Cortisol levels in hair are altered in irritable bowel

Journal of Psychosomatic Research 93 (2017) 69–75

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Journal of Psychosomatic Research

Cortisol levels in hair are altered in irritable bowel syndrome - A case control study in primary care A.-K. Norlin a,⁎, S. Walter b, E. Theodorsson c, V. Tegelstrom d, E. Grodzinsky e,f, M.P. Jones g, Å. Faresjö a a

Division of Community Medicine, Department of Medicine and Health Sciences, Linköping University, Sweden Division of Gastroenterology, Department of Clinical and Experimental Medicine, Linköping University, Sweden Division of Clinical Chemistry, Department of Clinical and Experimental Medicine, Linköping University, Sweden d National board of forensic medicine, Sweden e Department of Social and Welfare Studies, Linköping University, Norrköping, Sweden f Division of Drug research, Department of Medicine and Health Sciences, Linköping University, Sweden g Psychology Department, Macquarie University, Sydney, NSW, Australia b c

a r t i c l e

i n f o

Article history: Received 7 June 2016 Received in revised form 13 December 2016 Accepted 14 December 2016 Available online xxxx Keywords: Irritable bowel syndrome Stress Cortisol in hair HPA-axis Primary care

a b s t r a c t Objective: Stress is an important component in the pathophysiology of irritable bowel syndrome (IBS). Long term Hypothalamus Pituitary Adrenal (HPA)-axis activity can be studied by measuring hair cortisol concentrations (HCC). Some previous studies have indicated a dysregulated HPA-axis in IBS patients, but cortisol levels in hair have not yet been studied. We investigated whether HCC and self-reported stress differentiate IBS patients from controls. Methods: In a cross-sectional study within 10 Swedish Primary Health Care Centers we compared patients in working age with active IBS to patients without GI complaints. The participants donated hair samples and completed questionnaires including a scale of self-reported perceived stress (PSS). 169 Rome III-fulfilling IBS patients and 316 non-IBS patients were available for final analyses. Results: IBS patients had significantly lower HCC, median = 16.3 pg/mg, IQR = 26.9 pg/mg, compared to non-IBS patients, median = 22.8 pg/mg, IQR = 29.1 pg/mg. There was also a difference in the distribution of HCC quintiles between the two groups, with 30.2% IBS patients and 14.2% of non-IBS patients in the lowest quintile of HCC. PSS was higher among IBS patients with a mean (SD) total score of 25.3 (8.0) compared to controls 21.4, (7.5). Quintiles of HCC and PSS stayed significantly but very weakly related to IBS (B = −0.332, Std error = 0.146, p b 0.005) in multivariable analyses. Conclusion: This study suggests a possible suppression of the HPA-axis activity in a considerable portion of IBS patients. © 2016 Published by Elsevier Inc.

1. Introduction Irritable bowel syndrome (IBS) is a symptom-based diagnosis and affects 10–25% of the general population. It has a female predominance and the majority is diagnosed in primary care [1,2]. Quality of life among IBS patients is impaired with the illness seriously affecting everyday working and social life in addition to an increased use of health care resources [3]. Stress plays a major role in the complex pathophysiology of IBS [4– 6]. Early adverse life events are associated with a vulnerability to develop IBS [7]. Later on in life stress plays crucial parts in the first onset of IBS and also in the severity of gastrointestinal (GI) symptoms [8].

⁎ Corresponding author at: Division of Community Medicine, Department of medicine and Health Sciences, Linköping University, SE-581 83, Linkoping, Sweden. E-mail address: [email protected] (A.-K. Norlin).

http://dx.doi.org/10.1016/j.jpsychores.2016.12.009 0022-3999/© 2016 Published by Elsevier Inc.

The individual's response to physical and psychological stress includes activation of the hypothalamic pituitary adrenal (HPA)-axis and the autonomic nervous system [9]. Corticotrophin releasing factor involved in the HPA-axis, and also expressed in the gut, modulates inflammation, visceral hypersensitivity, gut permeability and motility [10–11]. The reverse is also true and the gut influences processes in the brain [12]. This reciprocal relationship has also been suggested regarding everyday perceived stress and gastrointestinal (GI) symptoms [13]. Hypocortisolism is thought to be an important but not yet completely understood maladaptive consequence of chronic stress exposure and HPA-axis dysfunction resulting in a vulnerability to develop stress-related bodily disorders [14–15]. Exposure to chronic stress initially activates the HPA axis producing elevated secretion of cortisol but over time the activity subsides and cortisol secretion falls below normal levels [15]. A dysregulated HPA-axis in IBS-patients has previously been suggested [16]. A recent study showed lower morning cortisol levels as

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A.-K. Norlin et al. / Journal of Psychosomatic Research 93 (2017) 69–75

well as a reduced total release of cortisol in the morning in females with IBS compared to controls [17]. In a meta-analysis from 2011 there was no statistically significant difference in baseline levels of cortisol in IBS patients compared to controls [18]. However baseline levels of cortisol in studies are often measured just before sigmoidoscopy or rectal extensions, which may elicit acute anticipatory responses with a hyperactive HPA axis [19]. In previous studies of IBS, cortisol was measured in blood, saliva or urine. These are acute, point-in-time measures that therefore can only relate to a short preceding time interval of stress exposure [20]. However those point-in-time measures have been used to study changes in the normal diurnal rhythm and response to psychological and physiological stress [16–17]. Recently a method of measuring cumulative concentration of cortisol for longer periods has been developed through the measurement of hair cortisol concentrations (HCC) [20–21]. Cortisol is retained in the hair strands [20] that grows at an approximate rate of 1 cm per month [22]. Through extracting cortisol an index of cumulative exposure over an extended period is provided. The method has been demonstrated to be valid and reliable [23]. To date, cortisol in hair levels as a measure of HPA axis activity averaged over a period of time has not been studied in IBS. Since stress and other psychological factors, which have been shown to be relevant to IBS, are dispositional or trait characteristics, it is expected that HCC will be a relevant marker of physiologic stress response and add further information on the previously suggested dysregulated HPA-axis in IBS. We hypothesized that hair cortisol levels in IBS patients would be lower than those found in non-IBS controls and that IBS patients would demonstrate higher levels of self-reported stress.

2. Material and methods The study adopted a case-control design in a defined region in south-east Sweden (The County Council of Östergötland). Ten Primary Health Care centers (PHCs), in the three major cities of the region, joined the study. These PHCs are responsible for primary care of a population of around 150, 000 inhabitants (about 1/3 of the region). Of the three cities, one could be labeled as a white-collar city (academic) and two as blue-collar cities (industrial) [24]. The selected ten PHCs were chosen to ensure diversity concerning socioeconomic status, age of the population and number of immigrants. 2.1. Selection and inclusion process As illustrated in Fig. 1, IBS and non-IBS control groups were identified in a two-step process. They were initially identified with ICD-10 diagnoses in the medical registers of the PHCs, then verified as meeting the ROME III criteria for IBS or not. Patients within the normal working age range (18–65 years) with a clinical IBS diagnosis identified with ICD-10 diagnoses in the medical registers of the selected PHCs were invited by mail to participate in the study. Potential controls were identified as other patients at the selected PHCs. They were similar in terms of sex and age and had sought care for other complaints not associated with gastrointestinal (GI) symptoms and had no earlier GI diagnoses found in the patient register for the previous two years. The individuals who agreed to participate were given an appointment at the PHC, where trained staff cut a hair

Fig. 1. Flow chart of drop-outs and exclusions.


sample and handed out questionnaires alongside a pre-paid envelop to return. All IBS patients that were included reported active symptoms during the 2 years prior to inclusion. Recruitment was conducted between March 2010 and February 2014. A total of 400 potential IBS-patients and 462 potential non-IBS controls agreed to participate. 191 persons did not complete the questionnaire and of the remaining patients, 31 (27 males and 4 females) failed to leave enough hair for analyses. In a second step in addition to the diagnose register and asking for symptoms, all individuals were asked to complete a questionnaire that included questions that enabled an IBS or control status using the ROME III criteria [25]. A total of 86 clinically diagnosed IBS patients who did not meet Rome III criteria were excluded from the study while four clinically-identified controls who did meet IBS criteria were classified as IBS (see Fig. 1). Finally answers regarding medication and comorbidities in the questionnaire revealed 3 IBS patients with an inflammatory bowel disease (IBD) and 6 controls with GI diagnoses. Those patients were excluded. Participants reporting topical or oral steroid medication were also excluded since they might interfere with the measurement of HCC (see Fig. 1). 2.2. Background data The questionnaire handed out at inclusion included demographic data and questions of general health, derived from The Swedish Living Conditions Survey of Health and Welfare [Statistics-Sweden. Employment, working hours and work environment 1994–95. Sveriges Officiella Statistik. Stockholm: Statistiska Centralbyrån (SCB), 1998.(In Swedish)]. The participants were also asked if they had experienced any serious life events during the last year. Comorbidities asked for in the questionnaire were divided into responses of “yes now” and “no never/earlier”. For analyses they were divided into categories of: major internal medical diseases (cardiovascular disease, hypertonia or diabetes), non-gastrointestinal pain disorders, sleeping disturbances and psychiatric disorders. Self-reported medication that could potentially affect the outcomes of PSS or HCC was classified into three categories; steroid inhalations, CNS-active medication and hormone medication (hormone replacement therapy, contraceptives and thyroid hormones). 2.3. Perceived stress scale For measuring perceived stress, we used the validated and widely used perceived stress scale-14 (PSS) [26]. It measures the degree to which the respondent has considered their lives to be unpredictable, uncontrollable and overloaded during the last month [26]. The respondents were asked how often they felt a certain way on a 5-point ordinal scale coded: never = 0, almost never = 1, sometimes = 2, fairly often = 3 and very often = 4. In the scoring algorithm positively worded items are reverse coded, consistent with recommended scoring methods [26]. The Swedish version as well as the original English has been demonstrated as reliable and valid [27]. 2.4. Analysis of cortisol concentration in hair Trained staff cut approximately 100 strands of hair from the posterior vertex area of the participants' heads in accordance with guidelines published by the Society of Hair Testing [22]. The hair was then enclosed in sealed plastic packages marked with identification numbers and stored in room temperature until analysis. All analyses were performed at the laboratory of clinical chemistry at the University of Linköping. We analyzed the first 3 cm of outgrowth for cortisol concentrations using a competitive radioimmunoassay in methanol extracts [28]. This hair segment reflects hair growth over the 3 months period prior to hair sampling [22,28]. At least 3 mg of hair was required for reliable measurements.

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Each sample was put into a 2 mL QiaGenRB sample tube with a 0.5 mm QiaGen stainless steel bead. The samples were weighed on Sartorius MC 210p microscale and homogenized using a Retch Tissue Lyzer II (20 HZ). The sample tubes were placed in aluminium cylinders and frozen in liquid nitrogen for 2 min and the hair samples were thereafter homogenized for 2 min, producing fine hair powder. 1 mL of methanol was added to each tube and the samples were extracted for 24 h on a moving board to keep the steel pellets in constant soft motion within the tubes. 0.8 mL of the methanol supernatant was pipetted off and lyophilized using a Savant Speed Vas Plus SC210A. The samples were dissolved in radioimmunoassay buffer and analyzed. Hair samples between 3 and 10 mg were required to maintain a total interassay coefficent of variation below 8% for hair extraction and measurement of cortisol by the radioimmunoassay. The intraassay coefficient of variation for the radioimmunoassay itself was 7% at 10 nmol/L. Antiserum cross-reacts by 137% with 5-alfa-dihydroxycortisol, 35.9% with 21-deoxycortisol and 35.9% with prednisolone, but b1% with endogenous steroids. The method is fully described elsewhere [24]. 2.5. Power and sample-size A sample size calculation regarding HCC was made using Altman's nonogram. The power was set to 0.80 and statistical significance at 0.05 (two-tailed). Previous studies revealed a standardized difference of approximately 0.3 between exposed and not-exposed [29–30] which yielded a total sample-size requirement across IBS cases and controls of approximately 350 individuals. Since our sample size exceeds that number we conclude that it has adequate statistical power. 2.6. Statistical analysis All data were stored in a common database and statistically analyzed using the SPSS version 21.0-22.0 software (SPSS Inc., Chicago, IL, USA). IBS cases and non-IBS controls were compared regarding demographic, socioeconomic and health-related background variables. Qualitative variables were reported through percentages within the group and the groups were compared using a chi square-test. Quantitative variables were reported as means and standard deviations and were compared using student's t-test. PSS-14 had a normal distribution and the total scores were reported in means and standard deviations and compared between groups with student's t-test. Hair cortisol concentrations were skewed towards higher values. 7 participants had hair cortisol values that could be considered extreme, above 900 pg/mg and their medical records were therefore reviewed. Out of those two cases and two controls were found to have topic or oral steroid medication, not reported in the questionnaire, and were hence excluded. One IBS patient with a HCC of 8520.5 pg/mg and two controls with HCC:s of 3690.4 and 6076.4 pg/mg were kept in the study. Original values are presented in median and interquartile range. Due to the skewness, HCC were logarithmic transformed before further analysis. Mean and standard deviation are presented. For an alternate, illustrative view we also calculated quintiles of hair cortisol i.e. the population studied divided into fifths. Differences between IBS patients and controls with respect to the distribution of quintiles were analyzed using chi-square test. Correlations were calculated using Pearson's correlations. Correlations are reported as very weak (b 0.3), weak (0.3–0.5), moderate (0.5–0.7) or strong (N0.7). Analysis of covariance, implemented via multiple linear regression was used to adjust for potential confounders among all background variables. Unstandardized coefficients and standard errors are presented. Statistically independent predictors of cortisol were selected by backward elimination and reported in Table 3. The multiple regression section of Table 3 should be read column-wise as indicating which IBS and background variables are statistically significantly and independently associated with each dependent variable. To adjust for the nonnormal distribution of cortisol we used the nonparametric bootstrap for formal statistical inference. The potential for multicollinearity was evaluated through variance inflation factor (VIF) analyses and there

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was no multicollinearity problem in the regression model. As is common in epidemiological studies, we interpret our findings concerning correlates of stress (the physiologic response and self-reported) as providing evidence to support our hypotheses rather than definitively proving them to be correct. For this reason we have not adjusted for the number of hypothesis tests performed and interpret any p-value b0.05 (two-tailed) as statistically significant. A response bias analyse was made regarding drop-outs that did not complete the questionnaire or provide hair for analysis. This revealed that the non-responders were more frequently male and younger than the responders in both groups (Supplementary table 1). Participants with missing values were excluded pairwise in the statistical analyses. 10 IBS patients and 9 controls that were included did not give complete answers to the PSS questionnaire. There was only a small fraction of missing values in the background variables. That is b1% in all variables except for hormone medication (2.7%) and CNS-active medication (2.9%). There was no difference in age between responders of PSS and non-responders. 2.7. Ethical considerations The study was approved by the Regional Ethical Research Committee at Linköping University (Dnr M41-09). Written informed consent was supplied by all participants. 3. Results 3.1. Sociodemographic and clinical characteristics IBS patients were older than non-IBS patients. Regarding gender and psychosocial environment the two groups were more comparable. IBS patients tended to live more alone and work less than non-IBS patients and more IBS-patients were born abroad. IBS patients were more likely to report extra intestinal pain disorders, sleeping disturbances and psychiatric comorbidities than non-IBS patients, but the major internal medical diseases were similarly reported in both groups. IBS patients reported more CNS-active medication, and hormone medication. Steroid inhalations and smoking were similarly frequent reported in both groups. IBS patients also had somewhat lower education, however not statistically significant. Finally IBS patients were more likely to report severe life events during the last year (Table 1). 3.2. HCC and PSS in IBS patients compared to non-IBS controls IBS patients had statistically significantly lower HCC and higher PSS scores compared with non-IBS patients. Correspondingly, there was also a statistically significant difference in the distribution of the quintiles of HCC between IBS and non-IBS patients. The largest proportion of IBS patients was found in the lowest hair cortisol quintile. The opposite was seen in non-IBS patients, where the smallest proportion was found in the lowest quintile (Table 2, Fig. 2). Hair cortisol concentrations and perceived stress scores in the different categories of all background variables are presented in the Supplementary table 2. 3.3. Multivariable associations of HCC and PSS Univariate and multivariable associations between the three dependent stress variables on the one hand, and IBS and other relevant background variables on the other, are shown in Table 3. PSS and quintiles of HCC remained significantly, but very weakly related to IBS in the final regression model. The correlation between IBS and HCC was very weak and negative while the correlation of IBS and PSS was very weak and positive. For the study population as a whole, several, generally very weak, associations regarding background variables and the dependent variables were identified. Older participants had somewhat lower

Table 1 Background characteristics. Background characteristics

IBS patients

Non-IBS patients

p

Age

51.5 (11.6)

45.6 (14.2)

b0.0001

86% (146) 14% (23)

85% (269) 15% (47)

0.706

29% (48) 72% (121)

17% (55) 83% (261)

0,005

56% (95) 44% (74)

54% (172) 46% (144)

0.707

37% (62) 24% (41) 39% (65)

52% (164) 21% (67) 27% (85)

0.005

15% (26) 46% (77) 39% (66)

9% (28) 44% (139) 47% (147)

0.059

82% (139)

92% (289)

0.002

43% (73)

46% (146)

0.506

17% (28) 57% (96) 37% (63) 23% (39)

14% (46) 39% (126) 19% (61) 5% (17)

0.557 b0.0001 b0.0001 b0.0001

9% (15) 37% (59) 20% (32)

7% (23) 16% (51) 13% (39)

0.533 b0.0001 0.036

53% (89)

39% (122)

0.003

Gender Women Men Subtype of IBS Constipation-predominant IBS Diarrhea-predominant IBS Mixed IBS Unspecified IBS Marital status Living alone/with parents Married de facto Psychosocial environment Living in a blue-collar city Living in a white-collar city Employment status Full-time work Part-time work Unemployed Educational level Low Medium High Native Born in Sweden Smoking habits Ever been a regular smoker Self-reported comorbidities (current) Cardiovascular disease, hypertonia or diabetes Other pain disorders Sleeping disturbances Psychiatric disease Self-reported medication Steroid inhalations CNS-active medication HRT, oral contraceptives and thyroid hormones Severe life event during the last year

12% (21) 27% (46) 51% (87) 9% (15)

Categorical data is presented with percentages (counts within brackets) and compared between groups with a chi-squared-test. Age is presented with means and s.d and compared between groups with student's t-test.

HCC and higher PSS. The quintiles of HCC were also very weakly correlated with gender and the psychosocial environment. More females and participants from the blue collar city were in the lowest quintile. Smokers had somewhat lower HCC but there was no significant correlation between smoking and PSS. Psychiatric comorbidity was very weakly associated with lower HCC and weakly associated with higher PSS. Further some variables were associated with PSS but not HCC. There was a very weak positive correlation between PSS score and sleeping Table 2 Outcome measures. Outcome measures

IBS patients

Non-IBS patients

p

HCC (pg/mg) HCClog(pg/mg) Quintiles of HCC Low Quite low Medium Quite high High PSS-14

16.3 (26.9) 3.0 (1.1)

22.8 (29.1) 3.3 (1.0)

0.022

30.2% (n = 51) 19.5% n = (33) 15.4% (n = 26) 17.8% (n = 30) 17.2% (n = 29) 25.3 (8.0)

14.2% (n = 20.3% (n = 22.8% (n = 21.2% (n = 21.5% (n = 21.4 (7.5)

45) 64) 72) 67) 68)

0.001

0.000

Original hair cortisol being not normally distributed is presented in median and IQR. The log transformed hair cortisol and PSS-14 are presented with mean and s.d. Comparison between groups are done with student's t-test. Quintiles of hair cortisol are presented in percentages (counts within brackets) and a chi-squared test was applied. HCC = hair cortisol concentrations, PSS = perceived stress scale.

A.-K. Norlin et al. / Journal of Psychosomatic Research 93 (2017) 69–75 35% 30% 25% 20% IBS-patients (%, n=169) 15%

Controls (%, n=316)

10% 5% 0%

1 (low)

2

3

4

5 (high)

Fig. 2. Quintiles of hair cortisol concentrations. Distributions (percentage) of cortisol levels (pg/mg) divided into quintiles. 1. (low) 3.1–11.4 (median = 8.00), 2. 11.5–16.2 (median = 14.0), 3. 16.3–25.3 (median = 20.8), nIBS = 30, nnon-IBS = 67, 4. 25.6–47.2 (median = 32.4), 5 (high) 47.3–8520.5 (median = 84.4)

disturbances, CNS-active medication and severe life events during the last year. Also PSS score was higher among unemployed and participants with low education. No associations were seen between the dependent stress variables and major internal diseases, steroid inhalations or hormone medication. 4. Discussion The most important finding of this study is a possible overrepresentation of low HCC individuals among IBS patients. At the same time IBS patients reported higher perceived stress than non-IBS patients, confirming a well-established relationship between psychosocial stress and IBS. This is, to our knowledge, the first study with HPA axis activity averaged over a period of time (3 months) in IBS. Our findings suggest that a portion of IBS patients could suffer from hypocortisolism, due to a suppressed HPA-axis. This supports the findings in some previous work of a dysregulated HPA-axis in IBS [16–17] and in general in stress-related bodily disorders [14]. One explanation for the low HCC in some IBS patients could be experiences of negative early life events. Several studies have shown associations between childhood trauma and an altered HPA-axis later in life

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[31–32]. Childhood trauma has also been associated with IBS [7] and HPA-axis responsiveness [33]. In further research on HCC and IBS the question of early life events is of great interest. In the present study IBS patients reported more severe life events during the last year than controls. That however had no correlation to HCC, but a positive correlation to PSS. In a study of post-traumatic stress syndrome it has been described that HCC changes from initially elevated levels relative to controls, to later becoming suppressed [34]. This study was not designed to capture such changes. A possible explanation to the overrepresentation of serious life events during the last year among IBS patients in the present study could also have been a selective memory for negative material as previously reported in IBS patients [35]. Another possible explanation for the suggested hypocortisolism in a portion of the IBS patients could be the gastrointestinal symptoms themselves being stressful and through negative feedback mechanisms in the long run causing a suppressed or dysregulated HPA-axis. In this matter of distinguishing order of events, a longitudinal study would be required. It could further also be interesting to study associations between symptom burden and HCC. A strength of the present study is that we controlled for many important potential confounders related to HCC and PSS with multivariable regression analyses (Table 3). In the regression analyses there was no multicollinearity problem indicating that none of the statistically significant predictor variables could be purely a reflection of other independent variables that we measured. There could of course be other predictors of importance beyond those measured in this study. In the regression model there is an interesting very weak association between the social environment and HCC. The participants living in a blue collar city were, like IBS patients, also overrepresented in the lowest quintile of HCC. It is likely that early life events are more common in the blue collar city with more psychosocial problems [36]. However there was no association between the psychosocial environment and PSS. We also found that patients with self-reported psychiatric comorbidities were overrepresented in the lowest quintile of HCC and were weakly associated with higher PSS. Our study, like earlier studies, shows an overrepresentation of psychiatric comorbidity in IBS [37]. The multiple regression analyses showed that the associations between IBS and the dependent stress variables were independent of that. The results

Table 3 Univariate and multivariable analyses. Univariate correlations (r)

Irritable bowel syndrome Age Gender (male vs female) Marital status (single vs married de facto) Psychosocial environment (white-collar vs blue-collar town) Native (born in Sweden vs born abroad) Employment status (Full-time, part-time, unemployed) Educational status (low, medium, high) Smoking habits (ever been a smoker vs never been a smoker) Cardiovascular disease/Hypertonia/Diabetes (yes vs no) Other pain disorder (yes vs no) Psychiatric comorbidity (yes vs no) Sleeping disturbances (yes vs no) Steroid inhalations (yes vs no) CNS-active medication (yes vs no) Hormone medication (yes vs no) Severe life event the last year (yes vs no)

Multiple regressions (B (Std error))

HCClog

Quintiles of HCC

PSS-14

−0.107⁎ 0.115⁎

−0.146⁎⁎ 0.101⁎ −0.103⁎

0.232⁎⁎ −0.191⁎⁎ 0.029 −0.080 −0.003 −0.137⁎⁎ 0.106⁎ −0.110⁎ 0.024 −0.044 0.145⁎⁎ 0.353⁎⁎ 0.209⁎⁎

−0.085 0.075 −0.047 −0,007 0.027 −0.002 −0.104⁎ 0.022 −0.017 −0.111⁎ −0.034 0.023 −0.034 −0.066 −0.022

0.077 −0.097⁎ −0,000 0.022 −0.012 −0.115⁎ 0.031 −0.041 −0.124⁎⁎ −0.052 0.005 −0.082 −0.041 −0.005

−0.037 0.171⁎⁎ 0.070 0.122⁎⁎

HCCloga

Quintiles of HCCb

PSS-14c

−0.136 (0.102) p = 0.226 0.008 (0.004)⁎ −0.271 (0.125)⁎

−0.332 (0.146)⁎

1.647 (0.759)⁎ −0,089 (0.027)⁎

−0.418 (0.173)⁎ −0.304 (0.126)⁎

−0.342 (0.127)⁎

−0.265 (0.135) p = 0.053

−0.420 (0.197)⁎

6.975 (1.125)⁎⁎ 2.691 (0.811)⁎

R2 = 0.186

R2 = 0.063

R2 = 0.179

Pearson's correlation coefficients are presented. For the multivariable regression analyses the models were selected through a backward elimination exercise. The values for the variables of the final models are presented. For quintiles of and logarithmic HCC, the unstandardized coefficients in the multivariable regressions were estimated through bootstrapping. HCC = hair cortisol concentrations, PSS = perceived stress scale. ⁎ p b 0.05. ⁎⁎ p b 0.001. a df = 4, F = 4302, p = 0.002. b df = 5, F = 6412, p = 0.000. c df = 4, F = 25.051 p = 0.000.

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however indicate that there could be common pathophysiologic mechanisms in IBS and at least some psychiatric comorbidities. Low HCC has previously also been seen in general anxiety disorder [38]. In our study we did not distinguish between different psychiatric disorders, which would be of interest in future studies. As seen in some previous studies we found an association between lower HCC and female gender [20]. Female gender is also associated with IBS, but the sex distribution was similar in the control group and gender was further controlled for in the multiple regression model. As seen in Supplementary table 1, a great proportion of non-responders were male. This was however similar in both groups and with IBS being a female-predominant disorder we believe the conclusion is still valid. This study has some limitations. Most importantly in the regression analyses all significant associations were very weak or weak, which may indicate that many factors each very weakly affect the outcome. The comorbidities being self-reported are another weakness. Some factors which have previously been reported to be associated to HCC were not elucidated. These were alcoholism, pregnancy or breastfeeding and BMI or abdominal obesity. However this is not likely to be a serious limitation since, to our knowledge, there are no associations between these factors and IBS. Considering the mean ages of the participants, not many would likely have been pregnant or breastfeeding. Further, rising BMI has been suggested to be a consequence and not a cause of high cortisol levels [20]. Neither were cortisol-related endocrine disorders specifically asked for. These are however rare conditions without, to our knowledge, known associations with IBS. Another weakness of the study is the somewhat older IBS cases than controls. However HCC is known to increase with age [20], which suggests that the association between low HCC and IBS could have been even stronger with an equal age distribution. In both groups also responders were older than nonresponders, which is a limit and our findings are most certain regarding female patients in the older working age. This study lines up with recently many previous in pointing out that measuring HCC plays an important role in the research of stress-related disorders [20,21,23,31,34,38,39]. To our knowledge the highest possible physiologically plausible HCC is unknown and no clinical standards or reference values have so far been presented. An international interlaboratory process is ongoing towards establishment of benchmark reference values [40]. The biological samples of HCC that could be considered extreme values in this study were replicated and analyzed on two independent occasions giving the same results. The medical records were reviewed and even though not reported in the questionnaire, four participants were seen to have cortisone treatment, which required them to be excluded. In order not to risk introduction of selection bias the remaining three participants with HCC that could be considered extreme, were kept in the study even though the associations we present would have been stronger with all possible outliers omitted from the study. The cortisol values were logarithmic transformed in the statistical analyses to reduce the variation possibly caused by extreme values and also bootstrapping was used to further correct for non-normality. A strength of the study is that it was undertaken in a primary care population where most IBS patients are diagnosed [2]. We also believe that our selection of controls, being other patients without GI complaints is a strength in order to find associations to IBS itself and not factors involved in being a primary care patient. Another strength is that we had access to all participants' medical records and the material was checked thoroughly when needed. 5. Conclusion For the first time HCC has been measured in IBS and revealed a possible suppression of the HPA-axis activity in a considerable portion of IBS patients. Hence IBS-related symptoms in these patients could be associated with hypocortisolism. This study also supports previous findings that IBS is associated with psychosocial stress. However all significant associations in this study were very weak or weak. Further

prospective studies regarding IBS, HCC, early life events and gastrointestinal symptom burden would add important information in order to understand the role of a dysregulated HPA-axis in the complex pathophysiology of IBS and the gut-brain axis. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jpsychores.2016.12.009. Acknowledgments, funding and disclosure The authors wish to thanks all the Primary Health Care Centers and patients which participated in the study. This study was partly funded by a grant from FORSS (Research fund in South of Sweden). ÅF, EG and SW participated in the study design and coordination, AKN and VT completed the data collection. ÅF, EG, VT, AKN, SW, ET and MPJ analyzed the data and drafted the manuscript. All authors contributed to analysis and interpretation of data, and read and approved the final manuscript. No competing interests declared. Funding This study was supported by the research council of Southeast Sweden (FORSS). Competing interest The authors declare that they have no competing interest. Financial disclosure The authors have no conflicts of interest relevant to this article to disclose. References [1] W.G. Thompson, K.W. Heaton, C. Smyth, Irritable bowel syndrome in general practice: prevalence, characteristics and referral, Gut 46 (2000) 78–82. [2] L. Agreus, K. Svardsudd, O. Nyren, et al., Irritable bowel syndrome and dyspepsia in the general population: overlap and lack of stability over time, Gastroenterology 109 (1995) 671–680. [3] N.J. Talley, R. Spiller, Irritable bowel syndrome: a little understood organic bowel disease? Lancet 360 (2002) 555–564. [4] E.A. Mayer, The neurobiology of stress and gastrointestinal disease, Gut 47 (2000) 861–869. [5] E.A. Mayer, J.S. Labus, K. Tillisch, et al., Towards a systems view of IBS, Nat. Rev. Gastroenterol. Hepatol. 12 (2015) 592–605. [6] L. Chang, The role of stress on physiologic responses and clinical symptoms in irritable bowel syndrome, Gastroenterology 140 (2011) 761–765. [7] K. Bradford, W. Shih, E.J. Videlock, et al., Association between early adverse life events and irritable bowel syndrome, Clin. Gastroenterol. Hepatol. 10 (2012) 385–390. [8] W.E. Whitehead, M.D. Crowell, J.C. Robinson, et al., Effects of stressful life events on bowel syndrome compared with subjects without bowel dysfunction, Gut 33 (1992) 825–830. [9] G. Chrousos, P. Gold, The concepts of stress and stress system disorders, JAMA 267 (1992) 1224–1252. [10] Y. Taché, B. Bonaz, Corticotropin-releasing factor receptors and stress-related alterations of gut motor function, J. Clin. Invest. 117 (2007) 33–40. [11] C. Wallon, P.C. Yang, A.V. Keita, et al., Corticotropin-releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro, Gut 57 (2008) 50–58. [12] K. Saito, T. Kasai, Y. Nagura, et al., Corticotropin-releasing hormone receptor-1 antagonists blocks brain-gut activation induced by colonic distention in rats, Gastroenterology 129 (2005) 1533–1543. [13] E.B. Blanchard, J.M. Lackner, J. Jaccard, et al., The role of stress in symptom exacerbation among IBS patients, J. Psychosom. Res. 64 (2008) 119–128. [14] C. Heim, U. Ehlert, H. Dirk, et al., The potential role of hypocortisolism in the pathophysiology of stress-related bodily disorders, Psychoneuroendocrinology 25 (2000) 1–35. [15] G.E. Miller, E. Chen, E.S. Zhou, If it goes up, must it come down? Chronic stress and the hypothalamic-pituitary-adrenocortical axis in humans, Psychol. Bull. 133 (2007) 25–45. [16] L. Chang, S. Sundaresh, J. Elliot, et al., Dysregulation of the hypothalamic-pituitaryadrenal (HPA) axis in irritable bowel syndrome, Neurogastroenterol. Motil. 21 (2009) 149–159. [17] C. Markert, K. Suarez-Hitz, U. Ehlert, et al., Endocrine dysregulation in women with irritable bowel syndrome according to Rome II criteria, J. Behav. Med. 39 (2016) 519–526.

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