AIherosclerosis, 85 (1990) 41-54 Elsevier Scientific Publishers Ireland,
ATHERO
47 Ltd.
04548
Effect of vitamin C and E supplementation on susceptibility of plasma lipoproteins to peroxidation induced by acute smoking D. Harats *, M. Ben-Naim
*, Y. Dabach I, G. Hollander and Y. Stein 1
I, E. Havivi ‘, 0. Stein *
’Department of Medicine B, Lipid Research Luboraiory, Hadassah University Hosprral, ’Department of Experimental Medicrne and Cancer Research, Hebrew Uniuersity - Hadassah Medical School, and 3 Department of Nutntlon, Minrstty of Health, Jertualem (Isruel) (Received 30 April, 1990) (Revised, received 25 June, 1990) (Accepted
3 July, 1990)
Summary The effect of acute smoking on plasma lipoproteins was studied in seventeen smokers. In study 1, 7 subjects were examined prior to and 2 weeks after supplementation with vitamin C. In study 2, the effect of acute smoking was first determined in 10 additional subjects and subsequently they were divided into 3 groups, 3 and 4 subjects were supplemented with vitamin C or E, respectively, for 4 weeks, and 3 remained untreated. Plasma and LDL TBARS were examined at time zero (i.e., 40-48 h after total abstention from smoking) and at 90 min after acute smoking (5-7 cigarettes). In all 17 subjects examined prior to vitamin supplementation, significantly higher TBARS values were found in plasma, native LDL and LDL conditioned with smooth muscle cells (SMC) when the 90 min values were compared to 0 time. The LDL isolated after 90 min and conditioned with SMC was metabolized more extensively by mouse peritoneal macrophages than its zero time counterpart. The differences between the 0 time and 90 min values were not seen after the subjects had been supplemented with vitamin C for 2 or 4 weeks or with vitamin E for 4 weeks. The present results indicate that acute smoking exerts an oxidative stress on plasma lipoproteins and that higher plasma levels of natural antioxidants, such as vitamins C and E have a protective role.
Key words:
Atherosclerosis; Smoking; muscle cells; Thiobarbituric
LDL modification; LDL peroxidation; Macrophages; acid reactive substances; Vitamin C; Vitamin E
Smooth
Introduction Correspondence to: Prof. Yechezkiel Stein, Lipid Research Lab., Dept. Medicine B, Hadassah University Hospital, P.O. Box 12000, Jerusalem 91120, Israel. Telephone: (02) 427427; Fax: (02) 434434.
0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland.
The mechanism by which smoking is linked to the development of accelerated atherosclerosis and Ltd.
48 acts as a risk factor for the development of coronary artery disease is unknown. One of the possibilities raised in previous studies [l-3] is that the deleterious effect of smoking is exerted via its action on plasma lipoproteins, changing them in a subtle way into a more atherogenic lipoprotein that is recognized by the macrophage scavenger receptor [4,5]. We have examined the effect of acute smoking on thiobarbituric acid reactive substances (TBARS) in LDL isolated from subjects asked to refrain from smoking for 24-40 h and reported a significant increase in TBARS, especially after conditioning of the LDL with cells [3]. Thus it appeared that smoking did render plasma lipoproteins more prone to peroxidative stress. The natural protectors against peroxidation of lipoproteins in plasma are vitamin C and vitamin E, the levels of which can be modified by dietary means. Plasma levels of vitamin C were reported to be lower in smokers than in nonsmokers [6-g], and could have contributed towards the greater susceptibility of smokers’ lipoproteins to peroxidation. In the present study, we proposed to investigate the putative role of vitamin C and E in protection of plasma lipoproteins during acute smoking. Materials and methods Human subjects Seventeen smokers (12 males, 5 females) volunteered to participate in this study with informed consent; they were examined over a period of 7 months. Study 1 included 7 smokers who were examined prior to and after 2 weeks of vitamin C supplementation (1 g/day). Study 2 included 10 smokers all of whom were examined prior to vitamin supplementation and then divided into 3 groups. Three smokers did not receive any vitamins, 3 were given 1.5 g vitamin C per day, and 4 were given 600 mg vitamin E per day for 4 weeks. The participants were asked to refrain from smoking for at least 40-48 h prior to blood collection. Blood was drawn into vacutainer tubes containing EDTA in the morning (0 time) and after 90 min. During the 90 min interval, they were asked to smoke 5-7 cigarettes. LDL was isolated by ultracentrifugation [9] at d = 1.019-1.063 g/ml from human plasma con-
taining 1 mg/ml EDTA. It was sterile filtered, aliquoted into plastic tubes and kept under N, at 4OC. Determination of thiobarbituric acid reactive substances was carried out immediately after isolation. Iodination of LDL was carried out by the iodine monochloride method [lo]. Tissue culture Bovine aortic smooth muscle cells (SMC) were prepared as described before and subcultured in Dulbecco-Vogt medium containing 10% fetal bovine serum [ll]. Peritoneal macrophages were obtained from mice 4 days after intraperitoneal injection of thioglycollate [12] and were cultured in 35-mm petri dishes with MEM containing 10% fetal bovine serum. Conditioning of LDL and rz51-LDL was carried out by incubation of the lipoproteins in Ham F10 medium without serum for 24 h, in the presence or absence of confluent cultures of SMC. At the end of conditioning the media were collected and centrifuged to remove cell debris. Media containing r2?-LDL were diluted with fresh FlO medium to give a final concentration of 33.3 pg LDL protein/ml and used for incubation with macrophages. For the determination of the effect of conditioning on oxidative modification unlabeled LDL, 50 pg protein/ml was incubated with or without SMC in FlO medium for 24 h. Thereafter, thiobarbituric acid reactive substances (TBARS) were determined. Metabolism of 12’I-LDL by macrophages On the day of the experiment, the medium was removed from the macrophages and the cells were washed twice by incubation for 15 mm at 37” C with 2 ml of FlO medium. Thereafter, 1 ml of conditioned or control medium (preincubated in the absence of cells) containing the labeled lipoproteins was added to the macrophages and incubation was carried out at 37O C in a CO, incubator for 4 h. Dishes without cells, containing conditioned or control medium, were incubated under the same conditions. At the end of the incubation, the medium was collected, the cells were washed, 0.05% trypsin was added and the cells were scraped with a rubber policeman. After inactivation of the trypsin with serum containing medium, the cells were pelleted by centrifugation
49 and the pellets were washed twice by recentrifugation with 4 ml buffered saline. The cell pellets were extracted with chloroform/methanol (1 : 1, v/v) to remove labeled lipids and protein radioactivity was determined. After precipitation of the medium with 50% trichloroacetic acid in the presence of carrier protein, non-iodide TCA-soluble radioactivity was determined [ll]. The non-iodide TCA-soluble radioactivity obtained in duplicate dishes without cells was subtracted from each experimental value. Chemical,
chromatographic
and radiochemical
prepared using a-tocopherol vitamin E and A, respectively.
and
P-carotene
for
Materials
Culture media and fetal bovine serum were obtained from Gibco (New York, NY). [‘251]Iodide was obtained from Amersham Int. (U.K.). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Vitamin E (DL-alphatocopherol acetate), 200 mg/tablet, was obtained from Teva (Israel). Vitamin C, 500 mg/tablet. was obtained from Rekah (Israel).
pro-
cedures
Peroxidation was measured by determining the amount of thiobarbituric acid reactive substances (TBARS) as modified by Lee [13]. Briefly, to 1 ml of plasma or 50 pg LDL protein in 1 ml, 0.5 ml 35% TCA was added and the solution was vortexed. Then, 1 ml 0.5% TBA was added and again vortexed. The solution was incubated for 90 min at 60” C in a shaking waterbath. After incubation, 1 ml 70% TCA and 2 ml chloroform were added, and the tubes were centrifuged for 20 min at 3000 t-pm. A standard curve was prepared using malondialdehyde and the intensity of fluorescence was determined at excitation of 515 nm and emission of 553 nm as employed by the method of Yagi [14]. The results were expressed as malondialdehyde equivalent content (nmol MDA/ml plasma or nmol MDA/mg LDL protein). Protein was determined according to Lowry et al. [15], and cholesterol and triacylglycerol were determined using a batch instrument (Vitatron FPS, Vital Scientific, Dieren, The Netherlands). Plasma high density lipoprotein cholesterol was determined by heparin-Mn2+ precipitation. Vitamin C was measured according to Denson and Bowers [16]. Vitamin E was measured by modification of the method of Hashin and Schuttringer [17]: to 1 ml plasma, 1 ml absolute ethanol was added, the solution was vortexed, 2 ml heptane was added, vortexed again, and the tubes were centrifuged for 5 min at 3000 rpm. To 1 ml supernatant, 0.2 ml 0.147% bathophenantroline was added, and vitamin A content was determined at 452 nm. Then, 0.1 ml 0.04% FeCl,, and 0.1 ml 6.23% H,PO, were added, and vitamin E was determined at 532 nm. A standard curve was
We examined the effect of acute smoking on plasma lipoproteins in 17 normal volunteers. In the first study, 7 subjects were examined prior to and after ingestion of vitamin C. In the second study, of the 10 subjects examined prior to administration of vitamins 3 received vitamin C, 4 vitamin E, 3 were not treated and were re-examined at the end of the experiment. As can be seen in Table 1, the 17 subjects were subdivided so that there were no differences in the initial plasma lipid values, BMI and smoking habits between the groups. Blood lipids were re-examined 4 weeks later and no change occurred in the subjects given vitamin C or E. The plasma levels of vitamin C, E and A are summarized in Table 2. In the first study in which 1 g/day of vitamin C was given for two weeks plasma levels of vitamin C doubled. The dose of vitamin C was increased to 1.5 g/day in the second study, and a slightly higher response was seen after 4 weeks. Administration of vitamin E resulted in an almost 3-fold increase in plasma vitamin E levels after 4 weeks of supplementation. No change occurred in plasma levels of vitamin A throughout the experimental period in all the 10 subjects examined. There was also no change in vitamin C or E plasma levels in control subjects (not given any vitamin) or those supplemented with vitamin E or C, respectively. Thiobarbituric acid reactive substances (TBARS) were determined on samples of plasma drawn at two time intervals; time 0 was after 40-48 h of total abstention from smoking. Thereafter, the subjects were allowed to smoke 557 cigarettes during 90 min and blood was drawn
50 TABLE
1
CHARACTERISTICS OF THE HUMAN SUBJECTS INVESTIGATED PRIOR TO TREATMENT WITH VITAMIN CORE Blood was drawn after overnight fast for lipid determination and other studies at the beginning of each study prior to administration of vitamins. The subjects were then divided into 3 groups which received either vitamin C (7 in study I, and 3 in study 2) or vitamin E (4 in study 2). or none (3 in study 2). Body mass index (BMI) = body weight (kg) divided by height (in m*); TG = triacylglycerol; HDL = high density lipoproteins. Values are means k S.E. Vitamin
No. of subjects Age (years) Sex: female male BMI Plasma cholesterol (mg/dl) Plasma TG (mg/dl) Plasma HDL cholesterol (mg/dl) Smoking habits: Packs (years) Cigarettes/day
C
Vitamin
E
No treatment
IO 28.8 4 6 23.2 + 2.4
4 31.0 1 3 24.6 f 3.1
3 30.3 0 3 22.2 rt 1.6
172.2 f 6.4
172.8 f 4.2
180.8 f 6.5
123.6*
114.2+
128.65
10.4
15.2
8.4
37.1*1.8
35.9 + 2.4
39.0 f 3.0
17.8+6.0 31.5 _+2.6
19.3 + 10.0 29.5 _+7.0
20.8 f 7.0 32.3 + 8.0
again (90 mm). In the first study, there was a significant rise in plasma TBARS after acute smoking in the subjects examined prior to vitamin C administration (Table 3). A slight, insignificant increase of TBARS occurred when the same subjects were re-examined after vitamin C administration. In the second study, plasma TBARS were
TABLE PLASMA
2 LEVELS
OF VITAMINS
C, E AND A BEFORE
Values are means + SE; n = number Study
1 2
determined at time 0 and after 90 min of smoking in 10 subjects (period I) and a significant increase was observed. Those subjects were re-examined after 4 weeks of either no treatment (None) or treatment with vitamin C or E. A similar increase in plasma TBARS between 0 and 90 min was seen in the non-treated subjects (period II). However, in those given vitamin C or E there was no increase in plasma TBARS at 90 min (Table 3). Essentially similar results were obtained when the TBARS were examined on LDL isolated from the subject’s plasma. Vitamin C and vitamin E ingestion prevented the rise in TBARS in LDL after acute smoking (Table 4). Next we examined the effect of conditioning of LDL in the presence of smooth muscle cells on TBARS formation. As expected, there was a very significant increase in the TBARS in conditioned LDL (Table 5). There was no difference between the 0 time values of all the subjects examined irrespective of the vitamin supplementation. However, comparison of the 90 rnin values shows that both vitamin C and vitamin E supplementation prevented the increase in TBARS in conditioned LDL after acute smoking. Similar results were obtained also when LDL was conditioned in the absence of cells (Table 5). Metabolism of the conditioned ‘251-LDL was examined in mouse peritoneal macrophages. In both experiments, essentially no differences were encountered in the 0 time value between the subjects of the different groups. Treatment with vitamin C or E affected only the 90 min values, i.e., at‘of contenuated the increase in metabolism ditioned ‘251-LDL caused by acute smoking (Table 6).
Vitamins given in period II
”
C None .E C
of subjects. Vitamin
(I) AND AFTER
For designation
C (mg/dl)
of study
(II) SUPPLEMENTATION
1 and 2, see Methods.
Vitamin
Vitamin
E (mg/dl)
A (pg/dl)
I
II
I
II
I
II
7
0.52 + 0.04
1.06+0.13
_
_
_
_
3 4 3
0.45 * 0.07 0.46 + 0.06 0.54 * 0.09
0.48 f 0.04 0.45 +0.05 1.26 + 0.06
0.61+ 0.01 0.61+ 0.01 0.59 f 0.01
0.64 f 0.04 1.7250.17 0.61& 0.01
5s+ 3.5 59 f 4.4 56 + 2.3
59+4.8 59 f 2.6 57 + 2.4
51 TABLE
3
TBARS
IN PLASMA
BEFORE
AND AFTER
SMOKING
IN SUBJECTS
TREATED
WITH
VITAMIN
C OR E
Blood was drawn from the volunteers 40-48 h after abstention from smoking, and 90 min after smoking 5 -7 cigarettes. Values are means + SE; n = number of subjects; each sample was examined in duplicate. In study 1, 7 smokers were examined before (period I) and after (period II) supplementation with 1 g vitamin C/day for 2 weeks. In study 2, 10 smokers were examined before treatment (period I). Thereafter (period II), 4 subjects were treated with 600 mg/day vitamin E and 3 with 1.5 g/day vitamin C for 4 weeks, 3 subjects were given no treatment and served as controls (none). TBARS were determined in plasma immediately after blood drawing. n
Study
7
None
7
Vitamin
10 3 3 4
None None Vitamin Vitamin
nmol MDA/ml
Period
Treatment
I
0.106 + 0.02
C
II
0.138+0.01
0.187 + 0.01 0.145 kO.01
c 0.01 “.\
C E
1 II II 11
0.141 f 0.003 0.106*0.014 0.107 f 0.021 0.119+0.013
0.217 * 0.02 0.197~0.009 0.118+0.020 0.123+0.018
< 0.005 c 0.01 n.s “..\
can be easily modulated by dietary means and are apparently not toxic even when given in large doses. Vitamin C levels were studied in smokers and compared to those of nonsmokers by several investigators [6,7] and were found to be lower. In a more recent study, it was reported that the lower plasma levels of vitamin C in smokers were independent of dietary intake [8]. Vitamin C was singled out by Frei et al. [18] in its effectiveness to protect lipids from peroxidative damage by aqueous oxidants. Thus, once ascorbate has been exhausted, lipid peroxidation occurred even though the other antioxidants, such as bilirubin, uric acid and vitamin E had not yet decreased markedly. Impaired serum antioxidant activity was indeed observed in smokers, but was related to an insufficient response of ceruloplasmin [21].
The two main findings of the present study were that in the 17 smokers who abstained from smoking for 48 h (a) a marked increase in plasma and LDL TBARS over the zero time value occurred after acute smoking for 90 min; (b) this increase could be prevented by dietary supplementation of either vitamin C or E. The 0 time and 90 min samples of plasma were processed together and thus the results appear even more significant as the 90 min sample was exposed for a shorter time interval after blood drawing to putative deleterious influences, such as air, light, etc. Of the multitude of natural antioxidants present in human plasma [18-201, we would like to focus on just two, vitamin C and E, since these compounds 4
TBARS IN NATIVE VITAMIN C OR E
LDL ISOLATED
FROM
Conditions as in Table 3. TBARS formation means + SE; n = number of subjects. Study
n
Treatment
PLASMA
BEFORE
was determined
Period
AND AFTER
immediately
SMOKING
after isolation
nmol MDA/mg
10 3 3 4
None None Vitamin Vitamin
C E
I II II II
0.584 0.683 0.627 0.705
+ f f +
0.026 0.02 0.03 0.04
IN SUBJECTS
TREATED
of the LDL from the plasma.
LDL protein
before smoking (0 time) 2
P
after smoking (90 min)
Discussion
TABLE
plasma
before smoking (0 time)
P
after smoking (90 min)
---
1.275+0.120 1.333kO.065 0.663 rt 0.05 0.733 _t 0.02
< 0.001 c 0.001 “.S. n.s.
WITH
Values
are
52 TABLE
5
TBARS IN LDL CONDITIONED VITAMIN C OR E
WITH
SMC,
BEFORE
AND
AFTER
SMOKING
IN
Conditions as in Tables 3 and 4. For conditioning, LDL 50 pg protein/ml medium was incubated smooth muscle cells. Values are means f SE; n = number of subjects. All data are from study 2. Treatment
n
Conditioned with cells None None Vitamin C Vitamin E
nmol MDA/mg
Period
SUBJECTS
TREATED
for 24 h in dishes with or without
LDL protein
P
before smoking (0 time)
after smoking (90 min)
10 3 3 4
I II 11 II
17.5+1.38 21.4k1.23 17.3kO.84 17.8+2.90
36.8k1.32 39.1* 1.03 18.9* 1.78 19.7 + 3.20
i 0.001 < 0.001 ns. n.s.
Conditioned without cells None 10 None 3
I II
1.66 + 0.21 2.31+ 0.40
Vitamin Vitamin
II II
1.48kO.23 2.17 + 0.37
3.98 k 0.66 5.42+ 1.30 1.59*0.30 2.53 k 0.30
i 0.005 < 0.05 n.s. n.s.
C E
3 4
In contradistinction to vitamin C, plasma levels of vitamin E in smokers were not found to be lower than those of nonsmokers [22,23]; in these studies, lower plasma levels of p-carotene were seen in smokers [22,23]. It is of interest to point out that while no difference in plasma vitamin E levels of smokers was seen as well by Pacht et al. [24], these authors reported a 7-fold decrease in vitamin E concentration in alveolar fluid obtained from smokers by bronchial lavage. Vitamin E sup-
TABLE EFFECT
WITH
plementation did raise the alveolar fluid vitamin E content, albeit not to normal levels, and the authors suggested that smoking may accelerate the oxidative metabolism of vitamin E at the alveolar surface [24]. In the plasma compartment, low density lipoproteins are an important potential target for lipid peroxidation. However, Esterbauer et al. [25] pointed out that while microsomes [26] and other cellular membranes undergo rapid autooxidation, this is not the case with LDL. The
6 OF VITAMIN
C OR E ON METABOLISM
OF CONDITIONED
LDL BY PERITONEAL
MACROPHAGES
by incubation in F10 medium with and without bovine aortic smooth muscle Conditions as in Tables 3-5. ‘*‘I-LDL was conditioned ‘251-LDL was then incubated for 4 h with mouse peritoneal macrophages or in the absence of cells. cells for 24 h. The conditioned ‘*‘I-degradation products in medium after ‘*‘I-LDL metabolized is the sum of uptake = cell associated ‘251-protein and non-iodide subtraction of no cell values. Values are means + SE; n = number of subjects. Study
1
2
n
Period
Treatment
7 7
None Vitamin
10 3 3 4
None None Vitamin Vitamin
‘*‘I-LDL
metabolized
(pg/mg
cell protein/4
before smoking (0 time)
after smoking (90 mitt)
h)
P
C
I II
2.15 f 0.29 1.99f0.02
3.90+0.60 2.28 rlO.17
i 0.05 n.s.
C E
I II II II
2.64 1.95 3.16 2.05
7.49 + 0.46 8.09 f 0.27 3.30+0.60 2.31 kO.27
< 0.001 < 0.001 n.s. n.s.
* f f +
0.49 0.014 0.63 0.20
53 relative resistance of LDL to peroxidation has been shown to be due to the presence of vitamin E in the particle, as under experimental conditions lipid peroxidation of LDL is initiated only after its store of vitamin E had been exhausted [25]. In the present study when LDL of smokers had been conditioned with aortic smooth muscle cells, the lipoproteins obtained after 90 min of acute smoking were metabolized more extensively by macrophages than their 0 time counterpart. This increase was also prevented by vitamin C or E supplementation, supporting the notion that the increase in TBARS reflected also changes in apo B, which were seen on gel electrophoresis in the form of faster migrating bands. Even though it is well established that lipid peroxidation of LDL results in a change in the apoprotein B, which in turn renders the particle recognizable by the scavenger receptor, it is not clear which of the many products of oxidation are responsible for this process [27]. How can we reconcile the effect of vitamin C supplementation in increasing the resistance of the isolated LDL particle to peroxidation and the well-known partition of the vitamin into the aqueous phase? Here we have to invoke the findings of Doba et al. [28] and of Niki et al. [29,30], who have shown that vitamin C protects or spares vitamin E content under various experimental conditions. Moreover, vitamin C may help to regenerate cy-tocopheryl radicals present in the LDL lipid, thus generating a lipid soluble chain-breaking antioxidant [20,30]. The above-mentioned authors [28-301 also proposed an interaction between vitamin C and E in which both vitamins cooperatively inhibit oxidation of lipid. We would like to suggest that even though the plasma vitamin E levels in smokers supplemented with vitamin C did not increase, ‘sparing’ of vitamin E in the LDL particle might have occurred and contributed towards its lesser susceptibility to acute smoking. Such behavior could be envisaged if a redistribution of vitamin E occurred from HDL to LDL, as the latter is known to transport 39%58% [31] or 59% [32] of plasma vitamin E in males and 42% in females [32]. We plan to determine the vitamin E content of LDL particles in smokers exposed to acute smoking under different regimes of dietary antioxidant supplementation.
The findings of our studies fit well with the concepts enunciated by Gey, who showed an impressive inverse correlation of vitamin C and E levels and the mortality from ischemic heart disease (IHD) in various countries in Europe [33 -351. As smoking is one of the major risk factors for IHD, high levels of vitamin E may protect lipoproteins from peroxidation and thereby prevent their recognition by the scavenger receptor and thus render them less atherogenic. References 1 Yokode, M.. Kita, T., Arai. H.. Kawai, C.. Narumiya, S. and Fujiwara, M., Cholesterol ester accumulation in macrophages incubated with low density lipoprotein pretreated with cigarette smoke extract. Proc. Natl. Acad. Sci. LISA, 85 (1988) 2344. 2 Nadiger, H.A., Mathew, C.A. and Sadasivudu. B., Serum malondialdehyde (TBA reactive substance) levels in cigarette smokers, Atherosclerosis. 64 (1987)71. 3 Harats, D., Ben-Naim, M.. Dabach. Y.. Hollander, G., Stein. 0. and Stein, Y., Cigarette smoking renders LDL susceptible to peroxidative modification and enhanced metabolism by macrophages. Atherosclerosis, 79 (1989) 245. 4 Parthasarathy, S.. Prim, D.J., Boyd. D.. Joy. I. and Steinberg, D., Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor, Arteriosclerosis, 6 (1986) 505. 5 Steinbrecher, U.P., Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J. Biol. Chem.. 262 (1987) 3603. 6 Brook, M. and Grimshaw, J.J.. Vitamin C concentration of plasma and leukocytes as related to smoking habit, age. and sex of humans. Am. J. Clin. Nutr., 21 (1968) 1254. 7 Chow, C.K., Changchit, C., Bridges. R.B., Rehn. S.R.. Humble, J. and Turbek. J., Lower levels of vitamin C and carotenes in plasma of cigarette smokers. J. Am. (‘011. Nutr., 5 (1986) 305. 8 Schectman, G., Byrd, J.C. and Gruchow, H.W.. The influence of smoking on vitamin C status in adults, Am. J. Pub. Hlth.. 79 (1989) 158. 9 Havel, R.J.. Eder. H.A. and Bragdon. J.H.. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. .I. Clin. Invest.. 34 (1955) 1345. IO Bilheimer. D.W.. Eisenberg, S. and Levy, R.I.. The metabolism of very low density lipoprotein proteins. I. Preliminary in vitro and in vivo observations. B&him. Biophys. Acta. 260 (1972) 212. 11 Bierman, E.L.. Stem. 0. and Stein. Y.. Lipoprotein uptake and metabolism by rat aortic smooth muscle cells in tissue culture, Circ. Res.. 35 (1974) 136. 12 Friedman, G.. Chajek-Shaul, T.. Gallily, R., Stein, O., Shiloni. E., Etienne. J. and Stein. Y.. Modulation of lipo-
54
13 14 15
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23
protein lipase activity in mouse peritoneal macrophages by recombinant human tumor necrosis factor, Biochim. Biophys. Acta, 963 (1988) 201. Lee, D.M., Malondialdehyde formation in stored plasma, B&hem. Biophys. Res. Commun., 95 (1980) 1663. Yagi, K., A simple fluorimetric assay for lipoperoxide in blood plasma, Biochem. Med., 15 (1976) 212. Lowry, O.H., Rosebrough, N.J., Fat-r, A.L. and Randall, R.J., Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. Denson, K.W. and Bowers, E.F., The determination of ascorbic acid in white blood cells. A comparison of WBC ascorbic acid and phenolic acid excretion in elderly patients, Clin. Science, 21 (1961) 157. Hashim, S.A. and Schuttringer, G.R., Rapid determination of tocopherol in macro- and microquantities of plasma, Am. J. Clin. Nutr., 19 (1966) 137. Frei, B., Stocker, R. and Ames, B.N., Antioxidant defenses and lipid peroxidation in human blood plasma, Proc. Natl. Acad. Sci. USA, 85 (1988) 9748. Esterbauer, H., Striegl, G., PubI, H. and Rotheneder, M., Continuous monitoring of in vitro oxidation of human low density lipoprotein, Free Radicals Res. Commun., 6 (1989) 67. Halliwell, B., How to characterize a biological antioxidant, Free Radicals Res. Commun., 9 (1990) 1. Galdston, M., Levytska, V., Schwartz, M.S. and Magnusson, B., Ceruloplasmin increased serum concentration and impaired antioxidant activity in cigarette smokers, and ability to prevent suppression of elastase inhibitory capacity of alpha,-proteinaise inhibitor’-3, Am. Rev. Respir. Dis., 129 (1984) 258. Comstock, G.W., Menkes, M.S., Schober, S.E., Vuilleumier, J.-P. and Helsing, K.J., Serum levels of retinol, beta-carotene, and alpha-tocopherol in older adults, Am. J. Epidemiol., 127 (1988) 114. Stryker, W.S., Kaplan, L.A., Stein, E.A., Stampfer, M.J., Sober, A. and Willett, W.C., The relation of diet, cigarette smoking, and alcohol consumption to plasma beta-carotene and alpha-tocopherol levels, Am. J. Epidemiol., 127 (1988)
283. 24 Pacht, E.R., Kaseki, H., Mohammed, J.R., Comwell, D.G. and Davis, W.B., Deficiency of vitamin E in the alveolar
25
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34 35
fluid of cigarette smokers. Influence on alveolar macrophage cytotoxicity, J. Clin. Invest., 77 (1986) 789. Esterbauer, H., Jurgens, G., Quehenberger, 0. and Koller, E., Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes, J. Lipid Res., 28 (1987) 495. Benedetti, A., Comporti, M. and Esterbauer, H. Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids, Biochim. Biophys. Acta, 620 (1980) 281. Steinbrecher, U.P., Lougheed, M., Kwan, W.-C. and Dirks, M., Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation, J. Biol. Chem., 264 (1989) 15216. Doba, T., Burton, G.W. and Ingold, K.U., Antioxidant and co-antioxidant activity of vitamin C. The effect of vitamin C, either along or in the presence of vitamin E, or a water-soluble vitamin E analogue, upon the peroxidation of aqueous multilamellar phospholipid liposomes, B&him. Biophys. Acta, 835 (1985) 298. Niki, E., Kawakami, A., Yamamoto, Y. and Kamiya, Y., Synergistic inhibition of oxidation of soybean phosphatidylcholine liposomes in aqueous dispersion by vitamin E and vitamin C, Bull. Chem. Sot. Jpn., 58 (1985) 1971. Niki, E., Interaction of ascorbate and a-tocopherol, Ann. N.Y. Acad., 498 (1987) 186. Bjomson, L.K., Kayden, H.J., Miller, E. and Moshell, A.N., The transport of a-tocopherol and p-carotene in human blood, J. Lipid Res., 17 (1976) 343. Behrens, W.A., Thompson, J.N. and Madere, R., Distribution of a-tocopherol in human plasma lipoproteins, Am. J. Clin. Nutr., 35 (1982) 691. Gey, K.F., Stahelin, H.B., Puska, P. and Evans, A., Relation of plasma level of vitamin C to mortality from ischemic heart disease, Ann. N.Y. Acad. Sci., 498 (1987) 110. Gey, K.F., On the antioxidant hypothesis with regard to arteriosclerosis, Bibl. Nutr. Dieta, 37 (1986) 53. Gey, K.F., Plasma vitamins E and A inversely correlated to mortality from ischemic heart disease in cross-cultural epidemiology, Ann. N.Y. Acad. Sci., 570 (1989) 268.
ATHERO
47 Ltd.
04548
Effect of vitamin C and E supplementation on susceptibility of plasma lipoproteins to peroxidation induced by acute smoking D. Harats *, M. Ben-Naim
*, Y. Dabach I, G. Hollander and Y. Stein 1
I, E. Havivi ‘, 0. Stein *
’Department of Medicine B, Lipid Research Luboraiory, Hadassah University Hosprral, ’Department of Experimental Medicrne and Cancer Research, Hebrew Uniuersity - Hadassah Medical School, and 3 Department of Nutntlon, Minrstty of Health, Jertualem (Isruel) (Received 30 April, 1990) (Revised, received 25 June, 1990) (Accepted
3 July, 1990)
Summary The effect of acute smoking on plasma lipoproteins was studied in seventeen smokers. In study 1, 7 subjects were examined prior to and 2 weeks after supplementation with vitamin C. In study 2, the effect of acute smoking was first determined in 10 additional subjects and subsequently they were divided into 3 groups, 3 and 4 subjects were supplemented with vitamin C or E, respectively, for 4 weeks, and 3 remained untreated. Plasma and LDL TBARS were examined at time zero (i.e., 40-48 h after total abstention from smoking) and at 90 min after acute smoking (5-7 cigarettes). In all 17 subjects examined prior to vitamin supplementation, significantly higher TBARS values were found in plasma, native LDL and LDL conditioned with smooth muscle cells (SMC) when the 90 min values were compared to 0 time. The LDL isolated after 90 min and conditioned with SMC was metabolized more extensively by mouse peritoneal macrophages than its zero time counterpart. The differences between the 0 time and 90 min values were not seen after the subjects had been supplemented with vitamin C for 2 or 4 weeks or with vitamin E for 4 weeks. The present results indicate that acute smoking exerts an oxidative stress on plasma lipoproteins and that higher plasma levels of natural antioxidants, such as vitamins C and E have a protective role.
Key words:
Atherosclerosis; Smoking; muscle cells; Thiobarbituric
LDL modification; LDL peroxidation; Macrophages; acid reactive substances; Vitamin C; Vitamin E
Smooth
Introduction Correspondence to: Prof. Yechezkiel Stein, Lipid Research Lab., Dept. Medicine B, Hadassah University Hospital, P.O. Box 12000, Jerusalem 91120, Israel. Telephone: (02) 427427; Fax: (02) 434434.
0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland.
The mechanism by which smoking is linked to the development of accelerated atherosclerosis and Ltd.
48 acts as a risk factor for the development of coronary artery disease is unknown. One of the possibilities raised in previous studies [l-3] is that the deleterious effect of smoking is exerted via its action on plasma lipoproteins, changing them in a subtle way into a more atherogenic lipoprotein that is recognized by the macrophage scavenger receptor [4,5]. We have examined the effect of acute smoking on thiobarbituric acid reactive substances (TBARS) in LDL isolated from subjects asked to refrain from smoking for 24-40 h and reported a significant increase in TBARS, especially after conditioning of the LDL with cells [3]. Thus it appeared that smoking did render plasma lipoproteins more prone to peroxidative stress. The natural protectors against peroxidation of lipoproteins in plasma are vitamin C and vitamin E, the levels of which can be modified by dietary means. Plasma levels of vitamin C were reported to be lower in smokers than in nonsmokers [6-g], and could have contributed towards the greater susceptibility of smokers’ lipoproteins to peroxidation. In the present study, we proposed to investigate the putative role of vitamin C and E in protection of plasma lipoproteins during acute smoking. Materials and methods Human subjects Seventeen smokers (12 males, 5 females) volunteered to participate in this study with informed consent; they were examined over a period of 7 months. Study 1 included 7 smokers who were examined prior to and after 2 weeks of vitamin C supplementation (1 g/day). Study 2 included 10 smokers all of whom were examined prior to vitamin supplementation and then divided into 3 groups. Three smokers did not receive any vitamins, 3 were given 1.5 g vitamin C per day, and 4 were given 600 mg vitamin E per day for 4 weeks. The participants were asked to refrain from smoking for at least 40-48 h prior to blood collection. Blood was drawn into vacutainer tubes containing EDTA in the morning (0 time) and after 90 min. During the 90 min interval, they were asked to smoke 5-7 cigarettes. LDL was isolated by ultracentrifugation [9] at d = 1.019-1.063 g/ml from human plasma con-
taining 1 mg/ml EDTA. It was sterile filtered, aliquoted into plastic tubes and kept under N, at 4OC. Determination of thiobarbituric acid reactive substances was carried out immediately after isolation. Iodination of LDL was carried out by the iodine monochloride method [lo]. Tissue culture Bovine aortic smooth muscle cells (SMC) were prepared as described before and subcultured in Dulbecco-Vogt medium containing 10% fetal bovine serum [ll]. Peritoneal macrophages were obtained from mice 4 days after intraperitoneal injection of thioglycollate [12] and were cultured in 35-mm petri dishes with MEM containing 10% fetal bovine serum. Conditioning of LDL and rz51-LDL was carried out by incubation of the lipoproteins in Ham F10 medium without serum for 24 h, in the presence or absence of confluent cultures of SMC. At the end of conditioning the media were collected and centrifuged to remove cell debris. Media containing r2?-LDL were diluted with fresh FlO medium to give a final concentration of 33.3 pg LDL protein/ml and used for incubation with macrophages. For the determination of the effect of conditioning on oxidative modification unlabeled LDL, 50 pg protein/ml was incubated with or without SMC in FlO medium for 24 h. Thereafter, thiobarbituric acid reactive substances (TBARS) were determined. Metabolism of 12’I-LDL by macrophages On the day of the experiment, the medium was removed from the macrophages and the cells were washed twice by incubation for 15 mm at 37” C with 2 ml of FlO medium. Thereafter, 1 ml of conditioned or control medium (preincubated in the absence of cells) containing the labeled lipoproteins was added to the macrophages and incubation was carried out at 37O C in a CO, incubator for 4 h. Dishes without cells, containing conditioned or control medium, were incubated under the same conditions. At the end of the incubation, the medium was collected, the cells were washed, 0.05% trypsin was added and the cells were scraped with a rubber policeman. After inactivation of the trypsin with serum containing medium, the cells were pelleted by centrifugation
49 and the pellets were washed twice by recentrifugation with 4 ml buffered saline. The cell pellets were extracted with chloroform/methanol (1 : 1, v/v) to remove labeled lipids and protein radioactivity was determined. After precipitation of the medium with 50% trichloroacetic acid in the presence of carrier protein, non-iodide TCA-soluble radioactivity was determined [ll]. The non-iodide TCA-soluble radioactivity obtained in duplicate dishes without cells was subtracted from each experimental value. Chemical,
chromatographic
and radiochemical
prepared using a-tocopherol vitamin E and A, respectively.
and
P-carotene
for
Materials
Culture media and fetal bovine serum were obtained from Gibco (New York, NY). [‘251]Iodide was obtained from Amersham Int. (U.K.). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Vitamin E (DL-alphatocopherol acetate), 200 mg/tablet, was obtained from Teva (Israel). Vitamin C, 500 mg/tablet. was obtained from Rekah (Israel).
pro-
cedures
Peroxidation was measured by determining the amount of thiobarbituric acid reactive substances (TBARS) as modified by Lee [13]. Briefly, to 1 ml of plasma or 50 pg LDL protein in 1 ml, 0.5 ml 35% TCA was added and the solution was vortexed. Then, 1 ml 0.5% TBA was added and again vortexed. The solution was incubated for 90 min at 60” C in a shaking waterbath. After incubation, 1 ml 70% TCA and 2 ml chloroform were added, and the tubes were centrifuged for 20 min at 3000 t-pm. A standard curve was prepared using malondialdehyde and the intensity of fluorescence was determined at excitation of 515 nm and emission of 553 nm as employed by the method of Yagi [14]. The results were expressed as malondialdehyde equivalent content (nmol MDA/ml plasma or nmol MDA/mg LDL protein). Protein was determined according to Lowry et al. [15], and cholesterol and triacylglycerol were determined using a batch instrument (Vitatron FPS, Vital Scientific, Dieren, The Netherlands). Plasma high density lipoprotein cholesterol was determined by heparin-Mn2+ precipitation. Vitamin C was measured according to Denson and Bowers [16]. Vitamin E was measured by modification of the method of Hashin and Schuttringer [17]: to 1 ml plasma, 1 ml absolute ethanol was added, the solution was vortexed, 2 ml heptane was added, vortexed again, and the tubes were centrifuged for 5 min at 3000 rpm. To 1 ml supernatant, 0.2 ml 0.147% bathophenantroline was added, and vitamin A content was determined at 452 nm. Then, 0.1 ml 0.04% FeCl,, and 0.1 ml 6.23% H,PO, were added, and vitamin E was determined at 532 nm. A standard curve was
We examined the effect of acute smoking on plasma lipoproteins in 17 normal volunteers. In the first study, 7 subjects were examined prior to and after ingestion of vitamin C. In the second study, of the 10 subjects examined prior to administration of vitamins 3 received vitamin C, 4 vitamin E, 3 were not treated and were re-examined at the end of the experiment. As can be seen in Table 1, the 17 subjects were subdivided so that there were no differences in the initial plasma lipid values, BMI and smoking habits between the groups. Blood lipids were re-examined 4 weeks later and no change occurred in the subjects given vitamin C or E. The plasma levels of vitamin C, E and A are summarized in Table 2. In the first study in which 1 g/day of vitamin C was given for two weeks plasma levels of vitamin C doubled. The dose of vitamin C was increased to 1.5 g/day in the second study, and a slightly higher response was seen after 4 weeks. Administration of vitamin E resulted in an almost 3-fold increase in plasma vitamin E levels after 4 weeks of supplementation. No change occurred in plasma levels of vitamin A throughout the experimental period in all the 10 subjects examined. There was also no change in vitamin C or E plasma levels in control subjects (not given any vitamin) or those supplemented with vitamin E or C, respectively. Thiobarbituric acid reactive substances (TBARS) were determined on samples of plasma drawn at two time intervals; time 0 was after 40-48 h of total abstention from smoking. Thereafter, the subjects were allowed to smoke 557 cigarettes during 90 min and blood was drawn
50 TABLE
1
CHARACTERISTICS OF THE HUMAN SUBJECTS INVESTIGATED PRIOR TO TREATMENT WITH VITAMIN CORE Blood was drawn after overnight fast for lipid determination and other studies at the beginning of each study prior to administration of vitamins. The subjects were then divided into 3 groups which received either vitamin C (7 in study I, and 3 in study 2) or vitamin E (4 in study 2). or none (3 in study 2). Body mass index (BMI) = body weight (kg) divided by height (in m*); TG = triacylglycerol; HDL = high density lipoproteins. Values are means k S.E. Vitamin
No. of subjects Age (years) Sex: female male BMI Plasma cholesterol (mg/dl) Plasma TG (mg/dl) Plasma HDL cholesterol (mg/dl) Smoking habits: Packs (years) Cigarettes/day
C
Vitamin
E
No treatment
IO 28.8 4 6 23.2 + 2.4
4 31.0 1 3 24.6 f 3.1
3 30.3 0 3 22.2 rt 1.6
172.2 f 6.4
172.8 f 4.2
180.8 f 6.5
123.6*
114.2+
128.65
10.4
15.2
8.4
37.1*1.8
35.9 + 2.4
39.0 f 3.0
17.8+6.0 31.5 _+2.6
19.3 + 10.0 29.5 _+7.0
20.8 f 7.0 32.3 + 8.0
again (90 mm). In the first study, there was a significant rise in plasma TBARS after acute smoking in the subjects examined prior to vitamin C administration (Table 3). A slight, insignificant increase of TBARS occurred when the same subjects were re-examined after vitamin C administration. In the second study, plasma TBARS were
TABLE PLASMA
2 LEVELS
OF VITAMINS
C, E AND A BEFORE
Values are means + SE; n = number Study
1 2
determined at time 0 and after 90 min of smoking in 10 subjects (period I) and a significant increase was observed. Those subjects were re-examined after 4 weeks of either no treatment (None) or treatment with vitamin C or E. A similar increase in plasma TBARS between 0 and 90 min was seen in the non-treated subjects (period II). However, in those given vitamin C or E there was no increase in plasma TBARS at 90 min (Table 3). Essentially similar results were obtained when the TBARS were examined on LDL isolated from the subject’s plasma. Vitamin C and vitamin E ingestion prevented the rise in TBARS in LDL after acute smoking (Table 4). Next we examined the effect of conditioning of LDL in the presence of smooth muscle cells on TBARS formation. As expected, there was a very significant increase in the TBARS in conditioned LDL (Table 5). There was no difference between the 0 time values of all the subjects examined irrespective of the vitamin supplementation. However, comparison of the 90 rnin values shows that both vitamin C and vitamin E supplementation prevented the increase in TBARS in conditioned LDL after acute smoking. Similar results were obtained also when LDL was conditioned in the absence of cells (Table 5). Metabolism of the conditioned ‘251-LDL was examined in mouse peritoneal macrophages. In both experiments, essentially no differences were encountered in the 0 time value between the subjects of the different groups. Treatment with vitamin C or E affected only the 90 min values, i.e., at‘of contenuated the increase in metabolism ditioned ‘251-LDL caused by acute smoking (Table 6).
Vitamins given in period II
”
C None .E C
of subjects. Vitamin
(I) AND AFTER
For designation
C (mg/dl)
of study
(II) SUPPLEMENTATION
1 and 2, see Methods.
Vitamin
Vitamin
E (mg/dl)
A (pg/dl)
I
II
I
II
I
II
7
0.52 + 0.04
1.06+0.13
_
_
_
_
3 4 3
0.45 * 0.07 0.46 + 0.06 0.54 * 0.09
0.48 f 0.04 0.45 +0.05 1.26 + 0.06
0.61+ 0.01 0.61+ 0.01 0.59 f 0.01
0.64 f 0.04 1.7250.17 0.61& 0.01
5s+ 3.5 59 f 4.4 56 + 2.3
59+4.8 59 f 2.6 57 + 2.4
51 TABLE
3
TBARS
IN PLASMA
BEFORE
AND AFTER
SMOKING
IN SUBJECTS
TREATED
WITH
VITAMIN
C OR E
Blood was drawn from the volunteers 40-48 h after abstention from smoking, and 90 min after smoking 5 -7 cigarettes. Values are means + SE; n = number of subjects; each sample was examined in duplicate. In study 1, 7 smokers were examined before (period I) and after (period II) supplementation with 1 g vitamin C/day for 2 weeks. In study 2, 10 smokers were examined before treatment (period I). Thereafter (period II), 4 subjects were treated with 600 mg/day vitamin E and 3 with 1.5 g/day vitamin C for 4 weeks, 3 subjects were given no treatment and served as controls (none). TBARS were determined in plasma immediately after blood drawing. n
Study
7
None
7
Vitamin
10 3 3 4
None None Vitamin Vitamin
nmol MDA/ml
Period
Treatment
I
0.106 + 0.02
C
II
0.138+0.01
0.187 + 0.01 0.145 kO.01
c 0.01 “.\
C E
1 II II 11
0.141 f 0.003 0.106*0.014 0.107 f 0.021 0.119+0.013
0.217 * 0.02 0.197~0.009 0.118+0.020 0.123+0.018
< 0.005 c 0.01 n.s “..\
can be easily modulated by dietary means and are apparently not toxic even when given in large doses. Vitamin C levels were studied in smokers and compared to those of nonsmokers by several investigators [6,7] and were found to be lower. In a more recent study, it was reported that the lower plasma levels of vitamin C in smokers were independent of dietary intake [8]. Vitamin C was singled out by Frei et al. [18] in its effectiveness to protect lipids from peroxidative damage by aqueous oxidants. Thus, once ascorbate has been exhausted, lipid peroxidation occurred even though the other antioxidants, such as bilirubin, uric acid and vitamin E had not yet decreased markedly. Impaired serum antioxidant activity was indeed observed in smokers, but was related to an insufficient response of ceruloplasmin [21].
The two main findings of the present study were that in the 17 smokers who abstained from smoking for 48 h (a) a marked increase in plasma and LDL TBARS over the zero time value occurred after acute smoking for 90 min; (b) this increase could be prevented by dietary supplementation of either vitamin C or E. The 0 time and 90 min samples of plasma were processed together and thus the results appear even more significant as the 90 min sample was exposed for a shorter time interval after blood drawing to putative deleterious influences, such as air, light, etc. Of the multitude of natural antioxidants present in human plasma [18-201, we would like to focus on just two, vitamin C and E, since these compounds 4
TBARS IN NATIVE VITAMIN C OR E
LDL ISOLATED
FROM
Conditions as in Table 3. TBARS formation means + SE; n = number of subjects. Study
n
Treatment
PLASMA
BEFORE
was determined
Period
AND AFTER
immediately
SMOKING
after isolation
nmol MDA/mg
10 3 3 4
None None Vitamin Vitamin
C E
I II II II
0.584 0.683 0.627 0.705
+ f f +
0.026 0.02 0.03 0.04
IN SUBJECTS
TREATED
of the LDL from the plasma.
LDL protein
before smoking (0 time) 2
P
after smoking (90 min)
Discussion
TABLE
plasma
before smoking (0 time)
P
after smoking (90 min)
---
1.275+0.120 1.333kO.065 0.663 rt 0.05 0.733 _t 0.02
< 0.001 c 0.001 “.S. n.s.
WITH
Values
are
52 TABLE
5
TBARS IN LDL CONDITIONED VITAMIN C OR E
WITH
SMC,
BEFORE
AND
AFTER
SMOKING
IN
Conditions as in Tables 3 and 4. For conditioning, LDL 50 pg protein/ml medium was incubated smooth muscle cells. Values are means f SE; n = number of subjects. All data are from study 2. Treatment
n
Conditioned with cells None None Vitamin C Vitamin E
nmol MDA/mg
Period
SUBJECTS
TREATED
for 24 h in dishes with or without
LDL protein
P
before smoking (0 time)
after smoking (90 min)
10 3 3 4
I II 11 II
17.5+1.38 21.4k1.23 17.3kO.84 17.8+2.90
36.8k1.32 39.1* 1.03 18.9* 1.78 19.7 + 3.20
i 0.001 < 0.001 ns. n.s.
Conditioned without cells None 10 None 3
I II
1.66 + 0.21 2.31+ 0.40
Vitamin Vitamin
II II
1.48kO.23 2.17 + 0.37
3.98 k 0.66 5.42+ 1.30 1.59*0.30 2.53 k 0.30
i 0.005 < 0.05 n.s. n.s.
C E
3 4
In contradistinction to vitamin C, plasma levels of vitamin E in smokers were not found to be lower than those of nonsmokers [22,23]; in these studies, lower plasma levels of p-carotene were seen in smokers [22,23]. It is of interest to point out that while no difference in plasma vitamin E levels of smokers was seen as well by Pacht et al. [24], these authors reported a 7-fold decrease in vitamin E concentration in alveolar fluid obtained from smokers by bronchial lavage. Vitamin E sup-
TABLE EFFECT
WITH
plementation did raise the alveolar fluid vitamin E content, albeit not to normal levels, and the authors suggested that smoking may accelerate the oxidative metabolism of vitamin E at the alveolar surface [24]. In the plasma compartment, low density lipoproteins are an important potential target for lipid peroxidation. However, Esterbauer et al. [25] pointed out that while microsomes [26] and other cellular membranes undergo rapid autooxidation, this is not the case with LDL. The
6 OF VITAMIN
C OR E ON METABOLISM
OF CONDITIONED
LDL BY PERITONEAL
MACROPHAGES
by incubation in F10 medium with and without bovine aortic smooth muscle Conditions as in Tables 3-5. ‘*‘I-LDL was conditioned ‘251-LDL was then incubated for 4 h with mouse peritoneal macrophages or in the absence of cells. cells for 24 h. The conditioned ‘*‘I-degradation products in medium after ‘*‘I-LDL metabolized is the sum of uptake = cell associated ‘251-protein and non-iodide subtraction of no cell values. Values are means + SE; n = number of subjects. Study
1
2
n
Period
Treatment
7 7
None Vitamin
10 3 3 4
None None Vitamin Vitamin
‘*‘I-LDL
metabolized
(pg/mg
cell protein/4
before smoking (0 time)
after smoking (90 mitt)
h)
P
C
I II
2.15 f 0.29 1.99f0.02
3.90+0.60 2.28 rlO.17
i 0.05 n.s.
C E
I II II II
2.64 1.95 3.16 2.05
7.49 + 0.46 8.09 f 0.27 3.30+0.60 2.31 kO.27
< 0.001 < 0.001 n.s. n.s.
* f f +
0.49 0.014 0.63 0.20
53 relative resistance of LDL to peroxidation has been shown to be due to the presence of vitamin E in the particle, as under experimental conditions lipid peroxidation of LDL is initiated only after its store of vitamin E had been exhausted [25]. In the present study when LDL of smokers had been conditioned with aortic smooth muscle cells, the lipoproteins obtained after 90 min of acute smoking were metabolized more extensively by macrophages than their 0 time counterpart. This increase was also prevented by vitamin C or E supplementation, supporting the notion that the increase in TBARS reflected also changes in apo B, which were seen on gel electrophoresis in the form of faster migrating bands. Even though it is well established that lipid peroxidation of LDL results in a change in the apoprotein B, which in turn renders the particle recognizable by the scavenger receptor, it is not clear which of the many products of oxidation are responsible for this process [27]. How can we reconcile the effect of vitamin C supplementation in increasing the resistance of the isolated LDL particle to peroxidation and the well-known partition of the vitamin into the aqueous phase? Here we have to invoke the findings of Doba et al. [28] and of Niki et al. [29,30], who have shown that vitamin C protects or spares vitamin E content under various experimental conditions. Moreover, vitamin C may help to regenerate cy-tocopheryl radicals present in the LDL lipid, thus generating a lipid soluble chain-breaking antioxidant [20,30]. The above-mentioned authors [28-301 also proposed an interaction between vitamin C and E in which both vitamins cooperatively inhibit oxidation of lipid. We would like to suggest that even though the plasma vitamin E levels in smokers supplemented with vitamin C did not increase, ‘sparing’ of vitamin E in the LDL particle might have occurred and contributed towards its lesser susceptibility to acute smoking. Such behavior could be envisaged if a redistribution of vitamin E occurred from HDL to LDL, as the latter is known to transport 39%58% [31] or 59% [32] of plasma vitamin E in males and 42% in females [32]. We plan to determine the vitamin E content of LDL particles in smokers exposed to acute smoking under different regimes of dietary antioxidant supplementation.
The findings of our studies fit well with the concepts enunciated by Gey, who showed an impressive inverse correlation of vitamin C and E levels and the mortality from ischemic heart disease (IHD) in various countries in Europe [33 -351. As smoking is one of the major risk factors for IHD, high levels of vitamin E may protect lipoproteins from peroxidation and thereby prevent their recognition by the scavenger receptor and thus render them less atherogenic. References 1 Yokode, M.. Kita, T., Arai. H.. Kawai, C.. Narumiya, S. and Fujiwara, M., Cholesterol ester accumulation in macrophages incubated with low density lipoprotein pretreated with cigarette smoke extract. Proc. Natl. Acad. Sci. LISA, 85 (1988) 2344. 2 Nadiger, H.A., Mathew, C.A. and Sadasivudu. B., Serum malondialdehyde (TBA reactive substance) levels in cigarette smokers, Atherosclerosis. 64 (1987)71. 3 Harats, D., Ben-Naim, M.. Dabach. Y.. Hollander, G., Stein. 0. and Stein, Y., Cigarette smoking renders LDL susceptible to peroxidative modification and enhanced metabolism by macrophages. Atherosclerosis, 79 (1989) 245. 4 Parthasarathy, S.. Prim, D.J., Boyd. D.. Joy. I. and Steinberg, D., Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor, Arteriosclerosis, 6 (1986) 505. 5 Steinbrecher, U.P., Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J. Biol. Chem.. 262 (1987) 3603. 6 Brook, M. and Grimshaw, J.J.. Vitamin C concentration of plasma and leukocytes as related to smoking habit, age. and sex of humans. Am. J. Clin. Nutr., 21 (1968) 1254. 7 Chow, C.K., Changchit, C., Bridges. R.B., Rehn. S.R.. Humble, J. and Turbek. J., Lower levels of vitamin C and carotenes in plasma of cigarette smokers. J. Am. (‘011. Nutr., 5 (1986) 305. 8 Schectman, G., Byrd, J.C. and Gruchow, H.W.. The influence of smoking on vitamin C status in adults, Am. J. Pub. Hlth.. 79 (1989) 158. 9 Havel, R.J.. Eder. H.A. and Bragdon. J.H.. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. .I. Clin. Invest.. 34 (1955) 1345. IO Bilheimer. D.W.. Eisenberg, S. and Levy, R.I.. The metabolism of very low density lipoprotein proteins. I. Preliminary in vitro and in vivo observations. B&him. Biophys. Acta. 260 (1972) 212. 11 Bierman, E.L.. Stem. 0. and Stein. Y.. Lipoprotein uptake and metabolism by rat aortic smooth muscle cells in tissue culture, Circ. Res.. 35 (1974) 136. 12 Friedman, G.. Chajek-Shaul, T.. Gallily, R., Stein, O., Shiloni. E., Etienne. J. and Stein. Y.. Modulation of lipo-
54
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protein lipase activity in mouse peritoneal macrophages by recombinant human tumor necrosis factor, Biochim. Biophys. Acta, 963 (1988) 201. Lee, D.M., Malondialdehyde formation in stored plasma, B&hem. Biophys. Res. Commun., 95 (1980) 1663. Yagi, K., A simple fluorimetric assay for lipoperoxide in blood plasma, Biochem. Med., 15 (1976) 212. Lowry, O.H., Rosebrough, N.J., Fat-r, A.L. and Randall, R.J., Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. Denson, K.W. and Bowers, E.F., The determination of ascorbic acid in white blood cells. A comparison of WBC ascorbic acid and phenolic acid excretion in elderly patients, Clin. Science, 21 (1961) 157. Hashim, S.A. and Schuttringer, G.R., Rapid determination of tocopherol in macro- and microquantities of plasma, Am. J. Clin. Nutr., 19 (1966) 137. Frei, B., Stocker, R. and Ames, B.N., Antioxidant defenses and lipid peroxidation in human blood plasma, Proc. Natl. Acad. Sci. USA, 85 (1988) 9748. Esterbauer, H., Striegl, G., PubI, H. and Rotheneder, M., Continuous monitoring of in vitro oxidation of human low density lipoprotein, Free Radicals Res. Commun., 6 (1989) 67. Halliwell, B., How to characterize a biological antioxidant, Free Radicals Res. Commun., 9 (1990) 1. Galdston, M., Levytska, V., Schwartz, M.S. and Magnusson, B., Ceruloplasmin increased serum concentration and impaired antioxidant activity in cigarette smokers, and ability to prevent suppression of elastase inhibitory capacity of alpha,-proteinaise inhibitor’-3, Am. Rev. Respir. Dis., 129 (1984) 258. Comstock, G.W., Menkes, M.S., Schober, S.E., Vuilleumier, J.-P. and Helsing, K.J., Serum levels of retinol, beta-carotene, and alpha-tocopherol in older adults, Am. J. Epidemiol., 127 (1988) 114. Stryker, W.S., Kaplan, L.A., Stein, E.A., Stampfer, M.J., Sober, A. and Willett, W.C., The relation of diet, cigarette smoking, and alcohol consumption to plasma beta-carotene and alpha-tocopherol levels, Am. J. Epidemiol., 127 (1988)
283. 24 Pacht, E.R., Kaseki, H., Mohammed, J.R., Comwell, D.G. and Davis, W.B., Deficiency of vitamin E in the alveolar
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34 35
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