Effects of smoking and abstention from smoking on ... - Clinical Science

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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2001 The Biochemical Society and the Medical Research Society

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K. A. Hunter and others

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 #


[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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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, #


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).


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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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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