Evaluation of gut dysbiosis using serum and fecal bile acid profiles.

Dysbiosis in the intestinal microflora can affect the gut production of microbial metabolites, and toxic substances can disrupt the barrier function of the intestinal wall, leading to the development of various diseases. Decreased levels of Clostridium subcluster XIVa (XIVa) are associated with the intestinal dysbiosis found in inflammatory bowel disease (IBD) and Clostridium difficile infection (CDI). Since XIVa is a bacterial group responsible for the conversion of primary bile acids (BAs) to secondary BAs, the proportion of intestinal XIVa can be predicted by determining the ratio of deoxycholic acid (DCA)/[DCA + cholic acid (CA)] in feces orserum. For example, serum DCA/(DCA+CA) was significantly lower in IBD patients than in healthy controls, even in the remission period. These results suggest that a low proportion of intestinal XIVa in IBD patients might be a precondition for IBD onset but not a consequence of intestinal inflammation. Another report showed that a reduced serum DCA/(DCA + CA) ratio could predict susceptibility to CDI. Thus, the BA profile, particularly the ratio of secondary to primary BAs, can serve as a surrogate marker of the intestinal dysbiosis caused by decreased XIVa.

Evaluation of the Risk of Clostridium difficile Infection Using a Serum Bile Acid Profile.

Since intestinal secondary bile acids (BAs) prevent Clostridium difficile infection (CDI), the serum BA profile may be a convenient biomarker for CDI susceptibility in human subjects. To verify this hypothesis, we investigated blood samples from 71 patients of the Division of Gastroenterology and Hepatology at the time of admission (prior to antibiotic use and CDI onset). Twelve patients developed CDI during hospitalization, and the other 59 patients did not. The serum unconjugated deoxycholic acid (DCA)/[DCA + unconjugated cholic acid (CA)] ratio on admission was significantly lower in patients who developed CDI than in patients who did not develop CDI (p < 0.01) and in 46 healthy controls (p < 0.0001). Another unconjugated secondary BA ratio, 3ß-hydroxy (3ßOH)-BAs/(3ßOH + 3αOH-BAs), was also significantly lower in patients who developed CDI than in healthy controls (p < 0.05) but was not significantly different between patients who developed and patients who did not develop CDI. A receiver operating characteristic (ROC) curve determined a cut-off point of DCA/(DCA + CA) < 0.349 that optimally discriminated on admission the high-risk patients who would develop CDI (sensitivity 91.7% and specificity 64.4%). In conclusion, a decreased serum DCA/(DCA + CA) ratio on admission strongly correlated with CDI onset during hospitalization in patients with gastrointestinal and hepatobiliary diseases. Serum BA composition could be a helpful biomarker for predicting susceptibility to CDI.

Western Diet Changes Gut Microbiota and Ameliorates Liver Injury in a Mouse Model with Human-Like Bile Acid Composition.

Western-style high-fat/high-sucrose diet (HFHSD) changes gut microbiota and bile acid (BA) profiles. Because gut microbiota and BAs could influence each other, the mechanism of changes in both by HFHSD is complicated and remains unclear. We first aimed to clarify the roles of BAs in the HFHSD-induced change of gut microbiota. Then, we studied the effects of the changed gut microbiota on BA composition and liver function. Male wild-type (WT) and human-like Cyp2a12/Cyp2c70 double knockout (DKO) mice derived from C57BL/6J were fed with normal chow or HFHSD for 4 weeks. Gut microbiomes were analyzed by fecal 16S ribosomal RNA gene sequencing, and BA composition was determined by liquid chromatography-tandem mass spectrometry. The DKO mice exhibited significantly reduced fecal BA concentration, lacked muricholic acids, and increased proportions of chenodeoxycholic and lithocholic acids. Despite the marked difference in the fecal BA composition, the profiles of gut microbiota in the two mouse models were quite similar. An HFHSD resulted in a significant increase in the BA pool and fecal BA excretion in WT mice but not in DKO mice. However, microbial composition in the two mouse models was drastically but similarly changed by the HFHSD. In addition, the HFHSD-induced change of gut microbiota inhibited BA deconjugation and 7α-dehydroxylation in both types of mice, which improved chronic liver injury observed in DKO mice. Conclusion: The HFHSD itself causes the change of gut microbiota due to HFHSD, and the altered composition or concentration of BAs by HFHSD is not the primary factor. On the contrary, the gut microbiota formed by HFHSD affects BA composition and ameliorates liver injury in the mouse model with human-like hydrophobic BA composition.

Current states of prevention of drug-induced gastroduodenal ulcer in real clinical practice: a cross-sectional study.

Non-steroidal anti-inflammatory drugs (NSAIDs) or low-dose aspirin (LDA) are the most common causes of drug-induced gastroduodenal ulcer and We investigated preventive treatment with use of concomitant anti-ulcer drugs and the clinical features of gastroduodenal ulcer in cases treated with these drugs. Patients with gastroduodenal ulcer and patients with bleeding were classified into 3 groups: LDA, non-aspirin NSAIDs, and those taking neither aspirin nor NSAIDs. Chronological changes over the past 16 years (1st-5th period) were investigated. The status of prevention of ulcer and clinical features were examined. From January 2002 to December 2018, the ratio of all patients taking NSAIDs and LDA increased significantly until 3rd period (p<0.05), but then started to decrease in 4th period; and the percentage of all patients taking NSAIDs and LDA decreased significantly (p<0.05) until 5th period. Among the 292 patients with gastroduodenal ulcer and the 121 patients with a bleeding ulcer taking NSAIDs and LDA, 16 (5.5%) and 9 (7.4%), respectively, were receiving preventive treatment with concomitant anti-ulcer drugs. The percentages of patients taking LDA and other antiplatelet drugs in patients with bleeding gastroduodenal ulcer were significantly higher than those in patients with non-bleeding. In conclusion, although the percentages of patients with gastroduodenal ulcer taking NSAIDs or LDA have not recently increased in real-world practice, preventive treatment in these patients is still low. This low rate of prevention suggests the need to enlighten physicians about preventive treatment because drug withdrawal of LDA has a high risk of cardiovasculr and cerebrovascular events.

Regulation of bile acid metabolism in mouse models with hydrophobic bile acid composition.

The bile acid (BA) composition in mice is substantially different from that in humans. Chenodeoxycholic acid (CDCA) is an end product in the human liver; however, mouse Cyp2c70 metabolizes CDCA to hydrophilic muricholic acids (MCAs). Moreover, in humans, the gut microbiota converts the primary BAs, cholic acid and CDCA, into deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. In contrast, the mouse Cyp2a12 reverts this action and converts these secondary BAs to primary BAs. Here, we generated Cyp2a12 KO, Cyp2c70 KO, and Cyp2a12/Cyp2c70 double KO (DKO) mice using the CRISPR-Cas9 system to study the regulation of BA metabolism under hydrophobic BA composition. Cyp2a12 KO mice showed the accumulation of DCAs, whereas Cyp2c70 KO mice lacked MCAs and exhibited markedly increased hepatobiliary proportions of CDCA. In DKO mice, not only DCAs or CDCAs but also DCAs, CDCAs, and LCAs were all elevated. In Cyp2c70 KO and DKO mice, chronic liver inflammation was observed depending on the hepatic unconjugated CDCA concentrations. The BA pool was markedly reduced in Cyp2c70 KO and DKO mice, but the FXR was not activated. It was suggested that the cytokine/c-Jun N-terminal kinase signaling pathway and the pregnane X receptor-mediated pathway are the predominant mechanisms, preferred over the FXR/small heterodimer partner and FXR/fibroblast growth factor 15 pathways, for controlling BA synthesis under hydrophobic BA composition. From our results, we hypothesize that these KO mice can be novel and useful models for investigating the roles of hydrophobic BAs in various human diseases.

The comparison of the intensity of human intestinal spirochetes between Brachyspira pilosicoli and Brachyspira aalborgi infections.

The agglutination titers of Brachyspira pilosicoli (B. pilosicoli) and Brachyspira aalborgi (B. aalborgi) were examined in colitis patients with human intestinal spirochetes. Among three cases of colitis patients, the titer of B. pilosicoli was extremely high in two cases while the titer of B. aalborgi was extremely high in one case. These three cases had symptoms of colitis, such as watery diarrhea, and we diagnosed the case as Brachyspira- related colitis. These findings suggest that the agglutination titers of Brachyspira may be useful in cases of Brachyspira- related colitis. Severe symptoms, such as abdominal pain and diarrhea, were observed in cases with high antibody titer of B. aalborgi, as well as B. pilosicoli, indicating that B. aalborgi could also cause symptomatic colitis.

Detection of Gut Dysbiosis due to Reduced Clostridium Subcluster XIVa Using the Fecal or Serum Bile Acid Profile.

Background: Dysbiosis, especially a reduced Clostridium subcluster XIVa (XIVa), has been reported in several gastrointestinal diseases. Since XIVa is thought to be the main bacterial cluster that metabolizes bile acids (BAs) in the human intestine, we hypothesized that the BA profile in feces, and possibly in serum, could be a convenient biomarker for intestinal XIVa activity. Methods: First, blood and feces were collected from 26 healthy controls and 20 patients with gastrointestinal diseases, and the relationships among fecal microbiomes and fecal and serum BA compositions were studied. Second, serum BA compositions of 30 healthy controls and the remission and exacerbation states of 14 Crohn's disease (CD) and 12 ulcerative colitis (UC) patients were compared. Fecal microbiomes were analyzed by terminal restriction fragment length polymorphism analysis, and BA compositions were quantified by HPLC-MS/MS. Results: The highest positive correlation was observed between the fecal XIVa proportion and fecal unconjugated deoxycholic acid (DCA)/(DCA+unconjugated cholic acid [CA]) (r = 0.77, P < 0.0001) or serum DCA/(DCA+CA) (r = 0.52, P < 0.001). Diurnal variation in serum XIVa candidate markers also showed that DCA/(DCA+CA) was most stable and not affected by the contraction of the gallbladder. Serum DCA/(DCA+CA) was not significantly different between remission and exacerbation states in either CD or UC patients, but was significantly reduced in those in the remission state of CD and the remission and exacerbation states of UC compared with healthy controls (P < 0.05). Conclusions: Decreased XIVa exhibits a strong correlation with reduced intestinal BA metabolism. Fecal and serum DCA/(DCA+CA) could be useful surrogate markers for the intestinal proportion of XIVa.

Effects of the Concomitant Use of Low-dose Clarithromycin with an Anti-TNFα Antibody in a Patient with Intestinal Behçet Disease.

A 66-year-old Japanese male with a history of Behçet disease exhibited oral and genital ulcers, and a round deep ileocecal ulcer. He was treated with a combination of mesalazine and 20 mg/day of prednisolone (PSL), but was only partially responsive to PSL and we were not able to reduce the steroid dosage. Adalimumab was also administered. However, the ulcer was not completely responsive, and weaning the patient off PSL remained impossible. In contrast, additional treatment with clarithromycin completely healed the refractory active ulcer and left only a scar. Furthermore, the ulcer has since maintained the scar stage despite successfully weaning the patient from PSL.

Simultaneous quantification of salivary 3-hydroxybutyrate, 3-hydroxyisobutyrate, 3-hydroxy-3-methylbutyrate, and 2-hydroxybutyrate as possible markers of amino acid and fatty acid catabolic pathways by LC-ESI-MS/MS.

We have developed a highly sensitive and specific method for quantification of salivary 3-hydroxybutyrate (3HB), 3-hydroxyisobutyrate (3HIB), 3-hydroxy-3-methylbutyrate (3HMB) and 2-hydroxybutyrate (2HB), which could be new non-invasive biomarkers for catabolic pathways of fatty acids/ketogenic amino acids, valine, leucine, and methionine/threonine/α-ketobutyrate, respectively. The four hydroxybutyrates (3HB, 3HIB, 3HMB, and 2HB) were extracted from 5 µl of saliva, converted to 2-pyridylmethyl (2PM) ester derivatives, and measured by liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode. [(13)C4]3HB was used as an internal standard. The detection limits for the 2PM esters were <1 pg (7.9-9.6 fmol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of the hydroxybutyrates were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements were calculated to be 0.45-5.28 and 0.54-3.45 %, respectively. Experiments performed using 5 µl of saliva spiked with 3.8-154.4 pmol of the four hydroxybutyrates gave recoveries of 98.5 to 108.8 %, with a mean recovery of 104.1 %. In vitro experiments in hepatocytes or skeletal muscle cells showed that addition of palmitic acid, valine, leucine or α-ketobutyrate to culture medium markedly increased the targeted hydroxybutyrate concentrations. The salivary concentration of each targeted hydroxybutyrate was positively correlated with that in serum, and the salivary levels were elevated in patients with liver cirrhosis, which is characterized by upregulated catabolism of lipids and amino acids. The proposed method is useful for quantification of salivary 3HB, 3HIB, 3HMB, and 2HB for monitoring of catabolic activities of amino acids and fatty acids.