Plasma cytokine levels in patients with chronic alcohol overconsumption: Relations to gut microbiota markers and clinical correlates.

BACKGROUND: Alcohol-related morbidity may involve changes in the gut microbiota and immune dysregulation. We have previously demonstrated alterations in gut microbiota composition and functions in patients with alcohol overconsumption, and now aimed to investigate possible associations between cytokine levels, gut microbiota, and clinical symptoms. METHODS: We included hospital inpatients with a history of chronic alcohol overconsumption. For comparison, we included control patients with a low alcohol intake. Cytokine levels (TGF-ß1, TNF-α, IL-10, IL-8, IL-6, IFN-γ, MCP-1, IL-1RA, IL-1ß, and IL-17) were determined using a customized V-plex assay. We then examined associations of cytokine levels with the abundance of Proteobacteria and Faecalibacterium, percentage of the short-chain fatty acid butyrate, psychiatric symptoms (Hospital Anxiety and Depression Scale), and biochemical liver variables. RESULTS: We included 28 patients with alcohol overconsumption (79% men), and 25 control patients (72% men). Patients with alcohol overconsumption had higher levels of IL-6 (p = 0.002), IFN-γ (p = 0.018), and MCP-1 (p = 0.006), and lower levels of TGF-ß1 (p = 0.017) compared with control patients. Inverse correlations were found between Proteobacteria abundance and TNF-α (Rs = -0.55, p = 0.02) and IL-8 (Rs = -0.58, p = 0.014), and between Faecalibacterium and MCP-1 levels (Rs = -0.56, p = 0.02) in the control patients, but not in patients with alcohol overconsumption. Patients with alcohol overconsumption reported more psychiatric symptoms, and these symptoms were inversely correlated with IL-10 levels. There were positive correlations between several of the assessed cytokines and biochemical liver variables, and negative correlations between cytokine levels and albumin. CONCLUSION: Patients with alcohol overconsumption had a cytokine profile suggestive of increased systemic inflammatory activity, with higher levels of pro-inflammatory cytokines (IL-6, IFN-γ, and MCP-1) and lower levels of anti-inflammatory cytokines (TGF-ß1). The findings may represent a link between alcohol use and alcohol-related morbidity.

Characterization of gut microbiota composition and functions in patients with chronic alcohol overconsumption.

Excessive alcohol intake can alter the gut microbiota, which may underlie the pathophysiology of alcohol-related diseases. We examined gut microbiota composition and functions in patients with alcohol overconsumption for >10 years, compared to a control group of patients with a history of no or low alcohol intake. Faecal microbiota composition was assessed by 16S rRNA sequencing. Gut microbiota functions were evaluated by quantification of short-chain fatty acids (SCFAs) and predictive metagenome profiling (PICRUSt). Twenty-four patients, mean age 64.8 years (19 males), with alcohol overconsumption, and 18 control patients, mean age 58.2 years (14 males) were included. The two groups were comparable regarding basic clinical variables. Nutritional assessment revealed lower total score on the screening tool Mini Nutritional Assessment, lower muscle mass as assessed by handgrip strength, and lower plasma vitamin C levels in the alcohol overconsumption group. Bacteria from phylum Proteobacteria were found in higher relative abundance, while bacteria from genus Faecalibacterium were found in lower relative abundance in the group of alcohol overconsumers. The group also had higher levels of the genera Sutterella, Holdemania and Clostridium, and lower concentration and percentage of butyric acid. When applying PICRUSt to predict the metagenomic composition, we found that genes related to invasion of epithelial cells were more common in the group of alcohol overconsumers. We conclude that gut microbiota composition and functions in patients with alcohol overconsumption differ from patients with low consumption of alcohol, and seem to be skewed into a putative pro-inflammatory direction.

Fecal Calprotectin: A Reliable Predictor of Mucosal Healing after Treatment for Active Ulcerative Colitis.

OBJECTIVES: Mucosal healing has become the new goal of treatment in ulcerative colitis. Fecal calprotectin has been demonstrated to differentiate between mucosal inflammation and mucosal healing. With this project, we investigated whether a reduction in f-calprotectin to <250 µg/g after medical treatment for active ulcerative colitis could predict mucosal healing. MATERIAL AND METHODS: After a baseline colonoscopy, 20 patients with active ulcerative colitis were followed with consecutive fecal calprotectin monthly until two measurements of fecal calprotectin < 250 µg/g or a maximum follow-up of 12 months. A flexible sigmoidoscopy was then performed and Mayo endoscopic subscore was used to evaluate degree of inflammation. Simple Clinical Colitis Activity Index was used for evaluation of clinical disease activity. RESULTS: A total of 16 patients achieved fecal calprotectin < 250 µg/g during follow-up, and all 16 patients had endoscopic mucosal healing (Mayo endoscopic subscore of ≤1) on the second endoscopy. The remaining four patients had persistently high f-calprotectin levels before the second endoscopy with Mayo endoscopic subscore corresponding to endoscopic mucosal healing in three out of four patients. CONCLUSIONS: Fecal calprotectin <250 µg/g after medical treatment for active ulcerative colitis is a reliable marker of endoscopic mucosal healing.

Chronic alcohol overconsumption may alter gut microbial metabolism: a retrospective study of 719 13C-D-xylose breath test results.

Objective: Alterations of gut microbiota composition or function may participate in the pathophysiology of several diseases. We aimed to explore the effect of chronic alcohol overconsumption on gut microbial metabolism, as assessed by evaluating 13C-D-xylose breath test results. Materials and methods: We investigated all 13C-D-xylose breath tests performed at Lovisenberg Diaconal Hospital during the years 2005 to 2011, using patient files for diagnosing the patients into one of three patient categories: alcohol overconsumption, coeliac disease and functional bowel disorder. In addition, a group of healthy controls was included. The time curves of 13CO2 excretion in breath samples were divided into two phases, evaluating small intestinal absorption (0-60 min) and colonic microbial metabolism (90-240 min), respectively. Results: A total of 719 patients underwent 13C-D-xylose breath testing during the inclusion period. Thirty-five had a history of alcohol overconsumption, 66 had coeliac disease, and 216 had a functional bowel disorder, while 44 healthy controls were included for comparison. The alcohol overconsumption group had similar small intestinal phase results as the group of patients with untreated coeliac disease. During the colonic phase, the group of patients with alcohol overconsumption differed from all the other groups in terms of 13C-xylose recovery, with significantly less 13CO2 excretion compared to the other groups. Conclusion: The results suggest that patients with a history of alcohol overconsumption suffer from both small intestinal malabsorption and impaired colonic microbial metabolism. The role of gut microbiota in chronic alcohol overconsumption should be investigated further.

Clinical importance of faecal calprotectin variability in inflammatory bowel disease: intra-individual variability and standardisation of sampling procedure.

OBJECTIVE: Faecal (f-) calprotectin is a biomarker of intestinal inflammation. Previous studies have described intra-individual day-to-day variability of this biomarker in patients with inflammatory bowel disease (IBD) and morning samples have been suggested for standardisation purposes. With this project, we investigated if day-to-day variability differed from diurnal variability. Additionally, we evaluated a new extraction method for f-calprotectin analysis. METHODS: Fifty patients provided three faeces samples from morning - evening - morning on two consecutive days. Nineteen patients provided two faeces samples from the same bowel movement, one conventional spot sample, and one sample with a device for patient-administered sampling and extraction. RESULTS: The two morning samples differentiated between mucosal inflammation and mucosal healing with same level of agreement as the two samples from the same day (kappa 0.76), using an f-calprotectin cut-off level of 259 µg/g. Although large intra-individual variation in f-calprotectin values, there were no significant day-to-day (p = 0.096) or diurnal variation (p = 0.78). Used by laboratory technicians, the new extraction device correlated significantly with the conventional extraction method (p < 0.001), Spearman's rank correlation coefficient 0.95. Of the 19 patients testing patient administered extraction, two patients provided samples leading to considerably higher f-calprotectin levels than conventional sampling procedure. CONCLUSIONS: The reliability of f-calprotectin morning samples is equal to the reliability of samples from different bowel movements on the same day. The new extraction method is reliable when used by laboratory technicians, but larger studies are recommended to evaluate patient administered extraction.

Prediction of endoscopic disease activity in ulcerative colitis by two different assays for fecal calprotectin.

BACKGROUND AND AIMS: As mucosal healing is the goal of treatment in inflammatory bowel disease, defining a fecal [f-] calprotectin cut-off level for mucosal healing is crucial. Previous studies have presented different cut-off levels. The aim of this study was to investigate the ability of two f-calprotectin assays to differentiate mucosal healing from inflammation in ulcerative colitis. METHODS: Sixty-two patients with ulcerative colitis underwent colonoscopy for classification of mucosal inflammation [Mayo endoscopic subscore]. The patients also submitted a fecal sample for f-calprotectin analysis using two different assays, Calpro ELISA and Buhlmann ELISA. RESULTS: The two assays correlated significantly, with a Spearman rank correlation coefficient of 0.86. Both assays showed significantly different f-calprotectin levels in patients with a Mayo endoscopic subscore of 0 [mucosal healing] and 1­3 [inflamed mucosa] [p <0.001]. Using ROC curve analyses, we selected the best cut-off levels for both assays with responding sensitivity and specificity [presented with 95% confidence intervals]; Calpro ELISA cut-off 61 µg/g, sensitivity 84.1% [75.0­93.2%], specificity 83.3 % [74.0­92.6%], and Buhlmann ELISA cut-off 96 µg/g, sensitivity 90.9 % [83.7­98.1%], specificity 83.3 % [74.0­92.6%]. Defining mucosal healing as a Mayo endoscopic subscore ≤1, cut-off levels increased: Calpro ELISA cut-off 110 µg/g, sensitivity 80.0%[70­90%], specificity 66.6 % [54.9­78.3%]; and Buhlmann ELISA cut-off 259 µg/g, sensitivity 83.3 %[74­92.6%], specificity 71.9 % [60.7­83.1%]. CONCLUSIONS: The study demonstrates the need for assay specific cut-off levels in clinical practice,as the f-calprotectin cut-off level for endoscopic disease activity differed in these two assays.

Small intestinal malabsorption in chronic alcoholism: a retrospective study of alcoholic patients by the ¹4C-D-xylose breath test.

OBJECTIVE: The ¹4C-D-xylose breath test was used at Ullevål University Hospital in the period from 1986 TO 1995 for malabsorption testing. The objective of this retrospective study was to reveal whether patients with chronic alcoholism may have intestinal malabsorption. MATERIALS AND METHODS: The consecutive ¹4C-D-xylose breath test database was reviewed and patients with the diagnosis of chronic alcoholism were identified. ¹4C-D-xylose breath test results of the alcoholic patients were compared with the results of untreated celiac patients and patient and healthy controls. In the ¹4C-D-xylose breath test, ¹4C-D-xylose was dissolved in water and given orally after overnight fast. Breath samples were taken at 30-min intervals for 210 min, and ¹4CO2 : ¹²CO2 ratios were calculated for each time point, presenting a time curve for ¹4C-D-xylose absorption. Urine was collected after 210 min and the fraction of the total d-xylose passed was calculated (U%). ¹4CO2 in breath and ¹4C-D-xylose in urine were analyzed using liquid scintillation. RESULTS: Both breath and urine analysis revealed a pattern of malabsorption in alcoholics comparable with untreated celiac patients, with significantly reduced absorption of d-xylose compared with patient and healthy controls. CONCLUSION: Alcoholic patients have a significantly reduced ¹4C-D-xylose absorption, comparable with untreated celiac patients. This indicates a reduced intestinal function in chronic alcoholism.

Follow-up of coeliac disease with the novel one-hour 13C-sorbitol breath test versus the H2-sorbitol breath test.

BACKGROUND: We recently developed a (13)C-sorbitol breath test ((13)C-SBT) as an alternative to the H(2)-sorbitol breath test (H(2)-SBT) for coeliac disease. In this study we compared the diagnostic properties of the H(2)-SBT and the (13)C-SBT in follow-up of coeliac disease. MATERIAL AND METHODS: Twenty-seven coeliac patients on a gluten-free diet (GFD) performed the breath tests. All had been tested before treatment in the initial study of the (13)C-SBT, in which 39 untreated coeliac patients, 40 patient controls, and 26 healthy volunteers participated. Five gram sorbitol and 100 mg (13)C-sorbitol were dissolved in 250 ml tap water and given orally. H(2), CH(4) and (13)CO(2) were measured in end-expiratory breath samples every 30 min for 4 h. Increased H(2) concentration ≥20 ppm from basal values was used as cut-off for the H(2)-SBT. Sixty minutes values were used as diagnostic index in the (13)C-SBT. RESULTS: (13)CO(2) levels at 60 min increased in 20/26 treated coeliac patients (77%) after GFD, but were significantly lower than in control groups. Out of 20 patients who had a positive H(2)-SBT before GFD, 12 had a negative H(2)-SBT after GFD. Peak H(2) concentrations were not correlated with (13)C-SBT results. CONCLUSION: The study confirms the sensitivity of a one-hour (13)C-SBT for small intestinal malabsorption. The (13)C-SBT has superior diagnostic properties compared with the H(2)-SBT in follow-up of coeliac disease.

Reduced ¹³C-D-xylose absorption in alcoholics is more likely caused by alterations in small intestinal mucosa than delayed gastric emptying.

OBJECTIVE: Alterations of the small intestinal absorptive surface are a probable cause of D-xylose malabsorption in chronic alcoholism. Delayed gastric emptying, however, may influence the (13)C-D-xylose breath test, which is used to study intestinal function in alcoholics. The aim of this study was to measure gastric emptying in alcoholics to elucidate whether retention of the test meal could explain the malabsorptive pattern of the (13)C-D-xylose breath test observed in alcoholics. MATERIAL AND METHODS: Fifteen alcoholics performed the (13)C-octanoic acid and the (13)C-D-xylose breath tests on consecutive days in a random order. The (13)CO(2) expired was measured every 30 or 15 min for 4 h in the (13)C-D-xylose and the (13)C-octanoic acid breath tests, respectively, using a mass spectrometer equipped with a gas chromatograph. Test meals consisted of 100 mg of (13)C-D-xylose and 5 g of unmarked D-xylose dissolved in 250 ml water and 91 mg (13)C-octanoic acid embedded in a one-egg omelette served with white bread with margarine, respectively. RESULTS: The alcoholic patients had a lower (13)C-D-xylose breath index compared with healthy controls (p < 0.0001). None of the (13)C-octanoic acid breath test variables, T(50%), T(max), T(lag), or GEC revealed any significant differences between the groups. CONCLUSION: The pathological (13)C-D-xylose breath test in this group of alcoholics is unlikely to be caused by delayed gastric emptying. Malabsorption is the probable cause of the pathological (13)C-D-xylose breath test results in alcoholics.

Intestinal malabsorption of D-xylose: comparison of test modalities in patients with celiac disease.

OBJECTIVE: The aim of the study was to compare three different D-xylose test modalities for small intestinal malabsorption, using patients with celiac disease and healthy persons as experimental models. MATERIAL AND METHODS: Ninety-one untreated celiac patients, 98 treated celiac patients, and 43 healthy subjects performed the (13)C-D-xylose breath test. 1-h plasma D-xylose levels were measured in 48 untreated patients, 41 treated patients and 41 healthy controls. 4-h urine D-xylose excretion was measured in 47 untreated patients, 51 treated patients and 42 healthy controls. 100 mg of (13)C-D-xylose and 5 g of D-xylose were dissolved in 250 ml tap water and given orally. (13)CO(2) was measured in breath every 30 min for 4 h. Blood was sampled after 1 h, and urine collected after 4 h. RESULTS: Test sensitivity/specificity for celiac disease was 88%/84% with the (13)C-D-xylose breath test, 65%/71% with the 1-h plasma D-xylose test, and 55%/74% with the 4-h urine D-xylose excretion test. Breath test results improved significantly in the treated celiac group compared to untreated patients, but were not normalized compared to healthy controls. No difference was found between 1-h plasma D-xylose levels and 4-h urinary D-xylose excretion in treated celiac patients and healthy controls. CONCLUSIONS: The (13)C-D-xylose breath test was superior to D-xylose testing in plasma and urine for assessment of small intestinal malabsorption with considerably higher sensitivity and specificity for untreated celiac disease.

Small intestinal malabsorption in chronic alcoholism determined by 13C-D-xylose breath test and microscopic examination of the duodenal mucosa.

OBJECTIVE: Diarrhea, weight loss and osteoporosis are prominent symptoms and clinical signs of alcoholism. One of several possible factors causing this clinical picture is small intestinal damage leading to malabsorption. The aim of this study was to prospectively evaluate small intestinal absorption in alcoholics using the (13)C-D-xylose breath test, and to relate the breath test results to morphological findings of the duodenal mucosa. MATERIAL AND METHODS: Sixteen alcoholics without liver failure or serious illness and presenting symptoms of dyspepsia, nausea or diarrhea were included. The (13)C-D-xylose breath test was performed in 14 of the included subjects. The breath tests of the alcoholics were compared to those of untreated coeliac patients and healthy subjects. Duodenal biopsy specimens were taken for assessment of epithelial morphology in 14 of the included subjects, using light- and electron microscopic techniques. RESULTS: Alcoholics had significantly reduced absorption of (13)C-D-xylose compared to healthy subjects. The time curve of (13)C-D-xylose absorption in the group of alcoholics was similar in appearance to that of untreated coeliac patients. Alcoholic patients had few light microscopic changes, but electron microscopic examination exposed morphological pathology in the majority of the patients, with a reduced surface area of microvilli as the main finding. CONCLUSIONS: Alcoholics have a pathological (13)C-D-xylose breath test with a time curve similar to that of untreated coeliac patients. This implies a condition of malabsorption. The morphological pathology found included a reduced absorptive area due to pathology of microvilli. These findings may explain our breath test results.

A novel one-hour 13C-sorbitol breath test versus the H2-sorbitol breath test for assessment of coeliac disease.

OBJECTIVE: The H(2)-sorbitol breath test (H(2)-SBT) has previously been suggested as a screening tool for coeliac disease. We developed an alternative (13)C-sorbitol breath test ((13)C-SBT). The aim of the study was to compare the diagnostic properties of the H(2)-SBT and the (13)C-SBT in a clinical setting. MATERIAL AND METHODS: Thirty-nine coeliac patients, 40 patient controls (mainly patients with irritable bowel syndrome) and 26 healthy volunteers underwent the breath tests. The patients were given an oral load of 5 g sorbitol and 100 mg (13)C-sorbitol dissolved in 250 ml tap-water. H(2), CH(4) and (13)CO(2) concentrations were measured in end-expiratory breath samples every 30 min for 4 h. Increased H(2) concentration > or =20 ppm from basal values was used as the cut-off for the H(2)-SBT. RESULTS: The H(2)-SBT had a sensitivity of 71%, a specificity of 46% versus healthy controls, and a specificity of 25% versus patient controls. Individuals with methane-producing intestinal flora had significantly lower peak H(2) concentrations than non-methane producers. The (13)C-SBT reached maximal combined sensitivity/specificity (74%/85%) for both control groups after 1 h. A diagnostic algorithm which stratified patients into high-, moderate- and low risk for coeliac disease was proposed. Following the algorithm, 62% of coeliac patients were detected with 100% specificity. The (13)C-SBT, but not the H(2)-SBT, correlated with age and serum IgA tissue-transglutaminase antibody levels in coeliac patients. CONCLUSIONS: The novel (13)C-SBT has superior diagnostic properties compared to the H(2)-SBT, which has unsatisfactory specificity in clinical practice. The 1-h (13)C-SBT may be a useful supplemental test when investigating for coeliac disease.

13C-xylose and 14C-xylose breath tests for the diagnosis of coeliac disease.

OBJECTIVES: Xylose absorption testing has traditionally involved measurement of serum xylose and/or measurement of excreted xylose in urine. However, by enriching xylose with a 13C- or 14C-isotope, absorption of an oral xylose load will be reflected in the time-dependent pattern of 13CO2 or 14CO2 exhaled in breath. Our objectives were to evaluate the diagnostic properties of 13C-xylose and 14C-xylose breath tests in coeliac disease, and to develop a diagnostic breath test index. MATERIAL AND METHODS: We reviewed data from 41 coeliac patients who underwent the 14C-xylose breath test before and after commencement of a gluten-free diet, and 60 coeliac patients who underwent the 13C-xylose breath test, 37 of whom repeated the test after starting a gluten-free diet. Coeliac patients were compared with healthy control subjects. RESULTS: Coeliac patients exhaled significantly less 13CO2 or 14CO2 than healthy controls during the first hour of the test and more isotope-labelled CO2 than control subjects after 3 h. Diagnostic accuracy was optimal with test duration of 210 min combining gas measurements at 30 min and 210 min in a simple fraction. This gas fraction index (30 min/210 min) distinguished between coeliac patients and healthy control subjects with 84-95% sensitivity and 87-94% specificity. After commencement of a gluten-free diet, the gas fraction index increased in most coeliac patients, but remained lower than that in healthy control subjects. CONCLUSIONS: 13C-xylose- and 14C-xylose breath tests discriminate between coeliac patients and healthy control subjects with high sensitivity and specificity. The stable isotope 13C-xylose breath test has comparable diagnostic accuracy to the radioactive isotope 14C-xylose breath test and should be the preferred alternative to traditional xylose absorption tests.