Associations between snus use and concentrations of CRP, 25(OH)D and testosterone: a population-based study

May combine two claims.

Among men, hs-CRP results were similar to the main results; among women, point estimates among snus users were lower and not statistically significant.

“Results from analyses on men were similar to our main results (Supplementary Table 4). Among women, the point estimates among snus users were lower and not statistically significant.”

May combine two claims.

There is no clear dose-response relationship between the amount of snus used and hs-CRP concentrations, but there is a trend of lower hs-CRP with longer duration of use.

“There was no clear dose-response relationship between the amount of snus used and hs-CRP-concentrations, but a trend of lower hs-CRP with longer duration of use (Supplementary Table 5).”

May combine two claims.

Male current snus users have a tendency towards lower odds of having a cfT concentration in the first quartile (OR 0.76, 95% CI 0.59;0.98, model 3), but this was non-significant in sensitivity analyses excluding occasional smokers.

“They had a tendency towards lower odds of having a cfT-concentration in the first quartile, OR 0.76 (95% CI 0.59; 0.98, model 3), but these results were non-significant in sensititivy analyses excluding occasional smokers (Supplementary Table 13).”

May combine two claims.

Analyses of cfT concentrations among women could not reveal any significant differences among current or former snus users compared to never-users, and there were no dose-response trends.

“Analyses of cfT-concentrations among women (Table 3b) could not reveal any significant differences among current or former snus users compared to never-users, and there were no dose-response trends (Supplementary Table 11b).”

May combine two claims.

Snus use for 10-20 years resulted in lower 25(OH)D concentrations compared to shorter use, but longer use beyond that did not further lower 25(OH)D concentrations.

“Snus use for 10-20 years resulted in lower concentrations compared to shorter use, but longer use beyond that did not further lower the 25(OH)D-concentrations (Supplementary Table 9).”

May combine two claims.

In dose-response analyses, use of 2-4 boxes/week and above resulted in lower 25(OH)D concentrations, supporting an exposure-related trend.

“In dose-response analyses, use of 2-4 boxes/week and above resulted in lower concentrations, supporting an exposure-related trend.”

May combine two claims.

Among female snus users, point estimates indicated increased cfT concentrations compared to never users, but the results were not statistically significant.

“Although our results were not statistically significant, possibly due to few non-smoking snus users, the point estimates indicated increased concentrations of cfT also among female snus users.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Smoking has been linked to increased testosterone concentrations in men.

“Smoking have been linked to increased testosterone concentrations in men [14].”

• cites [14]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Our research group performed analyses on cfT using radioimmunoassay in a subgroup of male participants (n=90) and compared them to BiomarCaRE results, showing differences in absolute values but good correlation in rank (Spearman 0.76), making quartile analyses more appropriate.

“Our research group has also performed analyses on cfT-concentrations using radioimmunoassay in a subgroup of male participants (n = 90) and compared them to the BiomarCaRE results.This comparison showed differences in absolute values (mean difference -0.07 nmol/L, standard deviation 0.09) but a good correlation in terms of rank (Spearman correlation coefficient 0.76), again making quartile analys”

Probably not a substantial claim: may not pass S3 (load-bearing).

Complete-case alcohol variable analyses only marginally changed the cfT results.

“Complete-case alcohol variable analyses only marginally changed the results (Supplementary Table 12).”

Classified as probably not a substantial claim.

We excluded the 99th percentile of hs-CRP (n=62, hs-CRP ≥21.26 mg/L) to eliminate subjects with highly inflammatory processes.

“To eliminate subjects with highly inflammatory processes, we excluded the 99 th percentile (n = 62, hs-CRP ≥21.26 mg/L).”

May combine two claims.

Snus users have lower concentrations of 25(OH)D and hs-CRP, irrespective of sex, while male users have higher cfT concentrations.

“snus users have lower concentrations of 25(Oh)D and hs-CRP, irrespective of sex, while male users have higher cft-concentrations.”

Classified as probably not a substantial claim.

We defined data-driven cut-off points based on the empirical distribution of the biomarker (quartiles) rather than relying on absolute clinical thresholds.

“We therefore defined data-driven cut-off points based on the empirical distribution of the biomarker in the study population (i.e.quartiles), in order to identify participants in the most extreme ranges rather than relying on absolute clinical thresholds.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Directed acyclic graphs were drawn to facilitate identification of possible confounding and mediating factors and were used to develop the multivariable models.

“We drew directed acyclic graphs (DAGs) to facilitate identification of possible confounding and mediating factors (Supplementary Figure 1) [28].The DAGs were used to develop our multivariable models.”

• cites [28]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Classified as probably not a substantial claim. May combine two claims.

All multivariable models were adjusted for age, sex and year of examination, and excluded participants reporting use of relevant medication.

“All multivariable models were adjusted for age, sex and year of examination, and excluded participants reporting use of relevant medication (e.g.anti-inflammatory medication in hs-CRP analyses).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Both low and high testosterone concentrations among men and women have been shown to increase CVD risk.

“Both low and high testosterone concentrations among men and women have been shown to increase CVD risk [13].”

• cites [13]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Snus use is associated with increased all-cause, cardiovascular and cancer-related mortality.

“There is growing evidence for an association between snus use and increased all-cause, cardiovascular and cancer-related mortality [2,3], but the underlying mechanisms for such associations are only partly explored [2,4].”

• cites [2]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [3]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Classified as probably not a substantial claim.

Measured or calculated free testosterone concentrations are considered to best reflect androgen status because only free or weakly bound testosterone can act on androgen receptors.

“Since only free or weakly bound testosterone can act on androgen receptors, measured or calculated free testosterone (cfT) concentrations are considered to best reflect androgen status [16].”

• cites [16]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Analyses including the dichotomized hypertension variable and complete case analyses of risky drinking did not alter the hs-CRP conclusions.

“Analyses including the dichotomized hypertension variable, and complete case analyses of risky drinking did not alter our con- clusions (Supplementary Tables 67).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Outcome biomarkers were measured with harmonized procedures at the BiomarCaRE laboratory in Germany between 2016 and 2018.

“Outcome biomarkers were measured with harmonized procedures at the BiomarCaRE (Biomarker for Cardiovascular Risk Assessment in Europe) laboratory in Germany between 2016 and 2018 [19,20].”

• cites [19]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [20]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Classified as probably not a substantial claim.

CRP is a marker of low-grade inflammation recognised as a contributing mechanism to major conditions including type 2 diabetes, cardiovascular diseases and cancer.

“One such emerging biomarker is C-reactive protein (CRP), a marker of low-grade inflammation which is recognised as a contributing mechanism to major conditions including type 2 diabetes, cardiovascular diseases (CVDs) and cancer [6].”

• cites [6]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Keeping 652 current and former occasional smokers in the sample was supported by sensitivity analyses showing only marginal change in estimates when excluded.

“652 current and former occasional smokers were kept in the sample after sensitivity analyses showed only marginal change in estimates when excluded.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Low 25(OH)D has been associated with CVD, cancer and increased mortality, although trials of vitamin D supplementation to prevent morbidity and mortality have not been successful.

“Low 25(OH)D has been associated with CVD, cancer and increased mortality, although trials of vitamin D supplementation to prevent morbidity and mortality have not been successful [9][10][11][12].”

• cites [9]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [10]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [11]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [12]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Cigarette smoking increases CRP concentrations.

“Previous studies have shown that cigarette smoking increases CRP-concentrations [7].”

• cites [7]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All models with continuous variables included at least 82% of the original cohort, except for cfT concentrations among women (70% in model 3).

“All models with continuous variables included at least 82% of the original cohort, except for cfT-concentrations among women (70% in model 3).”

May combine two claims.

The present study is considerably larger and excluded current and previous smokers, making these results more reliable than the previous study on snus use and CRP.

“The present study is considerably larger and excluded current and previous smokers, making these results more reliable.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The second model on testosterone included adjustment for BMI, adding physical activity, education and risky drinking to the third model.

“The second model on testosterone included adjustment for BMI, adding physical activity, education and risky drinking to the third model.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The mechanisms for lower 25(OH)D concentrations among smokers are unclear, but suggested to include disruption of parathyroid hormone and altered dermal vitamin D-conversion.

“The mechanisms for lower 25(OH) D-concentrations among smokers are unclear, but suggested to include disruption of parathyroid hormone and altered dermal vitamin D-conversion [9].”

• cites [9]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All results are based on complete case analyses except the alcohol variables, which were imputed based on median values within the same sex and age groups.

“All results are based on complete case analyses (Supplementary Table 3b), except the alcohol variables, that were imputed based on median values within the same sex and age groups.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The association between smoking and testosterone may be mediated through stimulation of gonadotropin-releasing and luteinising hormone, and inhibition of testosterone metabolism by cotinine.

“These associations may be mediated through stimulation of gonadotropin-releasing and luteinising hormone, and inhibition of testosterone metabolism by cotinine, the major nicotine metabolite present among all exposed to nicotine [30].”

• cites [30]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For logistic regression models, model fit was assessed using goodness-of-fit measures, and model discrimination was evaluated using the area under the receiver operating characteristic curve (AUC).

“For logistic regression models, model fit was assessed using goodness-of-fit measures, and model discrimination was evaluated using the area under the receiver operating characteristic curve (AUC).”

Probably not a substantial claim: may not pass S2 (checkable), S4 (based). May combine two claims.

The biomarker associations reflect underlying mechanisms that may differentially influence the elevated all-cause and cardiovascular mortality in snus users, with some augmenting risk and others attenuating it.

“These biomarker associations reflect underlying mechanisms that may differentially influence the elevated all-cause and cardiovascular mortality in snus users, with some augmenting risk and others attenuating it.”

• AssumptionRests on an assumption: Snus users have elevated all-cause and cardiovascular mortality.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Analyses of hs-CRP were further adjusted for education and physical activity in model 2, and for BMI, hypertension, DM and risky drinking in model 3.

“Analyses of hs-CRP were further adjusted for education and physical activity in model 2, and for BMI, hypertension, DM and risky drinking in model 3.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For linear regression models, assumptions of linearity, normality and homoscedasticity of residuals were assessed using residual plots and distributional diagnostics.

“For linear regression models, assumptions of linearity, normality and homoscedasticity of residuals were assessed using residual plots and distributional diagnostics.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Unmeasured confounding may have biased the estimates.

“As in all observational studies, unmeasured confounding may have biased the estimates.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Both low and high testosterone concentrations may increase cardiovascular risk.

“Previous research has indicated that both low and high concentrations may increase cardiovascular risk [13,31].”

• cites [13]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [31]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Among smokers, the association to lower 25(OH)D concentrations is well-studied.

“Among smokers, the association to lower 25(OH)D concentrations are well-studied [9].”

• cites [9]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

In a man with cfT of 300 pmol/L, a 4% increase corresponds to 12 pmol/L.

“In a man with cfT of 300 pmol/L, this increase corresponds to 12 pmol/L.”

May combine two claims.

The study had a large population-based sample, which enabled analyses on non-smoking men with solid statistical power.

“The main strength of this study was the large population-based sample, enabling us to perform analyses on non-smoking men with solid statistical power.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Testosterone excess in women has been linked to increased cardiovascular risk.

“Testosterone excess in women have been linked to increased cardiovascular risk [32].”

• cites [32]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

A recent Swedish study by Kimblad et al. found an increase of total testosterone by 3.0 nmol/L (p < 0.001), but no difference regarding cfT in snus users.

“This may be compared to the results from a recent Swedish study by Kimblad et al. who found an increase of total testosterone (tT) by 3.0 nmol/L (p < 0.001), but no difference regarding cfT in snus users [15].”

• cites [15]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Multicollinearity was assessed using variance inflation factors.

“Multicollinearity was assessed using variance inflation factors.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The BiomarCaRE laboratory takes several steps to prevent blood sample storage and handling effects, including immediate analyses after thawing and minimizing freeze-and-thaw cycles.

“However, the BiomarCaRE laboratory takes several steps to prevent this, including immediate analyses after thawing and minimizing number of freeze-and-thaw cycles.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Missing data was low in self-reported variables (0-1.6%) and biometric measurements ranged from 0.16 to 4.2%.

“Missing data was low in self-reported variables (0-1.6%,Supplementary Table 3a). Missing biometric measure- ments ranged from 0.16 to 4.2%.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Model 2 for 25(OH)D was additionally adjusted for Nordic origin and sampling month, while model 3 also included BMI, education and physical activity.

“Model 2 for 25(OH)D was additionally adjusted for Nordic origin and sampling month, while model 3 also included BMI, education and physical activity.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The biological pathways linking snus use, inflammation and cardiovascular disease remain incompletely understood.

“However, the biological pathways linking snus use, inflammation and cardiovascular disease remain incompletely understood [2].”

• cites [2]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Classified as probably not a substantial claim.

Smoking is associated with increased concentrations of free and total testosterone.

“Meanwhile, there is plenty of research on the association between smoking and testosterone, demonstrating increased concentrations of free and total testosterone [30].”

• cites [30]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Sporadic reports on testosterone and non-snus smokeless tobacco show increased tT concentrations among users.

“but sporadic reports on testosterone and non-snus smokeless tobacco show increased tT concentrations among users [14].”

• cites [14]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

A 12% lower hs-CRP among snus users would mean a lowering of 0.24 mg/L in a person with hs-CRP of 2 mg/L.

“The finding of a 12% lower hs-CRP among snus users would mean a lowering of 0.24 mg/L in a person with a hs-CRP of 2 mg/L.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Kimblad et al. adjusted for smoking rather than excluding smokers, as done in the present study, which may explain some of the differences.

“Kimblad et al. adjusted for smoking rather than excluding smokers, as done in the present study, which may explain some of these differences.”