Increased C-reactive protein levels in participants with frequent headache. The HUNT Study 2006-2008

Background Increased high sensitivity C- reactive protein (hs-CRP) levels have been found in many earlier studies on migraine, and recently also in persons with migraine and insomnia. The aim of this study was to see whether these ndings could be reproduced in a large-scale population-based study. Methods A total of 50,807 (54%) out of 94,194 invited aged ≥ 20 years or older participated in the third wave of the Nord-Trøndelag Health Study study performed in 2006-2008. Among these, 38,807 (41%) had valid measures of hs-CRP and answered questions on headache and insomnia. Elevated hs-CRP was dened as >3.0 mg/L. The cross-sectional association with headache was estimated by multivariate analyses using multiple logistic regression. The precision of the odds ratio (OR) was assessed with 95% condence interval (CI). Results In the fully adjusted model, elevated hs-CRP was associated with migraine (OR 1.14, 95% CI 1.04-1.25) and migraine with aura (OR 1.15, 95% CI 1.03-1.29). The association was stronger among individuals with headache ≥ 7 days/month for any headache (OR 1.19, 95% CI 1.08-1.31), migraine (OR 1.30, 95% CI 1.10-1.54), migraine with aura (OR 1.47, 95% CI 1.20-1.80), and other headaches (OR 1.16, 95% CI 1.03-1.30). No clear relationship was found between elevated hs-CRP and headache less than 7 days/month or with insomnia. Conclusions Cross-sectional data from a large-scale population-based study showed that elevated hs-CRP was associated with headache ≥ 7 days/month, especially frequent migraine with aura.

The association between headache and insomnia is well-established [27], but the impact of insomnia on the relationship between hs-CRP and migraine is unclear. Previously, a prospective study has demonstrated higher level of CRP in individuals with shorter sleep duration [28]. In a recent population-based cross-sectional study from Northern Norway, elevated hs-CRP was associated with migraine, but only among those with co-existing insomnia [29]. However, since the results on hs-CRP and migraine are con icting, and the relationship to insomnia only reported in one study, it is important to replicate the analyses in other population-based study with similar design.
The aim of this large-scale population-based study was to evaluate the cross-sectional association between hs-CRP and types of headache, and to see whether the relationship to insomnia could be reproduced.

Study design
The present population-based study of a large unselected adult population replicate the methodological design used in the Tromsø Study [29], including similar questionnaire-based diagnoses for headache and insomnia, identical categories of hs-CRP, and replication of the statistical analyses with adjustments for the same type of potential confounders.

The Nord-Trøndelag Health Study
The total population of the Nord-Trøndelag county aged ≥20 years has been invited four times between 1984 and 2019. All participant answered questionnaires and were invited to interview, clinical examination, and blood sampling [30].

Study population
In HUNT3 (2006-2008), a total of 94,194 individuals aged ≥ 20 years were invited, whereof 93,860 were eligible. Of these, 50,807 (54%) accepted the invitation. The diagnoses of headache and insomnia was questionnaire-based. The present study included data from 38,813 participants (41%) who had answered questions about headache and insomnia, and had hs-CRP measured from non-fasting blood samples (Fig. 1).

C-reactive protein
Hs-CRP was analyzed at the Central Laboratory, Levanger Hospital, using Architect cSystem ci8200, by latex immunoassay method. The detection limit was 0.03 mg/L, and samples without detectable hs-CRP were assigned this value. In the present study we replicated the strategy used in previous published studies from the Tromsø study [29,31] de ning normal CRP as 0-3.00 mg/L and elevated as 3.01-20.00 mg/L. Participants with hs-CRP values >20.00 mg/L, which probably indicate some acute or chronic disease [32], were excluded (n=527) (Fig.1).

Headache questionnaire
All details of the headache questionnaire have been published elsewhere [33], and all questionnaire-based headache diagnoses were identical to those presented in the Tromsø study [29]. Subjects who answered "no" to the screening question ("Have you suffered from headache during the last 12 months?") were included in the reference group without headache.
Participants who answered "yes" completed 12 additional questions, mainly to diagnose migraine according to the second version of the International Classi cation of Headache Disorders (ICHD-2) [34]. Regarding attack duration, <4 hours were accepted for those who reported visual disturbance before headache. Participants who ful lled the migraine diagnosis and reported visual disturbance prior to headache were classi ed as migraine with aura (MA), whereas the remaining with migraine had migraine without aura (MO). The remaining participants with headache were classi ed as having "other headache". The merged group of "any headache" consisted of participants with migraine or other headache. Furthermore, based to answer on the question about headache frequency, subjects with headache were subdivided in two groups; headache < 7 days/month or headache ≥7 days/month.
The validity of the questionnaire-based headache diagnoses has been published in a separate paper [39]. The agreement between the validation interview and questionnaire-based headache categories were very good for any headache (kappa value at 0.70, 95 % CI 0.61-0.79) and moderate for migraine (kappa value 0.50, 95 % CI 0.32-0.68), whereas the speci city for MA was high (95 %) (kappa value 0.44, 95 % CI 0.38-0.50) (33).

Questionnaire-based diagnosis of insomnia
The questionnaire-based diagnosis of insomnia was based on the DSM-V (Diagnostic and Statistical Manual of Mental Disorders, 5th ed.), and was nearly identical to the insomnia diagnosis used in the Tromsø study [29]. To ful l the proxy diagnosis of insomnia the participants had to answer "several times a week" to at least one out of the three questions, asking whether they had "di culties falling asleep at night", whether they "woke up repeatedly during the night", and/or whether they "woke up too early and could not get back to sleep" [36]. In addition, they also had to report "several times a week" to the question "felt sleepy during day" [36]. The overall agreement between questionnaire and interview for the rst three questions has previously been found to be moderate (kappa value 0.51, 95% CI 0.40-0.63) [37].

Ethics
This study was approved by the Regional Committee for Medical and Health Research Ethics, the Faculty of medicine, mailbox 8905, 7491 Trondheim. The approval number was #2018/2422/Rek Midt. The participants have given written informed consent.

Statistical analysis
The statistical method was identical to that presented in the Tromsø Study [29], and based on multivariate analyses using multiple logistic regression. The precision of the odds ratio (OR) was assessed with 95% con dence interval (CI). We present results for three different statistical models separated by number of confounders included; model 1 (age and sex), model 2 (age, sex and BMI), and model 3 (all other factors were tested). In model 3 we excluded factors if they did not change OR when evaluating each factor separately or when including several factors grouped together (i.e. self-reported diabetes, stroke/heart infarction, and hypertension). Potential interaction between two variables was evaluated by including the product of the variable in the logistic regression analyses, and the interaction was tested using Wald χ 2 statistics. Subjects with missing data of confounding factors (numbers reported in Table 1) were included in the analysis to reduce the impact of possible bias.
To evaluate insomnia as a modifying factor on the association between hs-CRP and types of headache, we repeated the multiadjusted analyses in model 3 in those with and without insomnia.
Analyses were performed with the IBM SPSS version 25 (SPSS, Chicago, Illinois, USA)

Results
Subjects with headache were more likely to be women, younger, and to be employed in paid work compared to those without headache ( Table 1).

The association between elevated hs-CRP and headache
In the multivariate analyses, adjusting for age and sex, elevated hs-CRP was associated with any headache (OR 1.11, 95% CI The in uence of insomnia diagnosis As demonstrated by Table 3, the relationship between elevated hs-CRP and headache was only found among subjects without insomnia, whereas no such association was found among those with insomnia.

Discussion
Cross-sectional population-based data based on a large number of participants showed that elevated hs-CRP was associated with headache ≥ 7 days/month, especially frequent migraine with aura. No relationship was found between elevated hs-CRP and headache less than 7 days/month or with insomnia.
In agreement with the present results, previous studies have demonstrated that women with migraine had elevated CRP compared to women without migraine [4], and that children and adolescents with headache were more likely to be in the highest CRP quintile [9]. In a Dutch case-control study, individuals with MA were more likely to have elevated hs-CRP in the unadjusted analyses, and a borderline tendency was found in the multi-adjusted model [24]. In contrast, in a population-based study from Iceland, no difference in CRP levels were found between participants with and without migraine [6].
In the present study, the nal multi-adjusted analyses showed that MA, but not MO, was associated with elevated hs-CRP. This is in contrast with a review of previous studies on this topic, where the association to MA was not found to be consistently stronger than to MO [26]. It should be highlighted that an association between hs-CRP and average number of aura attacks and total number of years with aura were reported in the Dutch study [24].

Interpretation
In accordance with ndings in the Tromsø 7 Study [29], MA was most consistently associated with elevated hs-CRP. It may be of relevance that hs-CRP is associated with risk of cardiovascular events hs-CRP [38], and that ischemic cardiovascular disease has a stronger association to MA than to MO [39].
In this large (n=38,813) population-based study with presumably high statistical power, the association between elevated hs-CRP and headache was strongly dependent on frequency of headache. Correspondingly, elevated hs-CRP was associated with migraine ≥7 days/month, but not migraine <7 days/month in the Tromsø study (n=20,486) [29]. This relationship with headache frequency and elevated hs-CRP has not been clearly demonstrated in other studies [3,4,6,7,8,14,[18][19][20][21][22], possibly because of limited statistical power. The results of the present study may be explained by the nding that hs-CRP is related to general pain sensitivity [31]. Alternatively, our ndings may be related to potential sterile in ammation in migraine [26] or other types of headaches, most prominent for those with frequent headache. However, whether sterile in ammation is an important pathological feature in migraine and other headaches is still debated.
We evaluated insomnia as a modifying factor on the association between hs-CRP and headache. In contrast to the results from the Tromsø 7 Study [26], no association was found between elevated hs-CRP and headache among those with insomnia. The reason for the contrasting nding is unclear. However, it may be a spurious nding in the Tromsø 7 study. An alternative explanation may be that there are differences between participants with insomnia in the Tromsø 7 study and in the HUNT3 study. It should be highlighted that the Tromsø Study was performed at almost 70 degree north, with midnight sun and winter darkness periods, whereas the HUNT3 study was performed in middle of Norway at 63-65 degrees north where the difference between summer and winter light is less extreme. The de nition of insomnia in the Tromsø 7 study was stricter [29] than applied in the HUNT3 study [36], demanding symptoms three or more days per week instead of "several", and with dissatisfaction with sleep quality as mandatory. Despite this, the prevalence of insomnia was higher in Tromsø 7 than in the HUNT3 study (8.1% vs. 6.9%, p<0.001) [29,36]. The prevalence reported in the present study is lower than other populationbased studies in Norway [40], and we cannot exclude that our ndings would have been different using another insomnia questionnaire.
Strengths and limitations of the study The present study share several strengths and limitations with the study from Tromsø [29]. The major strengths of this study are the population-based design and the possibility to perform adjustments for all potential confounding factors previously included in the Tromsø study [29]. The large number of included participants give high statistical power and small con dence intervals.
The most important study limitation is the low participation rate (41%). Therefore, generalization of results should be performed with caution. It should also be highlighted that the cross-sectional design does not permit any conclusions about causality.
Furthermore, it is a limitation that the diagnoses of headache and insomnia were questionnaire-based, which may have led to misclassi cation. On the other hand, the agreement between questionnaire-based diagnosis of migraine and interview has previously been found to be acceptable [33]. Insomnia was diagnosed by applying modi ed DSM-V criteria. DSM-V asks for symptoms three times or more per week [35], whereas the response option "several times per week" was used in the present study [36]. Furthermore, some types of daytime impairment is required in the DSM-V criteria [35]. In this respect it should be noted that our question "felt sleepy during the day?" may not have been optimal way to assess actual sleepiness, as it more probably re ects tiredness and fatigue [36].

Conclusions
Cross-sectional data based on 38,007 participants showed that elevated hs-CRP was associated with headache ≥ 7 days/month, especially frequent migraine with aura. Elevated hs-CRP was not associated with headache less than 7 days/month or with insomnia. Availability of data and materials: Part of the dataset supporting the conclusions of this article is available on request to the corresponding author. Some of the data are the property of HUNT Research Centre and can only be accessed through direct contact with the research centre.
Authors' contributions KH: Study concept and design, collection of data, analysis and interpretation of data, writing of manuscript. LJS, KBN, and BSW: Interpretation of data, critical revision of manuscript. All authors read and approved the nal manuscript.

Ethics approval and consent to participate
This study was approved by the Regional Committee for Medical and Health Research Ethics, the Faculty of medicine, mailbox 8905, 7491 Trondheim. The approval number was #2018/2422/Rek Midt. The participants have given written informed consent.
Competing interest: None of the authors have any competing interests in the manuscript.

Consent for publication Not applicable
Competing interest: None of the authors have any competing interests in the manuscript.  Table 3. Multi-adjusted* odds ratio (OR) with 95% confidence interval (CI) of elevated high sensitivity Creactive protein (hs-CRP) defined as >3.0-20.0 mg/l related to headache types and insomnia status

Without insomnia
With insomnia Total N OR (95% CI) N OR (95% CI) No headache