Background: Despite the substantial number of older adults suffering from gastrointestinal (GI) symptoms little is known regarding the character of these complaints and whether they are associated with an altered intestinal barrier function and psychological distress. Our aim was to explore the relationship between self-reported gut health, intestinal permeability and psychological distress among older adults. Methods: Three study populations were included: 1) older adults with GI symptoms (n = 24), 2) a group of older adults representing the general elderly population in Sweden (n = 22) and 3) senior orienteering athletes as a potential model of healthy ageing (n = 27). Questionnaire data on gut-health, psychological distress and level of physical activity were collected. Intestinal permeability was measured by quantifying zonulin in plasma. The level of systemic and local inflammation was monitored by measuring C-reactive protein (CRP), hydrogen peroxide in plasma and calprotectin in stool samples. The relationship between biomarkers and questionnaire data in the different study populations was illustrated using a Principal Component Analysis (PCA). Results: Older adults with GI symptoms displayed significantly higher levels of both zonulin and psychological distress than both general older adults and senior orienteering athletes. The PCA analysis revealed a separation between senior orienteering athletes and older adults with GI symptoms and showed an association between GI symptoms, psychological distress and zonulin. Conclusions: Older adults with GI symptoms express increased plasma levels of zonulin, which might reflect an augmented intestinal permeability. In addition, this group suffer from higher psychological distress compared to general older adults and senior orienteering athletes. This relationship was further confirmed by a PCA plot, which illustrated an association between GI symptoms, psychological distress and intestinal permeability. Keywords: Older adults, Gastrointestinal symptoms, Intestinal barrier function, Psychological distress * Correspondence: firstname.lastname@example.org Nutrition and Physical Activity Research Centre, Department of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ganda Mall et al. BMC Geriatrics (2018) 18:75 Page 2 of 9 Background Ethical considerations In the last decades lifespan has increased dramatically, The study obtained ethical approval by the Uppsala but even though a longer lifespan is a success story in it- Regional Ethics Review Board (Dnr. 2012/309, 2013/37 self, it cannot be neglected that old age correlates with and 2015/357). All procedures were carried out in an increased need for health care . Thus, it is import- accordance with the declaration of Helsinki and written ant to identify areas through which a healthy ageing informed consent was acquired from all participants. process can be promoted in order to increase the pro- portion of independent free-living older adults . Questionnaires Gastrointestinal (GI) symptoms are common among Gastrointestinal Symptoms Rating Scale (GSRS) was older adults. Fifty to seventy % of older adults report used as an initial instrument to assess and define the symptoms of constipation [3, 4] and 4-14% experience degree of GI symptoms (Table 1). Reliability and validity problems with diarrhoea [5–7]. In addition, older adults of the GSRS is well documented . The recruitment suffering from diseases of the digestive system are at risk process was based on the GSRS score for the domains of a higher total symptom burden. . Moreover, it has depicting diarrhoea and constipation (average score ≥ 3 been established that a well functioning gut is essential of the domains for diarrhea and/or constipation) as these in order for older adults to experience life-satisfaction conditions are two of the most common GI complaints and meaningfulness in everyday life [9, 10]. Thus, among the elderly [24, 25]. The Hospital anxiety and gut health represents an important area through which depression scale (HADS) is a widely used validated health and wellbeing might be promoted. In support of instrument for the evaluation of psychological distress in this, we recently showed that senior orienteering ath- medical settings, as well as in older adults [26, 27]. The letes, a new potential model of healthy ageing, experi- Frändin-Grimby Activity Scale (FGAS) was used to ence less GI symptoms than general older adults . describe the level of physical activity . Questionnaire Even though age-associated GI symptoms are common details can be found in Table 3. in the older population, knowledge regarding the mecha- nisms behind these symptoms remains poor. Increased Biological samples intestinal permeability is a hallmark of many GI diseases Blood samples [12–14] but has not been investigated in older adults After an overnight fast, the participants were instructed who self-report GI symptoms. The intestinal epithelium not to exercise in the morning, before collection of the is the major interface with the external environment and blood samples. Plasma from senior orienteering athletes while absorbing nutrients and water it simultaneously was separated using the Ficoll-paque PLUS (GE Health- restrict the free movement of luminal material to the care Life Sciences, UK). For older adults suffering from underlying mucosa [15, 16]. A disruption of the intes- GI problems and general older adults, plasma was sepa- tinal barrier may result in the passage of microbial anti- rated using standard operating procedures used in the gens and toxins , and are associated with intestinal daily routine at the Örebro University Hospital. Blood inflammation as well as neurological diseases [18, 19]. samples were centrifuged at 2200 g for 10 min and Furthermore, elevated levels of reactive oxygen species plasma was collected in Eppendorf tubes. Blood samples (ROS) (e.g. oxidative stress) have been suggested to drive from 10 individuals were collected using both separation intestinal inflammation . Understanding the charac- procedures and subsequently analysed to evaluate the teristics of GI symptoms in older adults and their under- difference between the two techniques; in the following lying pathophysiology is important in order to facilitate analyses the difference was controlled for. diagnosis and treatment. Here, we aim to investigate Systemic inflammation. C-reactive protein (CRP) is how self-reported GI symptoms among older adults an acute-phase plasma protein and a marker of correlate to plasma zonulin (an indicator of increased in- inflammation. CRP levels were assessed by the high- testinal permeability) [21, 22] and psychological distress. sensitivity immunoturbidimetric assay, CardioPhase™, and analysed on the ADVIA 1800 chemistry system (SIEMENS Healthcare Diagostics Inc., NY, USA). A nor- Methods mal CRP was defined as ≤2 mg/L. Study participants Oxidative stress. Oxidative stress was estimated by The study population was comprised of three groups of the FORT (Free Oxygen Radicals Test) colorimetric older adults; individuals suffering from GI symptoms assay (Callegari, Parma, Italy), as previously described (n = 24), senior orienteering athletes (n = 27) and gen- . eral older adults (n = 22). The demographic data and Intestinal permeability. To examine zonulin levels in recruitment process are outlined in Table 1.Comor- plasma, as an indirect indicator of small intestinal per- bidities and medications are displayed in Table 2. meability , a zonulin ELISA kit was used following Ganda Mall et al. BMC Geriatrics (2018) 18:75 Page 3 of 9 Table 1 Data for all the descriptive parameters, displayed for all three groups of older adults, presented as median (IQR) a b c III General older adults Senior orienteering athletes Older adults with GI symptoms p-value n =22 n =27 n =24 I I I Age: 70 (67-72) Age: 68 (66-71) Age: 73 (69-75) Age at onset of GI symptoms: II 59 (47-65) - Demographic data - Female/Male 11/11 12/16 18/6 Recruitment Advertisement in local Orienteering clubs within the Örebro Advertisement in local newspaper method newspaper County III Inclusion criteria Age ≥ 65 yrs Age ≥ 65 yrs Actively competing in Age ≥ 65 yrs GSRS* diarrhoea/constipation orienteering score ≥ 3 Exclusion criteria Diagnosed GI disease Prescribed Diagnosed GI disease Prescribed Diagnosed GI disease Prescribed inflammatory regulating drugs inflammatory regulating drugs inflammatory regulating drugs - Questionnaire data - GSRS (total score) 1.5 (1.2 – 2) 1.3 (1.1 – 1.6) 2.6 (2.2 – 3.1) Diarrhoea 1.3 (1 – 1.4) 1.3 (1 – 1.7) 3.3 (1.7 – 3.8) Constipation 1.3 (1 -2.7) 1.3 (1 – 1.7) 3.3 (2 – 4.7) Indigestion 1.9 (1.3 – 2.6) 1.5 (1.25 – 2) 3 (2.3 – 3.6) Abdominal pain 1.3 (1 – 1.8) 1 2.3 (1.7 – 3.3) Reflux 1 (1 – 1.5) 1 1.5 (1 – 2.5) ab, bc HADS* (total 4 (1.5 – 6) 1 (0 – 3) 5 (2.5 – 10.5) < 0.05 score) FGAS* 4 (3-4) 4 (4-5) 4 (3-4) - Biomarker data - CRP (mg/L) 0.5 (0.2 – 1.4) 0.9 (0.2 – 1.3) 1.3 (0.7 – 2.2) H O mmol/L 2 (1.7 – 2.2) 2 (1.7 – 2.4) 2.1 (1.8 – 2.5) 2 2 Calprotectin (μg/ 0(0 – 35.9) 0 (0 – 102.5) 0 (0 – 65) g) -CRP levels < 2 are considered normal - Calprotectin levels ≤ 50 μg/g are considered normal -H O mmol/L < 1.75 indicates no oxidative stress 2 2 -H O mmol/L 1.75-2.35 indicates intermediate oxidative stress 2 2 -H O mmol/L between 2.36-3.05 indicates oxidative stress 2 2 - H O mmol/L 3.05 < indicates strong oxidative stress 2 2 * Abbreviations, GSRS: Gastrointestinal symptoms rating scale, FGAS: Frändin-Grimby Activity Scale, HADS: Hospital Anxiety and Depression Scale, a cut-off score of ≥8 for either subscale indicates a significant level of depression/anxiety a = General population of older adults b = Senior orienteering athletes c = Older adults with GI symptoms I: age is presented with median and interquartile range (IQR) II: median age at onset of GI symptoms (IQR) III: p-value generated from pair-wise comparisons using the Mann-Whitney U-test manufacturer’s instructions (Cusabio, Baltimore, USA). according to the manufacturers information. In addition, In brief, plasma was diluted 1:2, and added to pre-coated the quantification and intra-assay precision (CV%) was plates in duplicate, together with controls and standards. < 8%. Primary biotinylated antibody, streptavidin-horseradish peroxidase, tetramethylbenzidine enzyme substrate, and Stool samples 2 M HCL were subsequently added. Absorbance was Material for faecal sample collection was provided to- measured at 450 nm in VERSAmax Tunable Microplate gether with comprehensive instruction one-week prior Reader (Molecular Devices, San Diego, CA, USA) using to collection. The sample was collected at the partici- Softmax pro 5 (Molecular Devices). The software gener- pant’s home, instantly frozen at -20 °C and transported ated a standard curve based on the standards, from to the laboratory within one week. Samples were stored which the concentrations of the samples were calculated. at -80 °C until analysis. The detection range of the kit was 0.625 ng/ml – 40 ng/ Intestinal inflammation. Faecal calprotectin was ml, with the lower limit of detection at 0.156 ng/ml measured to assess intestinal inflammation using the Ganda Mall et al. BMC Geriatrics (2018) 18:75 Page 4 of 9 Table 2 Distribution of comorbidities and medications used within the study groups Senior orienteering General older Older adults with GI athletes (n = 27) adults (n = 22) symptoms (n = 24) Comorbidities % % % Cardiovascular diseases 14.8 18.2 50.0 Gut symptoms 11.1 22.7 100.0 Psychological & neurodegenerative morbidities 3.7 13.6 8.3 Others (respiratory tract, urinary tract, musculo-skeletal, eyes) 25.9 22.7 33.3 Medications % % % Cardiovascular agents 25.9 18.2 50.0 -Blood pressure lowering substances 18.5 18.2 37.5 -Lipid-lowering medications - 18.5 - 0.0 - 12.5 -Anti-coagulants - 11.1 - 0.0 - 20.8 -Others - 3.7 - 0.0 - 12.5 Anti-inflammatory agents (NSAID, cortisone) 0 0 4.2 Gut motility regulating substances 3.7 18.2 29.2 -Anti-constipation medicine - 0.0 - 13.6 - 12.5 -Anti-diarrheal medicine -Other GI regulators (probiotics, fibres) - 7.4 - 4.5 - 4.2 -Proton pump inhibitors (PPI) - 0.0 - 0.0 - 4.2 Antibiotics 0 4.5 16.7 -Neurology drugs 3.7 13.6 12.5 -Anti-depressants - 3.7 - 13.6 - 0.0 -Hypnotics - 0.0 - 0.0 - 4.2 -Dopamine-agonists - 0.0 - 4.5 - 4.2 -Cholinesterase-inhibitors - 0.0 - 0.0 - 4.2 Others 18.5 27.3 29.2 Polypharmacy (5 or more drugs) 3.7 0 12.5 Other GI regulators (probiotics, fibres etc.) 0 4.5 8.3 Table 3 Questionnaire details The Gastrointestinal Symptoms The Hospital Anxiety and The Frändin-Grimby Rating Scale – GSRS Depression Scale - HADS Activity Scale - FGAS Subdomains Diarrhoea Anxiety Winter season Constipation Depression Summer season Indigestion Abdominal pain Reflux Nr of questions 15 14 6 Scale Score 1 = No problems Score 0-3 Score 1-6 Score 7 = Severe problems Score 3 being the highest symptom frequency Grading No problem = 1 point Normal = 0-7 points No physical activity = 1 Mild = 1-2 points Mild = 8-10 Intense regular exercise = 6 Moderate = 2-4 points Moderate = 11-14 Severe = 4-7 points Severe = 15-21 Total score Gut symptoms Psychological distress Physical activity Ganda Mall et al. BMC Geriatrics (2018) 18:75 Page 5 of 9 ELISA assay CALPRO® (CALPRO AS, Lysaker, Norway). Intestinal permeability Approximately 100 mg of faeces were extracted in 5 mL of extraction buffer and analysed using a pre-coated ns 96-well plate provided by the manufacturer. The enzyme ns reaction was detected at 405 nm. The normal range for faecal calprotectin is defined as < 50 μg/g. Statistical methods & data analysis All data are presented with median and IQR. Shapiro-Wilk test was used to assess normality. The non-parametric Kruskal-Wallis test was used to identify statistical differ- ences between the three study groups, followed by the non-parametric Mann-Whitney U-test for pair-wise com- parisons. The Bonferroni-Holm method was used to cor- rect for multiplicity. All statistical analyses were performed using GraphPad Prism version 6 for Mac (GraphPad Soft- ware, San Diego California, USA). For visualization of the data biplots of Principal Component Analysis (PCA) were created using R v3.3.0  with functions prcomp and ggbiplot. By convention all values below detection levels Fig. 1 Intestinal permeability as reflected by plasma zonulin levels in the different groups of older adults. The group of older adults with GI were replaced with 0 to facilitate the analysis. symptoms (n = 22) had significantly higher plasma zonulin levels than and general older adults (n = 21) but not senior orienteering athletes (n = 27). No significant difference was observed between senior Results orienteering athletes and general older adults. **p <0.01, *p <0.05 Intestinal permeability Assessment of zonulin, an indirect marker of intestinal permeability, showed significantly increased levels Comorbidities and medication among older adults suffering from GI symptoms, com- Comorbidities and medical use were reported in the case pared to general older adults (p < 0.05) but not to senior report forms and are presented in Table 2. The usage of orienteering athletes (Fig. 1). cardiovascular drugs (blood pressure lowering drugs, anti-coagulants etc) was most prominent in all groups. Fifty percent of older adults with GI symptoms reported Psychological distress cardiovascular disease and intake of cardiovascular Assessment of overall psychological distress (total HADS drugs. Hypertension was identified as the most common score) showed increased levels for both older adults with condition. Older adults with GI symptoms were also GI symptoms (p < 0.05) and general older adults (p <0.05) found to consume gut motility regulating substances when compared to senior orienteering athletes (Table 1). (29.2%) to a larger extent compared to the two other The level of probable depression was found to be signifi- study groups. Use of antibiotics was also found to be cantly higher among older adults with GI symptoms com- higher in the study group suffering from GI symptoms. pared to both general older adults (p < 0.01) and senior Polypharmacy (defined as intake of five or more medica- orienteering athletes (p <0.001) (Fig. 2a). Experience of tions simultaneously) was found to be more prominent anxiety showed a different pattern and was found to be in older adults suffering from GI symptoms (12.5%) significantly higher in both older adults with and without compared to both senior orienteering athletes (3.7%) GI symptoms (p < 0.05) compared to senior orienteering and general older adults (0%). The levels of zonulin and athletes (Fig. 2b). psychological distress was further found not be influ- enced by medication and comorbidities when confound- Biomarkers of inflammation and oxidative stress ing factors were controlled for. Biomarkers of inflammation and oxidative stress were assessed to monitor the inflammatory status of the study Illustration of the data using principal component participants. For all three populations the CRP levels analysis were found to be within the normal range, < 2 mg/L. The PCA revealed a separation between the 3 different Similarly, values of oxidative stress and faecal calprotec- study populations, based on GSRS scores and bio- tin were found to be within the normal range. These markers (Fig. 3). The highest degree of separation was data are presented as descriptive values in Table 1. observed between the senior orienteering athletes and Senior orienteering athletes General older adults Older adults with GI symptoms Zonulin (ng/mL) Ganda Mall et al. BMC Geriatrics (2018) 18:75 Page 6 of 9 ab Psychological distress Psychological distress Depression Anxiety *** ** ns p<0.08 ** * Fig. 2 Depression and anxiety assessed by HADS in the three different populations of older adults. a) The group experiencing GI symptoms (n =24) revealed significantly higher depression scores on the HADS scale than the other two groups. A trend towards a significant difference was observed between the senior orienteering athletes (n = 27) and general older adults (n = 22). b) Anxiety score was significantly higher in older adults with GI symptoms compared to the senior orienteering athletes. A significantly lower score was seen in senior orienteering athletes when compared to general older adults, with the latter showing no difference compared to group experiencing GI symptoms. *p < 0.05; **p < 0.01; ***p < 0.001, ns = non-significant older adults with GI symptoms. The general population regimens solely designed for age-associated GI symptoms of older adults were found to clearly overlap the other exist today, and knowledge regarding gut and intestinal two populations. This separation is expected and can be barrier function in older adults is poor. As a first step to explained by the higher GSRS score on the domains identify the mechanisms behind age-associated GI diarrhoea and/or constipation among older adults with symptoms we investigated the relationship between self-- GI symptoms. An association could be observed reported GI symptoms and intestinal permeability. Fur- between C-reactive protein, ROS and calprotectin. In thermore, the impact of GI symptoms on wellbeing was addition, GI symptoms appear to be associated with assessed, by investigating the level of psychological distress zonulin and psychological distress. in relation to experience of gut problems. As outlined in the results section, we identified that Discussion depression-like characteristics were more prominent To our knowledge this is the first study that investigated among older adults suffering from GI symptoms and less the characteristics of self-reported GI symptoms. This is common among senior orienteering athletes. The low important to facilitate treatment and diagnosis for older level of psychological distress among senior orienteering adults experiencing moderate GI symptoms. No treatment athletes is in line with our previous results . Senior Fig. 3 Principal component analysis displaying the relationship between all investigated biomarkers, HADS domains and GSRS domains in the 3 study populations. Both zonulin and HADS appear associated with the GSRS domains unlike ROS, CRP and calprotectin. The ellipsoid markings cover 95% of the total populations Senior orienteering athletes General older adults Older adults with GI symptoms Senior orienteering athletes General older adults Older adults with GI symptoms HADS Depression score HADS Anxiety score Ganda Mall et al. BMC Geriatrics (2018) 18:75 Page 7 of 9 orienteering athletes were also found to suffer from less fibres could be a potential therapeutic able to strengthen GI symptoms. Experience of anxiety did not differ be- the intestinal barrier in elderly individuals, however, this tween older adults with and without GI symptoms. An needs to be thoroughly investigated in pre-clinical and increased prevalence of anxiety disorders among older clinical settings. adults has been reported previously [31–33]. However, Zonulin is the only physiological mediator known to the levels of psychological distress estimated by HADS reversibly regulate intestinal permeability by modulating were below the cut-off value (≥ 8) for severe anxiety and intercellular tight junctions [21, 40]. Circulating zonulin depression. Hence, indicating that none of the study par- in serum/plasma is considered a useful marker of small ticipants suffered from severe psychological disease. It intestinal permeability [21, 41] and has been validated should also be taken into consideration that the results using lactulose/mannitol tests . However, zonulin as presented here are based on self-reported data, which a marker of small intestinal permeability has been, and relied on the respondents’ honesty, accuracy, and inter- is, under debate. Levels of zonulin have been found to pretation of the question asked. fluctuate over time making interpretation of the results GI symptoms were found to be associated with ele- difficult . Moreover, a recent study suggests that vated levels of plasma zonulin, which indicate that older circulating zonulin might not only be derived from the adults suffering from GI symptoms have increased small gastrointestinal tract but may be associated with obesity intestinal permeability. Moreover, the PCA analysis and hyperlipidaemia [43, 44]. However, the body mass showed that GI symptoms were found to be associated index (BMI) of the general older adults (25.6 ± 4.3 Std) with psychological distress and zonulin among older and older adults with GI symptoms (26.7 ± 5.0 Std) adults with GI symptoms. Females (n = 18) were further included in the present study was normally distributed overrepresented among elderly suffering from GI symp- and no significant differences were observed between toms (n = 24). This could reflect the normal distribution the two groups. In addition, stratification of the data of GI symptoms in the population as previous studies revealed no association between cardiovascular disease, report an increased frequency of GI symptoms, such as including hypertension, and increased zonulin levels. constipation, among women [34–36]. Stratification for Unfortunately, the BMI was not available for the senior gender did not reveal a significant difference between orienteering athletes in the present study. However, the men and women in regard to GI symptoms, zonulin BMI of eleven newly recruited senior orienteering levels and depression-like characteristics. Anxiety-like athletes enrolled in an additional study was found to be characteristics were, on the contrary, found to be normally distributed with a mean value of 23.8 ± 3.2 Std. significantly higher among elderly men with GI symp- This is in accordance with a recent study showing a toms (n = 6). However, this finding could be due to the lower BMI value of senior athletes compared to general small study population and further studies using larger older adults . Hence, these findings indicate that the sample sets will need to be performed in order to thor- increased zonulin levels in the present study were not a oughly elucidate the difference between men and women result of overweight/obesity or cardiovascular disease. in relation to psychological distress, GI symptoms and Nevertheless, the findings presented here needs to be intestinal barrier function. confirmed in future studies using more advanced tech- Increased intestinal permeability is a hallmark in the niques, such as the Ussing Chamber methodology. This pathophysiology of chronic inflammatory gastrointes- will allow for a thorough assessment ex vivo of the intes- tinal diseases, such as Crohn’s disease . An altered tinal barrier function in elderly using mucosal biopsies intestinal permeability has previously been associated [12, 39, 46] and hence add important information to the with psychiatric disorders such as depression and results presented here. anxiety . Thus, our results support the notion that Moreover, it is important to point out that older adults intestinal permeability might be an important target for with GI symptoms were found to suffer from more new treatment regimes for age-associated GI symptoms comorbidities and did also use more medications. Fifty that might have a positive impact on mental wellbeing. percent were identified to suffer from cardiovascular Moreover, elderly individuals are known to have a low disease, were hypertension was found to be the most fibre intake  that in addition to a disturbed intestinal common condition. A low dose (75 mg) of acetylsalicylic motility could alter the gut microbiota and result in a acid (ASA) is commonly prescribed to treat hyperten- diminished diversity that could have a negative impact sion [47, 48]. ASA is known to affect the intestinal bar- on the intestinal barrier function . Recently, we rier negatively and induce an increased permeability showed that a dietary fibre from yeast was able to at- . In addition, beta-adrenoceptor blocking agents tenuate stress-induced hyperpermeability ex vivo across (beta-blockers, mainly used to treat angina pectoris) small intestinal tissue from Crohn’s disease patients have been found to decrease bacterial translocation . mounted in the Ussing Chamber . Thus, dietary However, only three participants reported use of ASA Ganda Mall et al. BMC Geriatrics (2018) 18:75 Page 8 of 9 and two used beta-blockers. Moreover, the median of data files. In addition, we would like to acknowledge the FibeBiotics EU consortium and collaboration performed within this organization. plasma zonulin value in the participants taking medica- tions did not differ from the median of the whole study Funding group. Antibiotic use was also higher among the partici- This work was supported by Bo Rydins stiftelse (Grant ref.: F0514, principal investigator IS), the Knowledge Foundation (Grant ref: 20110225, principal pants suffering from GI symptoms (16.7%). Antibiotic investigator RJB) as well as The Faculty of Medicine and Health at Örebro use is known to influence the gut microbiota negatively University. Additional support was granted to ÅVK by Diarrheal Disease and can cause antibiotic associated diarrhoea [51, 52]. Research Centre, Linköping University. The funding bodies were not involved in the study design, collection of data, analysis or interpretation of data, Interestingly, three out of four participants suffered from writing of the manuscript or the decision to submit the manuscript for diarrhoea. However, none of these participants displayed publication. zonulin levels above the median for older adults suffer- Availability of data and materials ing from GI symptoms. Moreover, it is important to All data generated or analysed during this study are included in this point out that assessment of the gut microbiota was not published article and its supplementary information files. performed in the present study. Thus, we cannot ex- Authors’ contributions clude that the increased intestinal permeability is a con- LÖL, JPGM, DR2, RJB, ÅVK and IS are responsible for conception and design sequence of an altered gut flora with an increased number of the research. JPGM, LÖL, SA, DR1 and IS performed the study. JPGM, LÖL, of pathobionts. Nevertheless, assessment of confounding SA, DR1, DR2, ÅVK, CML and IS took part in the acquisition, analysis and interpretation of both questionnaire data and biological samples. JPGM, LÖL, effect showed that medication and comorbidities did not SA, DR and IS drafted the manuscript. JPGM, LÖL, SA, DR1, DR2, ÅVK, CML, influence intestinal permeability or psychological distress. RJB and IS edited and revised the manuscript critically. All authors reviewed In addition, assessment of biomarkers showed no and approved the final version of the manuscript and agreed to be accountable for all aspects of the work. change in inflammatory status in regard to GI symp- toms. Thus, confirming that none of the study partici- Ethics approval and consent to participate pants suffered from severe inflammation and infection. The study obtained ethical approval by the Uppsala Regional Ethics Review Board (Dnr. 2012/309, 2013/37 and 2015/357). All procedures were carried It should, however, be noted that in order to thoroughly out in accordance with the declaration of Helsinki and written informed assess low-grade inflammation pro-inflammatory cyto- consent was acquired from all participants before study start. kines, such as IL-6, TNF-α and IL-1β, should be investi- Consent for publication gated. Moreover, absence of intestinal inflammation was Not Applicable. confirmed by the low calprotectin levels, which fell within the normal range. Thus, indicating that the in- Competing interests The authors declare that they have no competing interests. creased intestinal permeability was not dependent on intestinal inflammation. Hence, our data suggest that Publisher’sNote moderate GI symptoms area associated with an altered Springer Nature remains neutral with regard to jurisdictional claims in intestinal barrier function and psychological distress. published maps and institutional affiliations. However, in future studies it will be important to perform Author details thorough analysis of the gut microbiota and assess intes- Nutrition Gut Brain Interactions Research Centre, Department of Medical tinal barrier function using more advanced technology. Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden. Nutrition and Physical Activity Research Centre, Department of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden. Conclusions 3 Department of Clinical and Experimental Medicine, Linköping University, Here we demonstrate that self-reported GI symptoms Linköping, Sweden. among older adults are associated with an altered intes- Received: 1 July 2017 Accepted: 12 March 2018 tinal barrier function as assessed by plasma zonulin levels. Furthermore, both GI symptoms and zonulin References levels are associated with increased psychological dis- 1. Cohen JE. Human population: the next half century. Science. 2003; tress. These findings suggest that even moderate levels 302(5648):1172–5. of GI symptoms can be reflected in a disturbed intestinal 2. Algilani S, Ostlund-Lagerstrom L, Kihlgren A, Blomberg K, Brummer RJ, Schoultz I. Exploring the concept of optimal functionality in old age. J barrier function and increased psychological distress. 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Published: Mar 20, 2018