derivatives, which can be compared with specific
LPO concentrations. Fluorescence intensity was measured on a spectrofluorometer (Hitachi F-3010; Hitachi, Ltd., Tokyo, Japan) with excitation
at 515 nm and emission at 553 nm.
Plasma levels of lipids were determined by using enzymatic methods. 14,is The levels of high-density lipoprotein cholesterol were determined
by the precipitation method, which involves the use of heparin and calcium, 16 and plasma levels of apolipoproteins were measured by using automated immunoturbidimetric analysis. 17 The plasma levels of vitamin C
were measured by the method of Roe and Kuether, is and the plasma levels
of vitamin E were measured by the method of Abe et al. 19 The coefficient
of variation was <5%.
All subjects had a baseline physical examination and were reexamined
at the end of the study. A urinalysis was also performed.
The results were reviewed by a panel of three medical physicians
under masked conditions so that the reviewers could assess the results
without bias.
Statistical Analysis
Data were analyzed by using the Mann-Whitney U statistic test and
Wilcoxon's signed rank test. A level of P < 0.05 was accepted as an indication of statistical significance.
RESULTS
Vitamin C significantly reduced the plasma levels of LPO and TBARS (P <
0.005, respectively) (table). Vitamin E significantly increased the plasma
levels of LPO (P < 0.05) and significantly reduced the plasma levels of
TBARS (P < 0.005). The plasma concentrations of vitamins C and E were
significantly higher after 4 weeks (P < 0.005, respectively) than at baseline. Plasma levels of lipids except LPO, TBARS, and apolipoproteins were
not affected by vitamins C and E (table). Both vitamin C and vitamin E
were well tolerated. No adverse effects were produced by the use of these
vitamins.
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VITAMIN C AND VITAMIN E EFFECTS ON LIPID PEROXIDATION
Table. Effects of vitamin C and vitamin E on plasma levels of lipid hydroperoxides (LPO),
thiobarbituric acid reactive substance (TBARS), lipids, apolipoproteins (Apo), and
vitamins.*
LPO (nmol/mL)
TBARS (nmol/rnL)
TC (mg/dL)
TG (mg/dL)
HDL-C (mg/dL)
Apo A-I (mg/dL)
Apo B (mg/dL)
Apo E (mg/dL)
Vitamin C (pmol/L)
Vitamin E (pmol/L)
Baseline
After
Vitamin C
Baseline
After
Vitamin E
2.47 ± 0.92
1.93 ± 0.31
233.5 ± 30.2
136.0 ± 105.3
57.4 ± 13.8
145.6 ± 21.9
114.5 ± 17.7
5.1 ± 0.9
22.7 ± 11.9
32.7 ± 15.3
0.96 + 0.32t
1.51 + 0.18t
235.3 ± 38.1
135.2 ± 119.2
59.2 ± 14.3
146.7 ± 29.9
113.7 ± 22.1
4.9 ± 0.8
40.9 ± 13.9t
40.9 ± 13.5
2.50 ± 1.00
1.97 ± 0.32
233.2 ± 26.8
130.9 + 53.5
59.8 ± 18.2
140.1 _+26.4
114.9 ± 23.7
5,0 ± 1.0
20.4 ± 8.5
34.6 ± 7.4
2.91 ± 1.38:~
1.61 ± 0.31t
226.6 ± 30.7
132.8 + 71.6
56.8 ± 16.9
143.6 ± 23.9
117.5 ± 22.4
5.3 + 1.4
21.6 ± 9.7
55.0 + 11.8t
TC = total cholesterol; TG = triglyceride; HDL-C = high-density lipoprotein cholesterol.
* Values are expressed as the mean + SD for 20 subjects in each group. To convert to millimoles per liter,
multiply cholesterol levels by 0.02586 and TG levels by 0.01129.
t P < 0.005 versus baseline.
P < 0.05 versus baseline.
DISCUSSION
In the present study, vitamin C significantly reduced the plasma levels of
LPO and TBARS, suggesting that vitamin C acts at a proximal step in the
peroxidation process, scavenging free radicals and protecting lipids from
significant peroxidative attack. These results suggest that vitamin C prevents the initiation of the chain of lipid peroxide propagation, reducing the
LPO level and, consequently, reducing the TBARS level. These results are
consistent with the results of previous studies in which Osteogenic Disorder Shionogi (ODS) mutant rats unable to synthesize vitamin C fed a diet
supplemented with vitamin C showed decreased LPO and TBARS levels
compared with ODS rats fed a vitamin C-free diet. 2°'21
Vitamin C is present in the aqueous phase of extracellular fluids and
within cells. It protects the lipids in lipoproteins and biomembranes from
peroxidative damage by reacting with oxidants generated in the aqueous
phase before they begin to attack the lipids. Vitamin E inhibits lipid peroxidation in biologic membranes and in serum LDL, acting primarily as a
scavenger of lipid peroxy radicals. 22-26 In the present study, vitamin E
significantly increased LPO levels and significantly reduced TBARS levels,
suggesting that lipid-soluble vitamin E breaks the chain of lipid peroxide
propagation following the formation of LPO. These findings suggest that
lipid-soluble vitamin E does not form the first line of defense against peroxidative injury in membranes or lipoproteins but acts as a point in the
lipid peroxidation cascade following the formation of LPO thereby preventing conversion of LPO to its derivatives. Studies have shown that plasma
levels of the products of lipid peroxidation, such as malondialdehyde, are
increased in patients with atherosclerotic disease, z-~
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N. SAKUMAET AL.
Epidemiologic studies suggest that there is a low prevalence of coronary artery disease in populations that consume large quantities of vitamins C and E. The present findings and the results of previous studies
suggest that vitamins C and E may prevent coronary artery disease by
decreasing the levels of TBARS in plasma in vivo. This is the first study to
demonstrate in humans that vitamin C can reduce LPO and TBARS levels,
and that vitamin E can increase LPO levels and reduce TBARS levels.
CONCLUSION
The results of this study suggest that vitamin C prevents both the initiation and propagation of lipid peroxidation, whereas vitamin E acts as a
chain-breaking antioxidant, inhibiting the propagation of lipid peroxidation following the formation of LPO.
Acknowledgment
Financial support for this study was provided by the Vitamin C Research
Committee, Tokyo, Japan.
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