Smoking history and serum cotinine and thiocyanate concentrations as determinants of rheumatoid factor in non-rheumatoid subjects

T. Korpilähde, M. Heliövaara, P. Knekt, J. Marniemi1, A. Aromaa and K. Aho

National Public Health Institute, Helsinki and 1 Social Insurance Institution and National Public Health Institute, Turku, Finland.

Correspondence to: T. Korpilähde, Tammelantie 43, 97220 Sinettä, Finland. E-mail: tuula.korpilahde{at}fimnet.fi


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Objectives. Smoking is associated with false-positive rheumatoid factor (RF). We explored the dose–response relationship of this association, using smoking history and serum cotinine and thiocyanate concentrations as measures of tobacco exposure.

Methods. A total of 6947 men and women aged 30 yr or over and free of clinical arthritis were included in the Mini-Finland Health Survey carried out between 1978 and 1980. Detailed histories of smoking and RF (sensitized sheep cell agglutination test) were obtained in the basic examination. In 2000, serum cotinine and thiocyanate were determined from serum samples collected at baseline and stored at –20°C. A cut-off point of 100 µg/l was used for serum cotinine and 10 µmol/l for thiocyanate to indicate active smoking.

Results. There was a close association between smoking and strongly positive RF. After adjustment for age, sex, coffee consumption and region, the odds ratios (95% confidence intervals in brackets) in current smokers and in those who had quit smoking were 3.94 (2.04–7.61) and 2.71 (1.33–5.53), respectively, compared with those who had never smoked. Among current smokers, the intensity, duration or tertiles of pack-years of smoking were not related to RF. No relationship between serum cotinine or thiocyanate and RF positivity was observed within the subgroups of current smokers and those who had quit. Among those who reported that they had never smoked but who nevertheless had serum cotinine levels at least 100 µg/l, the adjusted odds ratio of strongly positive RF was 4.48 (1.48–13.50) compared with people who had never smoked and whose serum cotinine levels were less than 100 µg/l.

Conclusions. The results are not in line with the hypothesis of a dose–response relationship between smoking exposure and RF positivity.

KEY WORDS: Cotinine, Rheumatoid factor, Smoking


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Items associated with rheumatoid factor (RF) reactions in the absence of clinical arthritis (false-positive RF) may be risk factors for the development of rheumatoid arthritis (RA), since RF can precede the onset of clinical disease by years [1]. Depending on the test system employed, 1–5% of healthy people may be RF positive and this prevalence of autoantibodies may increase to over 10% with advancing age [2–4]. Smoking may trigger the production of RF [5, 6] and false-positive RF reactions have been reported to occur twice as often in current and ex-smokers than in those who have never smoked [7]. Smoking has also been shown to be a risk factor for seropositive RA [5, 8, 9]. Recent studies have suggested a dose–response relationship between smoking and risk of developing RA, measured as pack-years of cigarette smoking [9–12]. In addition, pack-years of smoking have been associated with disease severity among RA patients [13].

The validity of self-reported smoking has been amply demonstrated. People may either over- or under-estimate the number of cigarettes they smoke daily, and the whole of their smoking history relies on memory. In addition, the amount of smoke products actually inhaled and absorbed tends to vary. Nicotine intake per cigarette ranges from 0.3 to 3.0 mg, depending on how cigarettes are smoked. Thus, self-reported measures in smokers, such as cigarettes smoked per day, are highly imprecise [14]. Black people are often reported to smoke fewer cigarettes per day, but still show higher levels of serum cotinine, which is the major plasma metabolite of nicotine. The underlying reason may lie in differing metabolism of cotinine or in higher intake of nicotine per cigarette by deeper inhalation among black people [15]. Although self-reported smoking status in Finland was concluded to be quite valid based on comparisons with serum cotinine levels [16], the possibility of recall bias remains and thus misclassification is possible.

Currently serum cotinine concentration is regarded as the best biomarker available for detection of exposure to tobacco smoke and for discriminating active smokers from non-smokers. A mean of 70–80% of nicotine is converted to cotinine, which has a half-life of about 17 h [14]. The cut-off points mostly used for serum cotinine in separating active smokers from non-smokers are between 100 and 200 µg/l [16]; active smokers usually have serum levels higher than 15 µg/l [17]. Passive smoking usually has only a slight effect on cotinine level. Yet some overlapping may exist between non-smokers highly exposed to environmental tobacco smoke and occasional smokers or those who inhale very little smoke [17]. Those non-smokers who are exposed to typical levels of environmental tobacco smoke have cotinine levels below 1 µg/l, whereas heavy exposure to environmental tobacco smoke produces cotinine levels up to 15 µg/l [17]. Another biomarker of tobacco exposure is serum thiocyanate; it is less specific than cotinine, but its advantage lies in its long half-life of about 7 days [18].

The close association between smoking and RF was previously established in the Mini-Finland Health Survey [7]. In the present study we re-analysed the association using more detailed information on intensity and duration of smoking and on the type of tobacco smoked. The cotinine and thiocyanate concentrations were determined from stored serum samples to estimate exposure to tobacco smoke. The main aim was to evaluate the dose–response relationship between smoking and RF.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The Mini-Finland Health Survey, which was carried out between 1978 and 1980, was designed to examine the epidemiology of major public health problems in a representative sample of the Finnish population aged 30 yr or over. The study sample consisted of 8000 people from 40 areas. The examinations were carried out by the mobile clinic of the Social Insurance Institution in two main phases: a screening phase and a diagnostic phase. A total of 7217 subjects (90%) participated in the screening phase.

In the present study, subjects with arthritis were excluded from the analyses. Arthritis was diagnosed based on medical history, symptoms, physical examination and hand radiographs. The methods and diagnostic criteria used for evaluating the presence of arthritides have been described in detail elsewhere [19].

The sensitized sheep cell agglutination (Waaler–Rose) test was performed by the time of the baseline survey for 6947 men and women free of arthritis. The test was carried out on microtitre plates, as described elsewhere [7, 19]. Titres ≥32 were regarded as positive and titres ≥128 as strongly positive. In 2000, serum cotinine and thiocyanate were determined from serum samples that had been stored at –20°C. The serum cotinine concentrations were determined with a method employing a modification of the Nicotine Metabolite radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA, USA) [20]. For serum cotinine a cut-off point of 100 µg/l was used to separate smokers from non-smokers [16]. The serum thiocyanate concentrations were determined colorimetrically, using ferric nitrate to generate ferric thiocyanate [21].

Smoking history was elicited using the following questions:

  1. Have you ever been a regular smoker for a total of at least 1 yr (regular smoking means smoking at least one cigarette, cigar, cigarillo or pipe every day or almost every day)?
  2. At what age did you begin smoking regularly?
  3. Do you smoke cigarettes regularly now?
  4. Have you smoked cigarettes regularly at some earlier period (and quit)?
  5. How long has it been since you last smoked cigarettes regularly (<1 month, 1–11 months, 1–9 years, ≥10 years)?
  6. How many cigarettes per day do you smoke on average (1–9 cigarettes/day, 10–19 cigarettes/day, 20–29 cigarettes/day, ≥30 cigarettes/day)?
  7. Do you smoke cigars or cigarillos regularly?
  8. Do you smoke a pipe regularly?
  9. Do you usually inhale when you smoke (or did you inhale, if you have quit)?

Pack-years of smoking were computed for current smokers. One pack-year was regarded as equivalent to 20 cigarettes smoked daily for 1 yr. A person who had smoked at least one cigarette, cigar or pipe daily for at least 1 yr was regarded as a current smoker. An ex-smoker had stopped smoking at least 1 month earlier.

The logistic regression model was used to estimate the strength of associations between smoking and false-positive RF reactions [22]; adjusted odds ratios with 95% confidence intervals were based on the model. Previous studies based on the Mini-Finland Health Survey have demonstrated regional differences in the prevalence of RF [23] and a linear relation between coffee consumption and RF [24]. Since geographical region and coffee consumption are also known to be associated with smoking habits, these factors were entered into the models as potential confounding factors in addition to age and sex. Effect modification was examined by also entering interaction terms into the models. The statistical significance of the interactions was tested, using the likelihood ratio test and expressed as P values.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
In the absence of arthritis, the prevalence of RF reactions (titres ≥32) was 2.1% and that of strongly positive (titres ≥128) reactions 1.0%. There was a close association between smoking and strongly positive RF and a modest association with a weaker titre of RF. The associations were statistically significant after adjustment for sex, age, coffee consumption and geographical region (Table 1). None of the interaction terms between smoking history and other determinants of RF proved to be statistically significant. For the interaction term of sex and smoking, the P value was 0.77.


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TABLE 1. Adjusteda odds ratios (OR) and 95% confidence intervals (CI) of positive rheumatoid factor reactions in titres ≥32 and ≥128

 
Men who smoked pipes or cigars in addition to cigarettes had a many-fold risk of developing strongly positive RF compared with those who had never smoked (Table 2). Since only six women smoked pipe or cigars, this analysis did not fit among women. Of the 60 men smoking cigarettes in addition to pipe or cigars, 18% smoked over 30 cigarettes per day, 27% smoked 20–29 cigarettes per day, 38% smoked 10–19 cigarettes per day and 17% smoked 1–9 cigarettes per day. The corresponding percentages among the men smoking only cigarettes were 12%, 35%, 38% and 15%, respectively.


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TABLE 2. Adjusteda odds ratios (OR) and 95% confidence intervals (CI) of rheumatoid factor reaction in titres ≥128 by smoking history among men

 
The distributions of serum cotinine and thiocyanate concentrations according to smoking history are given in Table 3. The levels were much higher in all groups of current smokers than in ex-smokers and those who had never smoked. The concentrations of cotinine and thiocyanate also correlated closely with each other (r = 0.63).


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TABLE 3. Serum cotinine (µg/l) and thiocyanate (µmol/l) concentrations according to smoking history

 
Those who reported that they had never smoked but whose serum cotinine concentrations were ≥100 µg/l showed a significantly increased risk of developing strongly positive RF (Table 4). Elevated serum thiocyanate concentrations were not associated with RF positivity.


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TABLE 4. Adjusteda odds ratios (OR) and 95% confidence intervals (CI) of rheumatoid factor reactions in titres ≥128 by serum cotinine and thiocyanate among those who have never smoked

 
Among ex-smokers, no association was found between serum cotinine or thiocyanate and RF (Table 5). The time elapsed since quitting appeared to be inversely proportional to RF positivity, but the association was not statistically significant (P for trend, 0.07).


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TABLE 5. Adjusteda odds ratios (OR) and 95% confidence intervals (CI) of rheumatoid factor ≥128 by serum cotinine, serum thiocyanate and time since quitting among ex-smokers

 
As presented in Table 6, self-assessed smoke inhalation or intensity, duration or tertiles of pack-years of smoking showed no association with RF. Serum cotinine or thiocyanate concentrations, analysed as tertiles, likewise showed no association with strongly positive RF among current smokers. All the indicators of intensity and duration of smoking were also analysed for their associations with RF reactions using a less stringent cut-off point (titres ≥32) within each stratum of smoking history, but no association was found.


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TABLE 6. Adjusteda odds ratios (OR) and 95% confidence intervals (CI) of rheumatoid factor ≥128 by serum cotinine and thiocyanate tertiles, self-assessed inhalation of smoke, intensity and duration of smoking, tertiles of pack-years smoked and cigar or pipe smoking among current smokers

 

    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
A close association between smoking and strongly positive RF reaction was demonstrated in the present population study. No evidence was found of a dose–response relationship between smoking and RF.

The subjects who reported that they had never smoked but whose serum cotinine still indicated active smoking showed a 4-fold risk of developing strongly positive RF. In men, smoking of pipe or cigars in addition to cigarettes appeared to be most strongly associated with RF. These findings were based on a few cases only and thus should be interpreted cautiously.

In the absence of arthritis the prevalence of strongly positive RF was 1.0%. Previously, Tuomi et al. [7] reported that false-positive RFs occur about twice as often among current and past smokers than among those who never smoked. In addition, they showed the proportion of smokers to increase with increasing RF titre, so that the prevalence of high titres (Waaler–Rose ≥500) was 4-fold among current smokers and 2-fold among ex-smokers. Jónsson et al. [6] reported an association between active smoking and combined elevation of IgM and IgA RF that was more persistent among smokers than non-smokers. In our study we found a close association between smoking and strongly positive RF among current smokers and a modest association among ex-smokers; these results are in agreement with previous studies [6, 7].

An interesting detail was noted among those who reported that they had never smoked but still had serum cotinine levels of at least 100 µg/l: an increased prevalence of RF positivity. Since heavy environmental exposure to tobacco smoke usually produces serum cotinine levels up to 15 µg/l [17], those subjects have obviously misrepresented their smoking status and actually been smokers. Rebagliato [17] has summarized 11 studies in which self-reported smoking status was compared with serum cotinine measurements and noted misclassification rates from 0.9 to 9.8%. Recall bias is also possible in our study because it relies on memory, yet it should be very easy to remember never having been a smoker. Although our finding was based on only four cases, it may support the hypothesis of a causal relationship between smoking and positive RF.

Serum thiocyanate showed no association with RF in any category of smoking history. It is less specific than serum cotinine as an indicator of active smoking, which may explain why it showed no association with RF in those who had never smoked.

It seemed contradictory that serum cotinine concentration was associated with RF among never smokers but not among ex-smokers. This may be due to the sharper comparison contrast between low and higher levels of serum cotinine among never smokers, because ex-smokers in this study had an almost three-fold risk of having strongly positive RF compared with those who reported as having never smoked.

The time elapsed since smoking appeared inversely proportional to the prevalence of RF among ex-smokers. The result is in parallel with a prospective cohort study concerning RA-associated RF in post-menopausal women [25] which showed that women who had quit smoking more than 10 yr before the study were no longer at increased risk of developing RA.

We found no association between intensity of smoking, duration of smoking and tertiles of pack-years smoked with RF positivity. These results are not in agreement with other recent studies on RA; the study mentioned above concerning post-menopausal women found both intensity and duration of smoking to be associated with RA [25]. Another retrospective cohort study concerning only women found duration, but not intensity, of cigarette smoking to be associated with a modestly increased risk of developing RA [26]. A population-based case–control study, using incident cases, found increased risk of developing RA after a long duration but merely with a moderate intensity of smoking and the risk remained for several years after smoking cessation [9]. A retrospective case–control study done in Sweden found smoking at any time to be associated with RA in both sexes. In men, an increase in risk was found with increasing pack-years smoked [11]. A case–control study done in the UK found an increasing association between increased pack-years smoked and RA [10], in which the risk of developing RA was 6-fold after 31–40 pack-years and 14-fold after 41–50 pack-years smoked. Thus, our results are not in line with the hypothesis of the association between cumulative exposure to tobacco smoke and RF positivity.

Instead of focusing on the association between smoking and RA, we focused on the strongly positive RF reaction sometimes occurring in non-rheumatoid subjects, which can precede clinical disease by years [1]. The study was representative for the Finnish population and the participation rate was high (90%). Recall bias is possible, because smoking history relies on memory. Yet we used serum cotinine and thiocyanate to discriminate active smokers from non-smokers and to quantify the exposure to tobacco smoke.

Newer RF determination methods were not used in the present study because RF had been measured at the baseline survey and at that time the sensitized sheep cell agglutination test was the method of choice. Previous studies focused on RF from the Mini-Finland Health Survey have been based on this method [7, 23, 24].

Cotinine and thiocyanate are stable compounds. In the present study their serum concentrations made a good distinction between never smokers and ex-smokers and current smokers. Thus, the serum determinations can be considered accurate.

It should be emphasized that although the dose–response relationship is lacking from our results, its absence does not imply falsification of the possible causation between smoking and RF positivity [27]. Biological plausibility is difficult to clarify, because the mechanisms governing how smoking may influence the immune system, leading to increased production of RF, remain unknown. The analogy can be tested; the number of cases of seropositive RA may increase among young women in the future due to the increase in smoking seen among young girls today.

Smoking continues having high interest as a risk factor for RF-positive RA. However, surrogate measures of smoking were not explored in previous studies. A limitation of the present study is that although it comprised almost 7000 participants, some of the findings were based on low numbers of cases. Thus, we cannot exclude the possibility that insufficient sample size and variation in the indicators of intensity and duration of smoking may explain the absence of a dose–response relationship between smoking and RF in this study. Due to the increased mortality related to smoking and RF, furthermore, a survivor effect may remain in a cross-sectional study like the current one.

In summary, the present study shows a close association between smoking and strongly positive RF. Yet, the results are not in line with the hypothesis of a dose–response relationship between smoking exposure and RF positivity.

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Submitted 27 January 2004; revised version accepted 9 July 2004.