Elevated plasma cotinine is associated with an increased risk of developing IBD, especially among users of combusted tobacco.

OBJECTIVE: Smoking has previously been associated with inflammatory bowel disease (IBD), but no study has reported on cotinine, an objective, biochemical measure of tobacco use. We aimed at testing the hypothesis that cotinine levels among healthy subjects are associated with an increased risk of developing IBD in later life.
DESIGN: We analysed plasma cotinine and evaluated corresponding lifestyle questionnaires that included tobacco habits in subjects (n = 96) who later developed late-onset IBD (70 ulcerative colitis (UC) and 26 Crohn's disease (CD)) and in sex and age-matched controls (n = 191).
RESULTS: Patients who later developed IBD had significantly higher plasma cotinine levels compared to controls. In multivariable analysis, higher log-cotinine was associated with a higher risk of developing IBD (OR 1.34 (95% CI 1.01-1.63)). After stratifying for time to diagnosis, the association was only significant in subjects with shorter time (< 5.1 years) to diagnosis (OR 1.45 (1.09-1.92)). The findings were similar for UC- and CD-cases, but did not reach statistical significance in CD-cases. Although plasma cotinine concentrations were higher in snuff users compared to combusted tobacco users, no increase in the risk of IBD and lower risk of developing IBD among subjects with shorter time (< 5.1 years) to diagnosis was seen among snuff users.
CONCLUSIONS: Cotinine, a biomarker of tobacco use, is associated with increased risk of developing late-onset IBD in general, and UC in particular. No increased risk among snuff users indicates that other components in combusted tobacco than nicotine may be involved in the pathogenesis of IBD among smokers.

Introduction

Tobacco is a major risk factor of a wide spectrum of diseases and in general the greatest preventable cause of premature death. Still, in the 21st century, a significant proportion of the world’s population is consuming tobacco. In Sweden, approximately 10.7% of inhabitants smoke daily, i.e. use combusted tobacco (2016–2017) [1].

Smoking has also been associated with the risk of inflammatory bowel disease (IBD) [2]. A recent systematic review concludes that the incidence of IBD is decreasing in countries where the smoking prevalence is decreasing, indicating a connection. The same study also reported that the proportion of never smokers developing IBD is increasing, indicating that smoking is not the only risk factor [3].

Smoking is the only proven environmental determinant for Crohn’s disease (CD) [4], and smokers with CD often have a more complicated course of disease than non-smokers [5].

The association between smoking and ulcerative colitis (UC) is possibly even more complex. Current smokers who stop smoking appear to be more prone to develop the disease, but there is no clear beneficial effect of tobacco smoking on the natural course of UC [6]. Recently, we showed that “never smokers”exhibit a lower risk for late-onset UC [7]. Specific components in smoked/combusted tobacco that may deteriorate IBD have not been identified so far. The same is true for possible protective factors of smoking that may modify the disease activity in patients with UC [8].

Smoking is associated with inflammatory activity. For example, a study on 1819 participants from the PLCO Cancer Screening Trial showed an association between biochemical markers of inflammation and smoking [9]. One study on peripheral blood mononuclear cells from 37 IBD patients and 17 controls showed that cytokine profiles differ in IBD patients compared to healthy controls and that these cytokine profiles may be modified by nicotine [10].

Cotinine is a metabolite of nicotine, with a longer half-life, which makes it suitable as a marker for long-term tobacco use [11, 12]. Cotinine concentrations in urine, saliva, serum and plasma are all associated with smoking intensity [13, 14]. Cotinine is considered a better marker for quantifying smoking than self-reporting of smoking [15].

Nicotine and cotinine have previously been analysed in one study on 51 UC patients who also were smokers [16]. Reported median serum concentrations of nicotine and cotinine were 8 ng/mL (49.3 nmol/L) and 180 ng/mL (1021.5 nmol/L), respectively. An increase in nicotine of 12.1 ng/mL (74.12 nmol/L) 2 minutes after smoking one cigarette was observed. While there are prospective studies on the associations between smoking risk and IBD [17], no study has assessed cotinine as a measure of smoking exposure in relation to IBD-risk.

In Sweden, the use of moist, smokeless, non-combusted tobacco (snuff) is common, with 12.9% of inhabitants using snuff daily (2016–2017) [1]. Smokeless tobacco, such as snuff, has been associated with higher levels of cotinine compared to smoking [18], Therefore, we have in this study chosen to analyse snuff use as well as smoking. Snuff use has not been associated with health risks in the same way as smoking has, and there was no association between snuff use and the development of IBD in a study on 1100 UC and 700 CD patients among Swedish construction workers [17].

The present study aimed to investigate whether pre-diagnostic cotinine levels predict the development of IBD in later life and to compare the predictive value of cotinine with self-reported smoking exposure and snuff use.

Materials and methods

Ethical approval

The regional ethical board, Umeå, Sweden (Dnr 06-024M, 2010-284-31 M), approved the study. Written informed consent was obtained from all participants before inclusion in the study.

Study population

Study participants were selected from patients with known IBD, confirmed by objective measures, i.e. colonoscopy, MRI or biopsy. All participants were recruited from the Västerbotten intervention project (VIP) or the Mammography screening project (MA), both within the Northern Sweden Health and Disease Study (NSHDS).

The VIP started in 1985 and is a health screening intervention project, which annually invites all inhabitants of the Västerbotten County in northern Sweden turning 40, 50 and 60 years to a health screening at their primary care centre. At these screenings, survey data and blood samples are collected and stored in a biobank that presently contains data from over 150 000 visits. The MA project was active between the years 1995–2006. Blood samples and survey data were collected in conjunction with mammography screening for women aged 18–82 years. The biobank contains about 54 000 samples.

Initially, all participants from the VIP or MA, with an ICD-10-diagnosis of IBD (K50.1–9 or K51.1–9) were included. All diagnoses were reaffirmed by reviewing medical records, and the participants with an incorrect diagnosis were excluded.

The time of IBD diagnosis was defined as the first objective finding (i.e. MRI, colonoscopy or biopsy) based on reviews of the patient’s medical records. Subjects diagnosed with IBD less than one year after participating in the NSHDS health examination were excluded from the study. Information about what type of IBD, UC or CD, was also collected from the medical records.

For each case, two controls, matched for age, gender and time and centre of health screening, were randomly selected among healthy participants from the NSHDS cohort. A total of 96 patients (70 UC and 26 CD) eligible for the study and 191 matched controls were available to be included in further analyses.

Lifestyle data

The participants smoking status and use of smokeless tobacco were obtained from questionnaire data.

The questionnaires were filled in, and the blood samples were collected at the same health screening. Length and weight were also measured during the visit. The questionnaire contained information on previous and current tobacco use, including type, amount (cigarettes per day for smokers and snuff boxes per week for snuff users), duration of use and time since quitting.

Participants smoking or using snuff when completing the questionnaire were defined as active users. Previous smokers or snuff users were categorized as non-smokers or non-snuff users. From the medical records the data on the present use of tobacco at the time of diagnosis was not accurate enough to be included in the study.

Sample collection and handling

Blood samplings were performed after overnight fasting, after resting for 15 minutes venous blood was collected in 10 mL Na-heparin tubes, centrifuged at 1500 G for 15 minutes, and aliquots of plasma were frozen within one hour and stored at -80°C.

Biochemical analyses

Plasma cotinine was analysed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) as reported previously [19]. The lower limit of detection was 1.0 nmol/L, and within-day and between-day coefficients of variation were 2–3% and 6%, respectively.

Statistical analyses

All statistical analyses were made using IBM SPSS statistics version 24.0 (IBM Corporation, New York, NY, USA). Differences between continuous variables were assessed using the Mann-Whitney U-test, and for categorical variables, the Chi2-test was used. Calculations were performed with IBD as outcome in the total study group and after stratification for the type of IBD (UC or CD).

Subjects with cotinine levels <85 nmol/L were defined as non-tobacco user and those with cotinine ≥85 nmol/L as tobacco users [20]. These subgroups were further divided into four categories; subjects with cotinine levels <5 nmol/L was defined as non- tobacco users, 5–85 nmol/L as passive tobacco users, >85–1700 nmol/L as tobacco users and >1700 nmol/L as heavy tobacco users [20].

For conditional logistic regression, log-transformed cotinine values were used as the distribution is highly skewed. Both univariable and multivariable conditional logistic regression were performed to assess the odds ratio (OR) for developing IBD. The multivariable model included log-cotinine, smoking and snuff use. For both univariable and multivariable models, the subjects were further stratified into two sub-groups based on the median time to diagnosis. When calculating p for trend, variables were treated as continuous variables in regression analysis.

P-values <0.05 were considered statistically significant.

Results

Baseline characteristics

Baseline characteristics are shown in Table 1. No differences between cases and controls were seen for age, gender, BMI or snuff use. Self-reported smoking was more common, and plasma cotinine concentrations were higher among those who developed IBD in later life compared to matched controls. For smoking, we observed similar results for UC and CD; however, cotinine was higher in UC, but not in CD-patients as compared to controls (S1 Table). In the CD group, self-reported number of cigarettes per day was higher compared to matched controls; no differences were seen for UC or all IBD subjects.

Table 1

Baseline characteristics for subjects who later developed IBD (case) and controls.

	Case	Control	p-value*	n Case/Control
Age, years	50.1 (40.1–59.8)	50.0 (40.1–59.7)	0.80	96/191
Time to diagnosis, years	5.09 (2.66–7.23)	n.a.	n.a.	96/n.a.
Gender, women (%)	50.0	53.4	0.68	96/191
BMI, kg/m2	25.1 (23.2–28.5)	25.4 (23.1–28.0)	0.83	87/172
Smoking (%)	31.3	19.4	0.041	87/170
Number of cigarettes/day (%)				83/164
0	61.5	70.7		59/135
1–4	4.2	2.6		4/5
5–14	13.5	9.9		13/19
15–25	7.3	2.6	0.15	7/5
Snuff use (%)	16.7	14.7	0.97	87/162
Cotinine, nmol/L	5.96 (1.32–1350)	1.45 (<1.00–917)	0.001	95/190
Cotinine categories (%)				95/190
Non-tobacco users	52.1	69.6		50/133
Tobacco users	46.9	29.8	0.006	45/57
Cotinine subcategories (%)				95/190
Non-tobacco users	49.0	63.4		47/121
Passive-tobacco users	3.1	6.3		3/12
Tobacco users	32.3	20.4		31/39
Heavy tobacco users	14.6	9.4	0.033	14/18
Cotinine, nmol/L (by type of tobacco)				86/160
Smokers	1060 (614.0–1425)	1185 (617.0–1495)	0.58	24/24
Snuff users	1670 (1370–2750)	1360 (1238–2080)	0.19	11/18
Smoking and snuff-use	2120 (1305–2400)	1770 (1108–2308)	0.68	5/10
Non-users	1.56 (0.75–2.22)	1.05 (0.00–1.88)	0.010	46/108

Values are median (25–75 percentiles) for continuous variables, and proportions for categorical variables.

*Calculated with Mann-Whitney for continuous variables and Chi2 for categorical variables.

Cotinine categories–Non-tobacco users: <85 nmol/L, tobacco users: ≥85 nmol/L. Cotinine subcategories–Non-tobacco users: <5 nmol/L, passive tobacco users: 5-<85 nmol/L, tobacco users: 85–1700 nmol/L, heavy tobacco users: >1700 nmol/L.

n.a.: Not applicable.

Using cotinine to define tobacco users /non-tobacco users showed a higher frequency of (cotinine-defined) tobacco users in the IBD-group compared to matched controls. This was also seen in the UC-, but not in the CD-group. Further sub-categorization based on cotinine for differentiation between passive and heavy tobacco users showed a significant difference between cases and controls in the IBD group as a whole, but this was no longer significant for UC or CD subjects.

Cotinine levels for different types of tobacco exposure are shown in Table 1, separately for cases and controls. There were no differences in cotinine levels between cases and controls for smokers, snuff users or users of both snuff and cigarettes (Table 1). Cotinine levels among non-users were higher among cases compared to controls (Table 1), also seen for UC, but not for CD cases (S1 Table).

There was a significant correlation between the self-reported number of cigarettes/day and categories of cotinine in plasma (Spearman correlation coefficient 0.62, p<0.001).

Plasma cotinine concentrations were higher in snuff users compared to smokers, median (25th-75th percentile) 1490 (1270–2230) vs 1145 (641–1435) nmol/L, p <0.001.

Univariable conditional logistic regression

There was a significant trend for developing IBD with a higher number of self-reported cigarettes per day (p = 0.023), but there was no significance in the risk for IBD when analysing each category of number of cigarettes (Table 2). After subdividing into UC and CD, this was only seen in the CD group (S2 Table).

Table 2

Conditional logistic regression, univariable odds ratios (OR) and 95% confidence intervals (CI) and p for trend across categories, for developing inflammatory bowel disease and categories of tobacco exposure.

Tobacco exposure	OR (95%CI)	p trend	n Case/Control
Number of cigarettes/day			83/156
0	Ref
1–4	1.37 (0.36–5.26)
5–14	1.89 (0.86–4.18)
15–25	3.18 (0.97–10.4)	0.023
Cotinine categories			95/188
Non-tobacco users	Ref
Tobacco users	2.00 (1.21–3.28)	0.006
Cotinine subcategories			95/188
Non-tobacco users	Ref
Passive-tobacco users	0.70 (0.17–2.84)
Tobacco users	1.97 (1.10–3.50)
Heavy tobacco users	1.82 (0.83–4.00)	0.013

Cotinine categories–Non-tobacco users: <85 nmol/L, tobacco users: ≥85 nmol/L. Cotinine subcategories–Non-tobacco users: <5 nmol/L, passive tobacco users: 5-<85 nmol/L, tobacco users: 85–1700 nmol/L, heavy tobacco users: >1700 nmol/L.

Cotinine-defined tobacco users showed a higher OR for developing IBD compared to controls (OR 2.00; 95% CI 1.21–3.28), and further dividing into cotinine-based sub-categories showed the same results (Table 2). When stratifying for IBD subtype, the same was seen for UC (OR 2.21; 95% CI 1.21–4.06), but not for CD cases (S2 Table).

A higher log-Cotinine, analysed as a continuous variable, was associated with a higher OR for developing IBD (OR 1.24; 95% CI 1.11–1.40), when stratifying for the median time from blood-sampling to diagnosis, the same was seen for both groups (Table 3). When stratifying for IBD subtype, the same was seen for UC (OR 1.33; 95% CI 1.14–1.55), but not for CD cases (S3 Table).

Table 3

Conditional logistic regression, univariable odds ratios (OR) and 95% confidence intervals (CI) for developing inflammatory bowel disease, subdivided by median time from data collection to diagnosis.

Tobacco exposure	OR (95%CI)	n Case/Control
All cases
log-Cotinine	1.24 (1.11–1.40)	95/188
Smoking	1.90 (1.06–3.42)	87/170
Snuff use	1.02 (0.49–2.16)	87/162
Data collected <5 years before diagnosis*
log-Cotinine	1.24 (1.05–1.45)	48/96
Smoking	1.59 (0.62–4.06)	44/87
Snuff use	0.78 (0.29–2.11)	44/84
Data collected >5 years before diagnosis*
log-Cotinine	1.25 (1.06–1.48)	47/92
Smoking	2.13 (<1.00†-4.55)	43/83
Snuff use	1.47 (0.48–4.46)	43/78

*5.09 years, the median time before diagnosis.

† 0.999975.

Self-reported smoking was associated with a higher OR for developing IBD (OR 1.90; 95% CI 1.06–3.42), although after stratifying by median time from sampling to diagnosis (5.09 years), this was no longer significant (Table 3). No association between self-reported smoking and risk for developing the disease was seen after subdividing cases in a UC and a CD group (S3 Table).

No associations were seen between snuff use and risk for IBD, not in the whole group, nor any of the sub-groups (Table 3; S3 Table).

Multivariable conditional logistic regression

In a multivariable model including log-Cotinine, smoking and snuff use, higher log-Cotinine was associated with a higher OR for developing IBD when analysing the whole group (OR 1.34; 95% CI 1.10–1.63) (Table 4). When sub-dividing by median time from blood-sampling to diagnosis, this was also seen for log-Cotinine in the group with data collected less than 5.09 years before diagnosis (OR 1.45 95% CI 1.09–1.92). In the same sub-group (samples collected less than 5.09 years before diagnosis), snuff use was associated with a lower risk for developing IBD (OR 0.23; 95% CI 0.06–0.95) (Table 4).

Table 4

Multivariable conditional logistic regression showing odds ratios (OR) and confidence interval (CI) for inflammatory bowel disease, subdivided by median time from data collection to diagnosis.

	OR (95% CI)	n Case/Control
All cases		86/158
log-Cotinine	1.34 (1.10–1.63)
Smoking	0.88 (0.39–1.97)
Snuff use	0.40 (0.15–1.02)
Data collected <5 years before diagnosis*		44/83
log-Cotinine	1.45 (1.09–1.92)
Smoking	0.42 (0.11–1.69)
Snuff use	0.23 (0.06–0.95)
Data collected >5 years before diagnosis*		42/75
log-Cotinine	1.30 (0.96–1.75)
Smoking	1.21 (0.43–3.47)
Snuff use	0.58 (0.15–2.31)

Three variables were included in all multivariable models: log-Cotinine, smoking and snuff use.

*5.09 years, the median time before diagnosis.

In the UC sub-group, higher cotinine was associated with a higher risk of developing the disease (OR 1.42; 95% CI 1.12–1.79). When stratifying for the median time from blood-sampling to diagnosis, this was only seen in the group with data collected less than 5.09 before diagnosis (OR 1.78; 95% CI 1.12–2.81) (S4 Table).

No associations were seen between tobacco exposure and risk for CD in the multivariable models (S4 Table).

Discussion

We found that cotinine levels were associated with increased risk of developing IBD, and UC in particular. Smoking has earlier been linked to IBD and is an established risk factor for CD, but its role in UC appears to be more complex. Up until now, all evidence of associations between smoking and IBD have been based on self-reported exposure. Self-reported data are prone to bias and objectifiable; quantitative data of smoking exposure in relation to IBD-risk would be desirable. This is the first study to report the potential impact of tobacco exposure as measured by plasma cotinine concentrations on the risk of developing IBD. Cotinine has earlier been demonstrated being an accurate estimate of nicotine exposure [15]. In this prospective cohort study, cotinine in plasma was associated with an increased risk of developing IBD in later life. Similar associations were found when using predefined [20] cotinine cut-offs for defining tobacco use. In addition to IBD, this association was also statistically significant in UC-cases, both before and after sub-grouping according to median time between blood sampling and IBD-diagnosis. Thus, our data confirm findings from earlier studies on associations of self-reported smoking with IBD.

The findings are in concordance with our previous study, showing for the first time that smoking status, based on questionnaires with self-reported smoking, was associated with a higher risk of developing UC, CD and IBD [7]. In the present study, these findings were confirmed and extended for cotinine, smoking and snuff use in multivariable analysis. Cotinine was associated with IBD risk in a model that differentiated between combusted and non-combusted tobacco use. In addition, the proportion of snuff users was lower among subjects developing IBD within five years from inclusion. The OR remained low in all models (although insignificant), which may indicate an elevated risk from combusted tobacco but not from non-combusted tobacco. This may also imply that nicotine (measured as cotinine) per se may not be a causal factor, but rather a proxy for other risk factors from combusted tobacco.

Many different pathophysiological factors associated with smoking, including vitamins, immunity, inflammation, antioxidants, vascular factors, gut permeability and motility [21], as well as effects on the gut microbiota [22] have been implicated in IBD.

The effects of nicotine in IBD is unclear. Nicotine has been shown to induce Neutrophil extracellular traps (NETs) in in vitro studies, which may be involved in inflammatory activity in IBD [23]. Based on biopsies, NETs were present in virtually all patients with IBD [24, 25]. In contrast, nicotine may protect against colitis by mechanisms involving micro RNA-124 and STAT3 in mice [26].

In humans there appear to be a risk of rebound effect upon smoking cessation, resulting in an increased risk of developing UC [8]. Changes in cytokines in IBD patients that smoke have been reported, i.e. IL-8 (inducing chemotaxis of granulocytes) being lower in smokers compared to non-smokers with IBD with contradictory findings among controls [27]. Nicotine may also have an effect on apoptosis and cell cycle regulation [10].

Cotinine was higher among snuff users compared to smokers in our study. This is in line with a previous study suggesting that the first-pass metabolism in the liver of swallowed nicotine from chewing tobacco and snuff results in higher plasma cotinine compared to nicotine from smoking [28]. This could explain the higher cotinine seen among snuff users compared to smokers. Still, there is a difference in the association of combusted and non-combusted tobacco with IBD risk in the present study, indicating that factors other than nicotine from combusted tobacco might contribute to the risk of IBD. The route of administration of nicotine or the tissue primarily affected may determine the type of immune response and thereby influence disease development.

There are some limitations to the present study. Firstly, this study mainly addresses patients with late-onset IBD due to the design of NSHDS. Late-onset IBD represents only about one-quarter of all IBD cases (23%) [29]. Therefore, the effect of cotinine in this study may not necessarily apply to younger patients. Secondly, not all participants reported their tobacco use; therefore, in analyses on self-reported data, the number of subjects was somewhat lower compared to subjects with plasma analyses. Thirdly, the data on the medical records on tobacco use was not accurate enough to be included in the analysis. Finally, the sub-group that later developed CD was small in number, which might explain the lack of a statistically significant association between cotinine and the development of CD.

The strength of the present study is its prospective nature; all data were collected years before diagnosis, reducing the risk for recall bias. The cases were thoroughly ascertained and matched with two controls. Blood samples were handled according to standardized pre-defined protocols, and cotinine in plasma was analysed with a gold standard LC-MS/MS method with high analytical performance. We were also able to separately study the effects of both combusted and non-combusted tobacco.

Conclusion

Exposure to cigarette smoking as determined by cotinine in plasma is a risk factor for late-onset IBD, including UC. The possible lower risk for IBD in users of non-combusted tobacco (snuff) in a multivariable model indicates that other components than nicotine may be involved in the pathogenesis of IBD in smokers.

Baseline characteristics for subjects who later developed ulcerative colitis and Crohn’s disease (cases) and matched controls.

Median (25–75 percentile) for continuous variables, proportions for categorical variables.

(DOCX)

Click here for additional data file.

Conditional logistic regression, univariable odds ratios (OR) and 95% confidence intervals (CI) and p for trend across categories, for developing ulcerative colitis and Crohn’s disease, and categories of tobacco exposure.

(DOCX)

Click here for additional data file.

Conditional logistic regression, univariable odds ratios (OR) and 95% confidence intervals (CI) for developing ulcerative colitis and Crohn’s disease, subdivided by median time from data collection to diagnosis.

(DOCX)

Click here for additional data file.

Multivariable conditional logistic regression showing odds ratios (OR) and confidence interval (CI) for ulcerative colitis and Crohn’s disease, subdivided by median time from data collection to diagnosis.

(DOCX)

Click here for additional data file.

13 Jan 2020

PONE-D-19-31583

Elevated plasma cotinine is associated with an increased risk of developing IBD, especially among users of combusted tobacco

PLOS ONE

Dear Dr. Lovisa Widbom,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by April 30, 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Stefanos Bonovas, M.D., M.Sc., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for including your ethics statement in your manuscript. We ask that you please additionally include that you obtained written informed consent from the participants of this study in your statement as well

3.  We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

5. Thank you for stating the following in the Competing Interests section:

'The authors have declared that no competing interests exist.'

We note that one or more of the authors are employed by a commercial company: Bevital AS.

Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I found this very interesting. The novel finding is that cotinine levels do not predict the risk of IBD developing in snuff users (higher levels of cotinine than smokers but not higher levels of IBD) suggesting some component of cigarette smoke other than nicotine is the causative factor increasing the risk of IBD.

However the manuscript does need significant revision and I do not think it can be accepted in its current form. The results section is very unclear and difficult to read. The authors describe their results in the text but do not put the actual statistics in the text. Rather they require the reader to look at "Table 2" to look up the relevant statistic. I think it would be better to follow the convention of having the statistic in brackets e.g. (OR x.xx, 95% CI x.xx-x.xx, Table 2).

I also find some of the results section misleading. For example "A higher number of self-reported cigarettes per day was associated with higher OR for developing IBD (Table 2)." When looking at table two I see that none of the ORs are statistically significant, all of the 95% CIs cross 1.0 but instead the authors are reporting a "p trend" on the values of the odds ratios themselves in relation to the number of cigarettes smoked. This is not described in the methods section and it should be clear exactly which statistical test was used.

My other major concern is that in supplementary table 2 it suggests the OR for developing Crohn's in a heavy smoker is 148631 relative to a non-smoker. This is clearly incorrect and likely results from the study being very under-powered. While I am not a statistician by training my experience is that such outlandish results are almost always incorrect and the statistical analysis has been performed incorrectly.

I also note that they have pre-defined cut-off levels of cotinine to determine smoking status. How did these concur with the patients' self reported smoking? Also when reading reference 20 I couldn't see these cut-off levels used but perhaps I missed this.

Finally the English, while intelligible and certainly better than my Swedish, is non-standard. In the first paragraph the authors refer to the 21th rather than the 21st century. They also refer to "medical journals" which to me would mean publications such as PLOS ONE but I believe they mean "medical records". I think it would be important to have a native English speaker to proof read the manuscript before re-submission.

Reviewer #2: Thank you for the opportunity to review the manusrcipt entitled "Elevated plasma cotinine is associated with an increased risk of developing IBD, especially among users of combusted tobacco". The topic is highly interesting but the manuscript would need a bit of a revision.

The Introduction is brief and clear.

In the Materials and Methods it should be added that the matching was not complete in a 1:2 maner.

Why were not previous smokers/snuff users as previous users rather than non-users? This may affect the findings.

Also, and even more important, there is a continuous unclearity throughout the manuscript (and tables) on cotinine levels being the cut off for smokers. Should this not be changed to tobacco users instead as smokers and snuff users later are analysed separately? Causes confusion in it's present form.

In the Results I would prefer to have cotinine levels evaluated first and then separate for smokers and snuff users.

Regarding all tables I would like to have them moved from a separate column at the end to be added in each column respectively. Further you should add % in the columns as this is unclear at the moment.

I do not get the numbers in the tables to add up as expected. E.g. in table 1 Smoking (tobacco user?) is 87/170 but regarding # of cigarettes/day 83/164. In table 2 # cig/day is only 83/156. Non-smokers in table 1 is 96/161 while in table 2 85/188. Same disparities apply for tables 3 and 4.

Was there any data on smoking status at time of diagnosis? Would be highly interesting, especially regarding the increased risk for UC patients.

In the discussion there I would like more discussion on the found risk for UC among smokers/tobacco users. Also there is a recent Swedish publication for incidence for late onset of IBD (Everhov et al) which might be more appropriate in a national epidemiological maner.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

20 May 2020

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future.

Response: The method has been published previously, ref 19 in the manuscript: Midttun Ø, Hustad S, Ueland PM. Quantitative profiling of biomarkers related to B-vitamin status, tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2009;23:1371-9.

Journal requirements

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response: Done.

2. Thank you for including your ethics statement in your manuscript. We ask that you please additionally include that you obtained written informed consent from the participants of this study in your statement as well

Response: Added under ethical approval: Written informed consent was obtained from all participants before inclusion in the study.

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly.

Response: All relevant data are within the paper and its Supporting Information files.

Individual data is not provided as it can be linked to individuals, in order to access individual data, a formal application to the northern Sweden health and disease study biobank can be made.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.

Response: Supporting information captions added to the manuscript and in-text citations updated.

5. We note that one or more of the authors are employed by a commercial company: Bevital AS.

* Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role

in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials,please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in

the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish,

or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in

development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following

statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.”.

If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this nformation has been declared.

Please include both an updated Funding Statement and Competing nterests Statement in your cover letter. We will change the online

submission form on your behalf.

Response: The text “Bevital AS provided support in the form of salary for author ØM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author are articulated in the 'author contributions' section.” has been added under funding.

The text “Author ØM was employed by Bevital AS: This does not alter our adherence to PLOS ONE policies on sharing data and materials. No other competing interests declared.” has been added under competing interests.

REVIEWER 1

The results section is very unclear and difficult to read. The authors describe their results

in the text but do not put the actual statistics in the text. Rather they require the reader to look at "Table 2" to look up the relevant statistic. I think it would be better to follow the convention of having the statistic in brackets e.g. (OR x.xx, 95% CI x.xx-x.xx,Table 2).

Response: For relevant significant results we have included p-values, OR and 95% CI in the text as suggested.

I also find some of the results section misleading. For example "A higher number of self-reported cigarettes per day was associated with higher OR for developing IBD (Table 2)." When looking at table two I see that none of the ORs are statistically significant, all of the 95%

CIs cross 1.0 but instead the authors are reporting a "p trend" on the values of the odds ratios themselves in relation to the number of

cigarettes smoked. This is not described in the methods section and it should be clear exactly which statistical test was used.

Response: We have included a clarification that there is a significant trend for IBD with increasing numbers of cigarettes. We have in the method section explained p for trend.

My other major concern is that in supplementary table 2 it suggests the OR for developing Crohn's in a heavy smoker is 148631 relative to

a non-smoker. This is clearly incorrect and likely results from the study being very under-powered. While I am not a statistician by

training my experience is that such outlandish results are almost always incorrect and the statistical analysis has been performed

incorrectly.

Response: We agree that the number 148631 is an obscure number, resulting from very few heavy smokers (n=4) and no controls. It is evident that this is not significant by looking at the confidence intervals. No conclusions can be drawn from this but we have included this to present all data of the study.

I also note that they have pre-defined cut-off levels of cotinine to determine smoking status. How did these concur with the patients' self reported smoking? Also when reading reference 20 I couldn't see these cut-off levels used but perhaps I missed this.

Response: The reason for the values being different in the reference is because of use of different units, the reference using ng/mL, whereas we have used mmol/L.

There was a significant Spearman correlation between self-reported number of cigarettes/day and categories of cotinine in plasma (p<0.001) in the total material of this study, although in some individuals the cotinine concentration does not correspond to number of cigarettes reported. The cotinine concentrations are a more accurate measure of smoking compared to self-reported smoking data, reducing bias from reporting. Added to results.

Finally the English, while intelligible and certainly better than my Swedish, is non-standard. In the first paragraph the authors refer to the 21th rather than the 21st century. They also refer to "medical journals" which to me would mean publications such as PLOS ONE but I believe they mean "medical records". I think it would be important to have a native English speaker to proof read the manuscript before re-submission.

Response: The English language has been corrected.

REVIEWER 2

In the Materials and Methods it should be added that the matching was not complete in a 1:2 maner.

Response: One control was lost due lack of subjects at one center. In the method section we added the sentence “..191 matched controls were available to be included in further analyses.”

Why were not previous smokers/snuff users as previous users rather than non-users? This may affect the findings.

Response: There were 67 subjects who were reported as previous smokers and 33 subjects who were reported previous snuff users. The questionnaires did not include information at what time stopped using tobacco neither for how longtime they used tobacco or degree of smoking. Therefore, we believe that these subjects were better handled in the group “non-smokers”. This was a conservative approach as this could reduce difference between groups. Despite this classification we showed a clear increased risk for smokers to develop IBD.

Also, and even more important, there is a continuous unclearity throughout the manuscript (and tables) on cotinine levels being the

cut off for smokers. Should this not be changed to tobacco users instead as smokers and snuff users later are analysed separately?

Response: Thank you for the suggestion, we have changed to tobacco users for categories defined by cotinine.

In the Results I would prefer to have cotinine levels evaluated first and then separate for smokers and snuff users.

Response: In table 1 we added cotinine-levels for smokers, snuff-users, users of both snuff and cigarettes and for those not using any tobacco.

Regarding all tables I would like to have them moved from a separate column at the end to be added in each column respectively. Further you

should add % in the columns as this is unclear at the moment.

Response: We have added ”%” in the tables. However, due to different missing subject in each category we think that a separate column is necessary to state number of subject in each analysis (see below).

I do not get the numbers in the tables to add up as expected. E.g. in table 1 Smoking (tobacco user?) is 87/170 but regarding # of

cigarettes/day 83/164. In table 2 # cig/day is only 83/156. Non-smokers in table 1 is 96/161 while in table 2 85/188. Same disparities apply for tables 3 and 4.

Response: The study includes different parameters for defining smoking. Unfortunately, there was no complete data for all the included parameters due to missing values in the questionnaires and one patient and one control subject was lost in the cotinine analysis.

1. Number of cigarettes: Based on questionnaires (n=83/166)

2. Smokers and non-smokers: Based on questionnaires (n=87/170)

3. Smokers defined by Cotinine measurements (n=95/190)

In the multivariate analysis the number drops.

Was there any data on smoking status at time of diagnosis? Would be highly interesting, especially regarding the increased risk for UC patients.

Response: We agree, but unfortunately, the data on tobacco use at the time of diagnosis of IBD was not accurate enough to be included in the study.

We have added a comment on this both in the method section and in the discussion section.

In the discussion there I would like more discussion on the found risk for UC among smokers/tobacco users. Also there is a recent Swedish publication for incidence for late onset of IBD (Everhov et al) whichmight be more appropriate in a national epidemiological maner.

Response: In the discussion section we have added Everhov et al as a reference.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

18 Jun 2020

Elevated plasma cotinine is associated with an increased risk of developing IBD, especially among users of combusted tobacco

PONE-D-19-31583R1

Dear Dr. Widbom,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Stefanos Bonovas, M.D., M.Sc., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I find that the authors have commented to all questions and changed the manuscript accordingly and improving the manuscript satisfactory.

Reviewer #3: Authors regurarly responded to all the questions, and the suggestions, posed by the previous reviewers. No major issues arised from this second revision.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No