Clinical Science (2001) 100, 459–465 (Printed in Great Britain)
Effects of smoking and abstention from
smoking on fibrinogen synthesis in humans
Kirsty A. HUNTER*, Peter J. GARLICK†, Iain BROOM‡, Susan E. ANDERSON§
and Margaret A. McNURLAN†
*Department of Land-based Studies, Nottingham Trent University, Southwell, Nottinghamshire NG25 OQF, U.K., †Department of
Surgery, Health Science Center T19, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A., ‡NHS Trust,
Aberdeen Royal Infirmary, Foresterhill, Aberdeen AB25 2ZN, Scotland, U.K., and §The Rowett Research Institute,
Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, U.K.
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Cigarette smoking and hyperfibrinogenaemia are both significant risk factors for the development of cardiovascular disease. Two studies are described here which aimed to establish
the metabolic mechanism responsible for the raised plasma fibrinogen concentration observed
in smokers. Chronic smokers had a significantly elevated absolute rate of fibrinogen synthesis
(ASR) compared with non-smokers (22.7p1.3 mg/kg per day versus 16.0p1.3 mg/kg per day ;
meanspS.E.M., P 0.01), with plasma levels of fibrinogen significantly correlated with
fibrinogen synthesis (r l 0.65, P l 0.04). Unlike fibrinogen, plasma albumin concentrations were
lower in smokers than in non-smokers (45p0.4 versus 47p0.7 g/l, P 0.05), but there was no
difference in rates of albumin synthesis between the two groups. Two weeks cessation from
smoking by previously chronic smokers was associated with a rapid and marked fall in plasma
fibrinogen concentration (from 3.06p0.11 g/l to 2.49p0.14 g/l, P 0.001), and a significant
reduction in ASR (a 33 % reduction, from 24.1p1.7 to 16.1p1.0 mg/kg per day, P 0.001). These
studies suggest a primary role for increased synthesis in producing the hyperfibrinogenaemia
associated with smoking. Moreover, abstention from smoking for a period of only 2 weeks
induces a significant decrease in the rate of fibrinogen synthesis by the liver, with a concomitant
reduction in the plasma fibrinogen concentration.
INTRODUCTION
The coagulation protein fibrinogen has emerged as an
important contributor in the development of coronary,
peripheral and cerebral vascular disease due to its
involvement in both atherogenesis and thrombosis [1,2].
Large-scale epidemiological studies have consistently
demonstrated that an increased plasma fibrinogen concentration is an independent risk factor for a future
cardiovascular event [3,4]. The Northwick Park Heart
Study [5], for example, reported that an elevation in
fibrinogen concentration of one S.D. (approx. 0.6 g\l)
was associated with an 84 % increase in the risk of
developing coronary heart disease in the next 5 years.
More limited data suggest that it can also predict future
mortality in survivors of myocardial infarction [6] and
stroke [7]. Despite these observations, the metabolic
mechanism(s) responsible for producing the hyperfibrinogenaemia associated with an increased risk of
cardiovascular complications is yet to be established. The
concentration of fibrinogen in the intravascular compartment is the result of a dynamic balance between
fibrinogen synthesis, catabolism and the intra- and extravascular distribution. Of these processes, it is generally
thought that the rate of fibrinogen synthesis and output
by the liver has the greatest regulatory influence on the
plasma fibrinogen level.
A group of the population which consistently exhibits
Key words: albumin synthesis, cardiovascular disease, cigarette smoking, fibrinogen synthesis, stable isotopes.
Abbreviations: ASR, absolute rate of fibrinogen synthesis; BMI, body mass index ; FSR, fractional rate of fibrinogen synthesis.
Correspondence: Dr Margaret McNurlan (e-mail mcnurlan!surg.som.sunysb.edu).
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an increased incidence of vascular problems is cigarette
smokers. Cigarette smoking has been estimated to double
fatalities from coronary heart disease [8], and is also an
independent risk factor for stroke [9] and peripheral
vascular disease [10]. A raised plasma fibrinogen concentration is one of the most common aberrations of the
haemostatic system found in smokers [5,11], and the
clinical significance of this observation has been highlighted by several investigators [12,13]. Kannel et al. [13],
for example, used data from the Framingham Study to
estimate that 50 % of the smoking associated with
ischaemic heart disease may be mediated through the
deleterious effects of fibrinogen.
Although considerable attention has been focused on
the relationship between smoking and fibrinogen, the
mechanism by which smoking increases the plasma
fibrinogen concentration has not been elucidated. In the
present paper, we describe two studies which use stable
isotope methodology to perform in vivo investigations
on the influence of cigarette smoking on fibrinogen
synthesis.
In the first study, rates of fibrinogen synthesis were
compared in groups of smokers and non-smokers,
whereas, in the second study, a group of chronic smokers
abstained from smoking for 14 days to determine if
short-term cessation affected fibrinogen synthesis. In
addition, it was possible to investigate the effect of
smoking on the synthesis of albumin, another plasma
constituent whose intravascular concentration is altered
in smokers.
METHODS
Study protocols were approved by the Joint Ethical
Committee of Grampian Health Board and the University of Aberdeen, Scotland, U.K. and participants gave
written informed consent before taking part. Participants
were recruited following appeals on local radio and in the
press. All participants were healthy and medication-free.
Exclusion criteria included diabetes, overt liver, kidney
or thyroid dysfunction, infection, routine consumption
of aspirin, lipid-lowering or fibrinolytic drugs, a
history of vascular disorders, obesity, hypertension and
hyperlipidaemia. Since an acute-phase response alters
fibrinogen metabolism [14], participants with plasma
concentrations of C-reactive protein 10 mg\l were
excluded. All measurements were performed after
participants had fasted for 12 h.
For Study 1, eight male smokers [mean age, 36.6p4
years ; body mass index (BMI), 24.4p1.2 kg\m# ; and
20 cigarettes smoked\day for at least 5 years] were
individually matched with eight male non-smokers (mean
age, 37.0p4 years ; BMI, 24.3p1.4 kg\m#). Matching for
sex, age and BMI was necessary since these variables are
thought to influence plasma fibrinogen concentration
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2001 The Biochemical Society and the Medical Research Society
[15,16] and might, therefore, affect fibrinogen synthesis.
To standardize smoking habits, smokers were asked to
refrain from smoking for 1 h before measurements were
made.
For Study 2, 11 male chronic smokers (mean age,
50.4p3 years ; BMI, 23.7p3.1 kg\m# ; and 20
cigarettes smoked\day for at least 5 years) were recruited.
Measurements were performed before and immediately
after a 2 week period of complete abstention from
smoking. For the first measurement, participants were
asked to refrain from smoking for 9 h. Compliance to the
non-smoking regimen was monitored by measuring the
urinary concentration of cotinine (a metabolite of nicotine) on two occasions during smoking abstinence. To
remove sources of nicotine other than cigarette tobacco,
which would also raise urinary levels of cotinine,
participants were asked to refrain from using products
containing nicotine such as patches or chewing gum.
Urinary cotinine levels were compared with a reference
range obtained from a group of non-smokers.
Participants whose urinary cotinine level exceeded the
maximum value of this range (55 µg\ml) were excluded
from the study. To assist participants during the abstention period, they attended a smoking cessation
support group run by a trained counsellor.
Measurement of the rate of fibrinogen
synthesis
Measurement of the rate of fibrinogen synthesis was
carried out according to the method of Ballmer et al.
[17], following injection of 43 mg of L-[#H ]phenyl&
alanine\kg body weight, enriched to 5–10 atoms %
excess (MassTrace, Woburn, MA, U.S.A. and Ajinomoto,
Tokyo, Japan). Blood samples were taken over 90 min to
determine the isotopic enrichment of the plasma free
amino acid and newly synthesized protein.
The enrichment of phenylalanine incorporated into
fibrinogen was determined from blood samples taken at
0, 30, 50, 70 and 90 min after infusion of isotope.
Fibrinogen was isolated from plasma by repeated
ammonium sulphate precipitation followed by
solubilization in sodium citrate [18]. Purity of isolated
fibrinogen was confirmed by SDS\PAGE. Amino acids
were liberated by acid hydrolysis and, after enzymic
decarboxylation of phenylalanine to β-phenylethylamine
followed by conversion into the heptafluorobutyryl derivative, isotopic enrichment was measured by GC–MS,
using a VG 12-253 quadrupole mass spectrometer
coupled to a Hewlett Packard 5890 gas chromatograph
under electron-impact ionization. Selective-ion recording
conditions were employed, and the ions at m\z 106 and
109, corresponding to the Mj2 and Mj5 ions respectively, were monitored [19,20].
Plasma free phenylalanine enrichment was determined
as described previously [20], following ion-exchange
Fibrinogen synthesis and smoking
chromatography and derivatization to the tertiary butyldimethylsilyl derivative. Measurement was made by
GC–MS, with monitoring of ions at m\z 336 and 341.
The rate of fibrinogen synthesis was expressed as both
the fractional and absolute rates. The fractional rate
(FSR), i.e. the percentage of the intravascular fibrinogen
pool synthesized per day, was calculated as the increase
in [#H ]phenylalanine enrichment in fibrinogen divided
&
by the area under the curve of the plasma free phenylalanine enrichment, multiplied by 100. The precursor
time-curve was adjusted for the secretion time, which
represents the period taken for the synthesis of fibrinogen
and its subsequent secretion into the blood. The absolute
rate of fibrinogen synthesis (ASR) was calculated as the
product of the FSR and the intravascular fibrinogen mass,
and expressed as mg of fibrinogen synthesized\kg of
body weight\day. The intravascular fibrinogen mass was
estimated as the measured plasma fibrinogen concentration multiplied by the plasma volume which was
estimated by nomogram [21].
Measurement of the rate of albumin
synthesis
Albumin synthesis was measured according to the
method of Hunter et al. [22]. Albumin was isolated from
serum by differential solubility in ethanol in the presence
of trichloroacetic acid [23]. Purity of isolated albumin
was verified by SDS\PAGE. Phenylalanine enrichment
in albumin, plasma free phenylalanine and the calculation
of albumin FSR and ASR were performed as described
for fibrinogen.
Analytical methods and statistics
Plasma fibrinogen and serum C-reactive protein concentrations were determined immunologically by
automated-laser rate nephelometry (Behring Laser
Nephelometer). Biochemical profiles were determined
using routine techniques in the Department of Clinical
Biochemistry, Aberdeen Royal Infirmary, Scotland, U.K.
Urinary cotinine was determined by GC, according to
the method of Beckett and Triggs [24]. Serum albumin
concentrations were measured by an automated version
of the Bromocresol Green method [25].
Unless stated otherwise, data are expressed as
meanspS.E.M. and were compared using Student’s t test
for unpaired (Study 1) or paired (Study 2) data.
Differences were considered significant if P 0.05.
RESULTS
Figure 1 FSR (%/day ; upper panel) and ASR (mg/kg per day ;
lower panel) in smokers and non-smokers from Study 1
ference failed to reach statistical significance. In Study 1,
the rate of fibrinogen synthesis expressed as the percentage of the plasma fibrinogen pool was higher in the
smokers (17.1p1.5 %\day) than in the non-smokers
(14.0p1.3 %\day ; Figure 1, upper panel), but this was
also not statistically significant. However, the absolute
rate of fibrinogen synthesis, i.e. the amount of fibrinogen
(in mg) synthesized by the liver per day was significantly
greater in smokers compared with non-smokers
(21.5p1.9 mg\kg per day compared with 16.0p1.4 mg\
kg per day in the non-smokers, P 0.05 ; Figure 1, lower
panel). Smokers also had significantly lower plasma
albumin concentrations (45p0.4 g\l) compared with
non-smokers (47p0.7 g\l, P 0.05). There was no difference, however, in albumin FSR (7.1p0.5 %\day in
smokers versus 6.9p0.6 %\day in non-smokers) or ASR
(143p9 mg\kg per day in smokers versus 146p13 mg\
kg per day in non-smokers).
Study 1
Smokers had a plasma fibrinogen concentration which
was on average 10 % higher than the non-smokers
(2.86p0.20 g\l versus 2.58p0.20 g\l), although this dif-
Study 2
Eight of the 11 participants, mean age 46p2 years, BMI
23.3p1.2 kg\m#, who had previously smoked 25p2
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2001 The Biochemical Society and the Medical Research Society
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K. A. Hunter and others
Figure 3 Relationship of fibrinogen in plasma (g/l) and ASR
(mg/kg per day) in smokers ($) and non-smokers (#)
Data from Study 1 and Study 2 combined. r l 0.652, P l 0.04.
Figure 2 Plasma fibrinogen concentration (g/l ; upper
panel), FSR (%/day ; middle panel) and ASR (mg/kg per day ;
lower panel) before and after abstention from smoking for 14
days
Data from Study 2 with P values from paired t test analysis.
cigarettes per day (assessed by a 7 day record of daily
cigarette consumption completed before the study began), successfully completed Study 2. Of the remaining
three participants, one developed a respiratory infection
and was excluded, one did not complete the smoking
abstention period and the other was excluded due to
elevated urinary cotinine levels (0.7 and 0.8 µg\ml
compared with the maximum value for non-smokers of
0.55 µg\ml). The mean urinary cotinine levels of the
successful abstainers was 0.20p0.03 µg\ml, which
suggested that they had complied with the non-smoking
regimen.
Cessation from smoking was associated with significant weight gain from a mean smoking value of
71.4p3.0 kg to an abstention value of 74.3p2.9 kg,
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2001 The Biochemical Society and the Medical Research Society
P 0.001. Accordingly, participants reported noticeable
changes in their dietary habits during abstinence including eating more in general, and increasing their
consumption of coffee, sugary snacks and soft drinks in
particular, as has been reported by other investigators
[26]. This weight change was significant to the present
study because it resulted in increased estimated plasma
volumes for use in the calculation of fibrinogen ASR.
In all participants, a fall in plasma fibrinogen concentration was observed from a mean value of
3.06p0.11 g\l while smoking, to 2.49p0.14 g\l after
abstention, an average reduction of 19p3 % (P 0.001 ;
Figure 2, upper panel). The mean fibrinogen FSR while
participants were still smoking was 17.3p0.8 %\day.
Cessation from smoking resulted in a reduction in FSR in
all participants to a mean value of 14.8p0.9 %\day, an
average decrease of 14 % (P l 0.02 ; Figure 2, middle
panel). Calculation of the ASR includes changes in plasma
fibrinogen concentration and plasma volume. The average reduction in fibrinogen ASR (FSRifibrinogen
concentrationiplasma volume) was 33 %, from a mean
value of 24.1p1.7 mg\kg per day while smoking, to
16.1p1.0 mg\kg per day following smoking cessation
(P 0.001 ; Figure 2, lower panel).
The subjects in Study 1 and Study 2 were not matched
for age. However, there was no difference in fibrinogen
concentration (2.86p0.20 versus 3.09p0.12), fibrinogen
FSR (17.05p1.53 versus 17.3p0.84) and fibrinogen ASR
(21.5p1.93 versus 24.1p1.71) between the smokers in
Study 1 and those in Study 2. Combining the smokers
of Study 1 with those in Study 2 indicates that fibrinogen synthesis in smokers was elevated relative to
non-smokers, both expressed as ASR (22.7p1.3 versus 16.0p1.34, P l 0.004) and as FSR (17.2p0.88 versus
14.0p1.25, P l 0.05). In addition, baseline plasma
concentrations of fibrinogen for smokers and nonsmokers were correlated with absolute fibrinogen synthesis (r l 0.65, P l 0.04 ; Figure 3).
Fibrinogen synthesis and smoking
Elevated plasma levels of C-reactive protein were not
detected in the smokers relative to the non-smokers, all
values were 10 mg\l.
DISCUSSION
Hyperfibrinogenaemia conveys an increased risk of
developing cardiovascular disorders by promoting a
multitude of atherogenic and thrombogenic processes,
and is widely thought to be as important a cardiovascular
risk factor as raised plasma cholesterol. The pathophysiological mechanisms by which fibrinogen and its
derivatives are thought to accelerate atherothrombogenesis include : the stimulation of vasoactivity [27], the
alteration of prostaglandin metabolism [28] and tissue
oxygenation [29], the promotion of platelet hyperactivity
[5] and erythrocyte aggregability [30], and the initiation
and sustained growth of atherosclerotic lesions [31]. As a
consequence, many researchers recommend that plasma
fibrinogen concentration is included during the assessment of cardiovascular risk [32]. Cigarette smoking is the
strongest known environmental influence on plasma
fibrinogen concentration [1] and has consistently been
linked to the development of elevated plasma fibrinogen.
Ernst et al. [11], for example, reported a dose–effect
relationship between the number of cigarettes smoked
per day and plasma fibrinogen concentration. Conversely, cessation from smoking results in a rapid
reduction in plasma fibrinogen [33,34], which subsequently may remain slightly elevated for several years
[35]. The present paper aimed to establish if the hyperfibrinogenaemia observed in smokers is accompanied by
an increased rate of synthesis and, conversely, whether
synthesis is reduced by short-term smoking cessation.
Both the higher plasma fibrinogen concentrations of
smokers compared with non-smokers (combined data
from Studies 1 and 2), and the significant fall in plasma
fibrinogen concentration with 2 weeks abstention from
smoking observed in Study 2, are in accordance with
previous findings [13,33,34]. Moreover, the elevated
levels of plasma fibrinogen concentration in smokers
were also significantly correlated with elevated rates of
fibrinogen synthesis (Figure 3 ; r l 0.65, P l 0.04). In
response to 2 weeks cessation in smoking, the rates of
fibrinogen synthesis were reduced to levels comparable
with those of the non-smokers (16.1p1.0 mg\kg per day
versus 16.0p1.4 mg\kg). The results from both studies
support the proposal that smoking induces fibrinogen
synthesis, and this effect can be reduced by abstention
from smoking.
Fibrinogen concentrations in the plasma are the result
of both the rate of synthesis of fibrinogen and the rate of
removal of fibrinogen from the plasma. Although the rate
of disappearance of fibrinogen from the plasma was not
measured in the present paper, the elevated rate of
fibrinogen synthesis in smokers suggests that fibrinogen
synthesis plays a role in the elevation of fibrinogen in the
circulation in smokers versus non-smokers. However,
the magnitude of the changes in the ASR (34 % in Study
1 and 33 % in Study 2) were greater than the observed
differences in plasma fibrinogen concentration between
smokers and non-smokers or abstainers (10 % in Study 1
and 20 % in Study 2), suggesting that the rate of
disappearance of fibrinogen from the plasma was also
elevated in smokers compared with non-smokers or
abstainers. From the current studies, it is not possible to
differentiate the loss of plasma fibrinogen through
degradation of the protein from the loss of fibrinogen
from plasma through deposition or increased transcapillary loss.
There have been relatively few evaluations of the rate
of fibrinogen synthesis in humans, and the present paper
is the first to investigate the effect of smoking on
fibrinogen metabolism. In general, the rates reported here
are similar to those observed by others in healthy
participants using a number of different isotopic techniques [36,37].
There are several possible biochemical mediators
which may be responsible for the difference in the
fibrinogen ASR of smokers and non-smokers (Study 1)
and the reduction in fibrinogen synthesis which occurs
with abstention from smoking (Study 2). It has been
suggested that chronic smokers exhibit a mild, but
sustained, acute-phase response, characterized by
increased plasma concentrations of positive acute-phase
proteins, such as fibrinogen and α -antitrypsin [38]. This
"
response probably develops as a result of the persisting
inflammatory insult to cells evoked by tobacco smoke
inhalation. The rise in the plasma concentration of
fibrinogen during this response has been attributed to an
increase in fibrinogen synthesis via a stimulation of
transcriptional activity [39]. The mediators of this response are thought to be cytokines, primarily interleukin6 [39] and it is possible, therefore, that interleukin-6 may
be responsible for the enhanced rate of fibrinogen
synthesis in smokers. The plasma concentration of
interleukin-6 has been shown to be elevated in smokers
[40].
The observation that the smokers in Study 1 had lower
plasma albumin concentrations than non-smokers also
suggests the induction of an acute-phase response to
smoking, since albumin is a negative acute-phase protein.
Lower plasma albumin concentrations in smokers agrees
with certain [41], but not all [38], epidemiological
findings, and in the present study, does not appear to
arise from an alteration in the rate of albumin synthesis.
It is probable that the mechanism responsible is an
accelerated transcapillary loss of albumin into the extravascular space [42]. Moreover, there may be dietary
effects on albumin synthesis, since smokers have been
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2001 The Biochemical Society and the Medical Research Society
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K. A. Hunter and others
shown to consume less protein and more energy than
non-smokers [26].
In addition to cytokines, catecholamines may also be
mediators of the smoking effect on fibrinogen synthesis.
Smoking has been shown to stimulate catecholamine
release [43], and studies in perfused liver have demonstrated that epinephrine may increase fibrinogen synthesis directly [44], possibly by enhancing mRNA
synthesis [45]. Indirect evidence suggests that fatty acids
may also have a role in increasing fibrinogen synthesis in
smokers, since incubation of human liver slices with fatty
acids accelerates incorporation of amino acids into
fibrinogen [46], and plasma non-esterified fatty acid
concentrations are elevated after smoking [47]. This
mechanism may be facilitated by thrombin, as injection
of thrombin into mice has been shown to result in raised
non-esterified fatty acid concentrations, and a stimulation
of fibrinogen production [48] and thrombin generation is
induced by smoking [49].
In conclusion, the present paper suggests that an
increase in the rate of fibrinogen synthesis is at least
partially responsible for the development of the hyperfibrinogenaemia observed in chronic smokers. Moreover,
a significant reduction in the rate of fibrinogen synthesis
occurs after only short-term cessation from smoking
(Study 2), and this is probably instrumental in the
concomitant fall in the intravascular fibrinogen concentration.
ACKNOWLEDGMENTS
We would like to thank all the volunteers who
participated in this study and applaud the efforts of those
who abstained from smoking. The skilled technical
assistance of Mr George Casella is, as always, most
appreciated. We would also like to acknowledge the
support of the Scottish Office Agriculture and Fisheries
Department, U.K., NIH grant MO1RR10710, and the
assistance of Grampian Health Promotions, Aberdeen,
Scotland, U.K. for organization of the smoking cessation
support group is also appreciated.
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Received 14 August 2000/11 December 2000; accepted 17 January 2001
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