From the Department of Medicine, University of Helsinki, Finland.
Correspondence to Timo Kuusi, MD, PhD, Department of Medicine, Helsinki University Hospital, Haartmaninkatu 4, 00290 Helsinki 29, Finland.
Methods and Results—Blood samples were collected during 2
ordinary working days from healthy, nonsmoking subjects (n=10) before
and after (up to 5.5 hours) spending half an hour in a smoke-free area
(day 1) or in a room for smokers (day 2). Passive smoking caused an
acute decrease (1.5 hours after exposure) in serum ascorbic acid
(P<.001) and in serum antioxidant defense
(P<.001), a decreased capacity of LDL to resist
oxidation (P<.01), and the appearance of increased
amounts of lipid peroxidation end products in serum
(P<.01). Finally, LDL isolated from subjects after
passive smoking was taken up by cultured macrophages at an
increased rate (P<.05).
Conclusions—Exposure of nonsmoking subjects to secondhand smoke
breaks down the serum antioxidant defense, leading to accelerated lipid
peroxidation, LDL modification, and accumulation of LDL
cholesterol in human macrophages. These data
provide the pathophysiological background for the
recent epidemiological evidence about the increased CHD risk among
passive smokers.
SHS is known to contain numerous oxidants and pro-oxidants that
are capable of producing free radicals and possibly initiating lipid
peroxidation.10 The free radicals entering the
body are first trapped by serum aqueous and lipophilic antioxidants,
which interact and provide greater protection against lipid
peroxidation than any antioxidant on its own. After a failure of this
antioxidant barrier, LDL lipid peroxidation can take place. LDL
oxidation, followed by LDL cholesterol accumulation in
macrophages, is generally accepted as a key event of
atherosclerosis.11 Therefore,
this could be one factor leading to the high incidence of CHD in
smokers, who also have decreased plasma levels of certain
antioxidants.12 However, only fractional and, to
some extent, controversial evidence concerning the connection between
CS and lipid peroxidation has been reported so far from studies
conducted with active smokers or under in vitro
conditions.13 14 15
To understand the pathogenesis underlining the connection between CHD
and passive smoking, we assessed the acute effect of passive smoking on
LDL metabolism. We started with the effect of SHS on the
antioxidant barrier protecting LDL and then continued to test the
effect of passive smoking on lipid peroxidation and finally on the
accumulation of LDL cholesterol in cultured human
macrophages.
Blood samples were collected at time 0 and 1.5 and 6 hours after the
beginning of the exposure into tubes kept on ice. Serum or plasma
containing 1 mg/mL ethylene diamine tetra-acetate
(Na2EDTA 1 mg/mL) was separated by
centrifugation at +4°C. Unless used immediately,
samples were stored at -80°C and used within a 2-month period. Serum
cholesterol, triglycerides, HDL
cholesterol, and uric acid were measured in a Cobas Mira-S
Centrifugal Analyzer (Roche Inc) with commercially available
reagents of Roche (catalogue No. 0736643, 0736805, 0720674, and
0736813, respectively). Serum lipid-soluble antioxidants (available
from seven subjects), aqueous antioxidants, and the combined capacity
of all serum antioxidants to resist artificially induced peroxidation,
ie, the TRAP, were determined as described
recently.16
LDL was isolated by rate-zone ultracentrifugation in a
density gradient. Before oxidation, EDTA was separated from LDL by use
of small dextran-sulfate affinity columns (Liposorber LA-15, Kaneka
Co). Lipid peroxidation was initiated by adding freshly prepared
CuSO4 solution to a final concentration of
10.4 µmol/L, and the formation of conjugated dienes was
monitored at a wavelength of 234 nm with standard techniques using a
computerized system.16 The resistance of LDL to
oxidation was derived from the length of the lag time (minutes) before
the propagation of the reaction. The TBARS in serum and LDL were
determined as described previously.17
Human monocytes (>95% pure) were obtained from healthy volunteers and
isolated in a discontinuous gradient at 1.065 g/mL by
centrifugation for 20 minutes at
2000g.18 Before this, the white cell
pellet was preconditioned according to Recalde19
at 320 mOsm/L. On average, 45 µg of cellular protein was added per
culture well, and the cells were cultured for 5 days, after which the
monocyte layers were washed and the culture medium changed to contain
300 µg/mL of LDL. Lipid accumulation in the monocyte
macrophages was determined by measuring the incorporation
of (1-14C)-oleate (52 mCi/mmol) into the cellular
cholesteryl oleate (ACAT assay) as described by Basu et
al.20 LDL isolated after passive smoking and
after the control period from each subject were studied in the same
cell culture plate simultaneously. The amount of cellular
protein was not influenced significantly by any type of LDL used in
this study.
Statistical analysis was done by use of the Systat statistical
package. Significances are given as an overall difference between the
values obtained after the subjects spent half an hour in a smoke-free
area or in a smoking room and were tested by ANOVA for repeated
measurements. The values are given as mean±SE.
The total serum antioxidant defense can be quantified by determining
the TRAP, which can be done either experimentally from the serum
samples or by calculating the sum capacity of all major antioxidants to
trap free radicals (Table 1
Passive smoking did not affect serum lipid concentrations, but
simultaneous with the deterioration of the antioxidant
defense, a significant (19%, P<.01) decrease in the
resistance of LDL to Cu2+-initiated oxidation was
found (Table 2
In the final step of the lipid oxidation hypothesis, the end
products of lipid peroxidation modify apolipoprotein B in LDL. This
leads to a more rapid and unregulated uptake of LDL
cholesterol by macrophages and to foam cell
formation. To study the influence of passive smoking on LDL
cholesterol uptake by macrophages, we isolated LDL
from the subjects before and after they spent half an hour in a
smoke-free area (day 1) or in a smokers' room (day 2). The
macrophages were incubated with 300 µg/mL of freshly isolated
LDL without further modification. Incubation of cells with LDL
separated 1.5 to 6 hours from the beginning of passive smoking induced
a 1.6- to 2.3-times-higher synthesis of cholesteryl oleate in
macrophages than LDL separated before the exposure
(P<.05; Fig 2
Oxidative stress induced by acute passive smoking significantly
decreased plasma ascorbic acid and caused a minor, nonsignificant
decrease in protein sulfhydryl groups, reflecting increased use of
these antioxidants. This is in line with previous studies demonstrating
that long-term exposure to SHS decreases plasma ascorbic acid in both
active and passive smokers.7 12 Furthermore, in
vitro data exposing human plasma to CS suggest that ascorbic acid is
the first antioxidant to be consumed.14 However,
adequate quantification of serum antioxidant defense can be obtained
only by measurement of the combined capacity of all antioxidants to
resist peroxidation, the TRAP.16 A one-third
reduction in the experimental TRAP values was evident after passive
smoking, but the values returned to close to normal at the end of the
follow-up. These changes were much larger than could be calculated from
changes in the serum levels of individual antioxidants. These findings
indicate changes in the cooperation between antioxidants, best known
between vitamin E and ascorbate, and contribution of some unknown
antioxidants.16 Part of this discrepancy can also
be explained by serum bilirubin, carotenoids, retinols, and serum
antioxidant enzymes, which are not included in the calculations,
because their contribution to TRAP and their action as chain-breaking
antioxidants have been suggested to be
negligible.16 Thus, in accordance with previous
reports, throughout this study, all the TRAP values measured directly
from the serum samples were up to 50% higher than those calculated as
the sum of the capacity of major antioxidants to trap free radicals
(Table 1
Simultaneous with the failure in the antioxidant defense, a
reduction in the resistance of isolated LDL to
Cu2+-stimulated oxidation after passive smoking
could be demonstrated. Previously, the reduction in the resistance of
isolated LDL to oxidation has been shown to correlate with the severity
of coronary
atherosclerosis.21 Because LDL is
separated from its aqueous surroundings, the lag period reflects the
lipophilic antioxidants in LDL, such as
The amount of conjugated dienes in freshly isolated LDL was 10% higher
after passive smoking than before the smoke exposure and 20% higher
than after the control period (P<.001, data not shown), but
no lipid peroxidation end products (TBARS) were detectable in LDL.
TBARS may have been removed as water-soluble aldehydes from LDL during
isolation, because they were significantly higher in serum after
passive smoking than after spending the same time in normal air. When
the oxidation process of LDL is continued by adding
Cu2+, TBARS are produced at an accelerated rate
in CS-modified LDL compared with control LDL. This indicates that the
peroxidation process has already been started during SHS exposure.
Thus, CS-modified LDL may resemble the minimally modified LDL and be
more susceptible to further peroxidation by Cu2+.
The minimally modified LDL has been shown to induce platelet
aggregation, to cause retraction of vascular smooth muscle cells, to be
cytotoxic to endothelial cells, and to cause induction
of macrophage chemotactic
cytokines.25 Notably, many of the above
effects are caused by passive smoking.3 4 5 8
Definitive evidence about the atherogenic nature of CS-modified LDL is
its interaction with cultured human macrophages, which store
cholesterol and develop to foam cells. The cholesteryl
esterification reaction reflects the amount of cholesterol
stored in these cells.20 Incubation of cells with
LDL, separated 1.5 to 6 hours after passive smoking without further
modifications, induced 1.6- to 2.3-times-higher synthesis of
cholesteryl oleate in macrophages than LDL separated before the
exposure, respectively. The CS-modified LDL may also stimulate the
macrophages to release free radicals, further modifying these
lipoproteins.13 Whatever the mechanism, the
results demonstrate that even after a short period of passive smoking,
increasing amounts of LDL cholesterol are taken up by human
macrophages. In accordance, 2 to 4 hours of exposure of rats to
environmental tobacco smoke has recently been shown to increase LDL
accumulation in perfused arteries.26
An increasing number of reports has documented the harmful effects of
environmental tobacco smoke.27 28 29 We found that
a short period of passive smoking changed LDL metabolism,
favoring the progression of atherosclerosis. The
present results demonstrate one mechanism by which SHS could
increase the risk of CHD, already shown in epidemiological and clinical
studies.
Received September 23, 1997;
revision received December 18, 1997;
accepted January 14, 1998.
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
Passive Smoking Induces Atherogenic Changes in Low-Density Lipoprotein
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—According to the American
Heart Association, passive smoking is an important risk factor for
coronary heart disease (CHD), but the mechanisms underlying
this association are not fully understood. We studied the acute effect
of passive smoking on the factors that influence the development of
CHD: the antioxidant defense of human serum, the extent of lipid
peroxidation, and the accumulation of LDL cholesterol in
cultured human macrophages, the precursors of foam cells in
atherosclerotic lesions.
Key Words: lipoproteins • coronary disease • smoking • atherosclerosis
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Clinical
and epidemiological studies have shown that involuntary exposure to
environmental tobacco smoke is an important cause of heart disease and
death, but only a few studies have provided
pathophysiological evidence underlying this
relationship.1 It has been suggested that acute
exposure to passive smoking deteriorates oxygen delivery and use in the
myocardium,2 causes mild
coronary vasoconstriction,3 increases
platelet activity in nonsmokers,4 and damages
the endothelium, indicated by the appearance of
anuclear endothelial cell carcasses in the
blood.5 Long-term exposure to passive smoking
reduces the serum level of ascorbic acid,6
impairs the arterial endothelial function
probably through impaired endothelial nitric oxide
activity,7 and increases the thickness of the
carotid wall.8 Furthermore, accelerated
atherosclerotic plaque development has been found in animals subjected
to CS.9
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Subjects
Blood samples were taken from 10 (5 female and 5 male)
nonsmoking, normolipidemic subjects who ranged from 23 to 39 years of
age, were taking no known medication, and were without evidence of
disease. All subjects lived in smoke-free homes, worked in smoke-free
environments, and were advised to avoid environmental smoke during
their free time for at least 48 hours before entering the study. The
samples were collected during 2 ordinary working days before (time 0)
and during 6 hours of follow-up from the beginning of the exposure. The
subjects spent half an hour in their normal office rooms, thus in a
smoke-free area (day 1) or in a room used specifically by active
smokers (day 2). Subjects had their normal breakfasts before the study
and lunches during the study. They were exposed to smoke for 30 minutes
in the 88-m3 ventilated room (ventilation rate,
600 s-1), where 16 cigarettes were consumed
during the exposure by active smokers. After passive smoking, the
subjects continued their normal working days in smoke-free
environments. All participants reported symptoms such as headache,
nausea, or palpitation after passive smoking. The study was approved by
the local ethics committee.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Passive smoking resulted in a major loss of serum antioxidant
defense in all the nonsmoking subjects. A one-third decrease in serum
ascorbic acid took place (P<.001), starting 1.5 hours from
the beginning of the exposure to smoke and lasting until the end of the
follow-up, ie, a total of 6 hours (Table 1
). Furthermore, protein sulfhydryl
groups (SH groups) decreased gradually for 6 hours by 26%
(P<.063) (Table 1). The smoke exposure did not affect serum
lipid-soluble antioxidants (vitamin E, retinol, and ß-carotene) and
uric acid (Table 1). No significant changes took place in any of these
parameters after the control period in a smoke-free
area.
View this table:
[in a new window]
Table 1. Serum Antioxidants and TRAP (Mean±SE) in Healthy
Subjects (n=10) Before and After Passive Smoking 
). Passive smoking caused a significant
(31%, P<.01; Fig 1) and a
minor (10%, nonsignificant) decrease 1.5 hours after the beginning of
the exposure in experimental and calculated TRAP values, respectively.
Notably, the mean values of both experimental and calculated TRAP
returned to close to baseline at the end of the follow-up in all
subjects. At baseline, the calculated TRAP values in these subjects
ranged from 474 to 929 µmol/L, which were 50% lower than the
measured TRAP values (931 to 1680 µmol/L) (Table 1). The
baseline TRAP values did not differ significantly between the 2 days of
the study (Table 1), and no significant change was found after the
control period in a smoke-free area (day 1) (Fig 1).
View larger version (30K):
[in a new window]
Figure 1. Effect of passive smoking on serum antioxidant
defense (measured TRAP) and on serum lipid peroxidation end
products (TBARS). Individual changes were noted after the subjects
spent half an hour in a smoke-free area or in a smoking room. Exposure
period was 30 minutes ().
] and Fig 2). This was first observed 1.5 hours
after the beginning of passive smoking, and the lag times remained
still 11% shorter than at baseline at the end of the follow-up.
Furthermore, the initial amount of conjugated dienes in freshly
isolated LDL, without further modifications, was significantly higher
after passive smoking than before the exposure to smoke or after the
control period (P<.001). Also, the end products of
lipid peroxidation in serum increased after passive smoking, indicated
by a significant increase in the small aldehyde substances, TBARS, that
are released from polyunsaturated fatty acids during their oxidation
(P<.01, Fig 1 and Table 2). The TBARS were not detectable
in LDL with no further modification but were 1.5 times higher in
Cu2+-oxidized LDL 1.5 to 6 hours after the
beginning of passive smoking than after exposure to normal air
(P<.05). No significant changes took place in these
parameters after the control period in a smoke-free
area.
View this table:
[in a new window]
Table 2. Indexes of Lipid Peroxidation and
Cholesterol Esterification Rate (ACAT Activity) in Cultured
Human Macrophages Before and After Passive Smoking
View larger version (28K):
[in a new window]
Figure 2. Effect of passive smoking on the capacity of LDL
to resist peroxidation (lag time) and on the accumulation of LDL
cholesterol in cultured human macrophages (ACAT
activity). Individual changes were noted after subjects spent half an
hour in a smoke-free area or in a smoking room. Exposure period was 30
minutes (). ). No significant difference in the cellular
cholesteryl oleate synthesis was observed at baseline between the 2
days of the study or after the control period in a smoke-free area
(Table 2).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Acute exposure of a nonsmoking subject to passive smoking resulted
in deterioration of serum antioxidant defense, accelerated lipid
peroxidation, and accumulation of LDL cholesterol in
cultured human macrophages. These events were observed even
after a very short period (30 minutes) of passive smoking. Indeed, it
has previously been suggested that the cardiovascular
system is extremely sensitive to the chemicals in environmental tobacco
smoke.1 Furthermore, the oxidative stress induced
by SHS may have a more prominent effect on a nonsmoking subject than on
an active smoker whose cardiovascular system has
adapted to CS.1 ). 
-tocopherol. In
vivo LDL will also interact with serum water-soluble antioxidants such
as ascorbic acid, which protects LDL from lipid peroxidation by
regenerating vitamin E from tocopheryl
radicals.22 Thus, the decrease in serum ascorbic
acid after passive smoking may increase the amount of tocopheryl
radicals, which can act as reducing agents and possibly accelerate
metal-ion–dependent oxidative damage.23
Furthermore, the low levels of ascorbic acid in
subendothelial spaces could make LDL more susceptible
to oxidative modification at this site in vivo.24
We have previously shown that the combined antioxidant capacity and the
resistance of LDL to oxidation can be increased by dietary
supplementation in healthy subjects with vitamin
E.16 The present results indicate that
impairment of vitamin E regeneration by the loss of ascorbic acid
during SHS-induced oxidative stress seems to decrease them both.
Selected Abbreviations and Acronyms
CHD
=
coronary heart disease
CS
=
cigarette smoke
SHS
=
secondhand smoke
TBARS
=
thiobarbituric acid reactive substances
TRAP
=
total peroxyl radical trapping potential of serum
Acknowledgments
This study was supported by Finnish Heart Research Foundation
and Aarne Koskelo Foundation.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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