Bile acid diarrhea is seen in approximately 10 million Irritable Bowel Syndrome (IBS) patients, and about 86% of Crohn’s patients. Furthermore, there are 90 million patients worldwide annually that have Bile Acid-Associated Diarrhea (BAD). Despite this high prevalence, the underlying mechanism associated with BAD is not completely understood. The primary goal of our research is to identify the mechanism behind bile acid-induced tight junction dysfunction that results in diarrhea. Bile is produced by the liver, released into the duodenum where it helps emulsify fat. Once its job is done, about 95% of bile acid is recycled back to liver, and less than 5% enters the colon. The primary bile acids enter the colon, where anaerobic bacteria convert the primary bile acids to secondary bile acids, and they aid with normal secretion of fluid. However, if the bile acid reabsorption is hindered, as in cases like Crohn’s disease and Celiac disease, there is an excess amount of bile acids that accumulate in the colon, resulting in fluid loss and diarrhea. It has been shown by us and others that bile acids with two hydroxyl groups, such as chenodeoxycholate (CDCA), can alter tight junctions when present in the luminal surface and stimulate chloride secretion when present in the basolateral surface to result in diarrhea. However, the bile acid with one hydroxyl group, lithocholate (LCA), does not alter secretion. In fact, it helps attenuate the barrier dysfunction caused by CDCA. So, how does CDCA travel from lumen to the basolateral surface? This summer, in collaboration with an organic chemist, Dr. Rubush, at my university, we tested the hypothesis that CDCA loosens tight junctions to travel paracellularly to reach basal surface. We synthesized and tested fluorescently tagged CDCA and LCA in order to visually track their movement in experiments. Our results thus far, show that there is a time dependent increase in CDCA content in the basolateral chamber of the transwell. I will continue to test if LCA behaves similarly. The reason for the yin and yang in bile acid action could be due to it structural variation, the lack of a hydroxyl group in the 7th position. Therefore, we are synthesizing 7, methyl CDCA to compare its effects on tight junctions with that of CDCA and LCA.
Being part of a research laboratory has made me realize that the process of doing science is filled with many unknowns and surprises. From having to deal with slow growing cells and lack of the expected band in a Western blot to problems getting the methyl group on the correct hydroxyl group and trying to increase the yield in chemistry lab, I was exposed to the challenges in day-to-day activities in a scientific lab. For example, a minor problem with the air conditioner in our school resulted in the decomposition of an intermediate reactant in the synthesis of methyl CDCA, which set us back by a week or so. Suddenly, our colon cell line started to grow slowly and this made us change the direction of our research temporarily and focus on performing ELISA assays on samples frozen previously.
My day-to-day life as a researcher consists of a combination of planned activities, such as making buffers, media and feeding cells every Monday, Wednesday, and Friday, and designing and running experiments on confluent cells at other times. At first, I thought research would be very repetitive with rigid timings and I would be alone doing experiments independently in a lab. To my surprise, my research timings were very flexible, and I collaborated with students and professors of different departments at my university. In fact, along with organic chemistry students in the summer research program at Benedictine University who synthesized the tagged bile acids, we gave a collaborative, interdisciplinary research presentation which was very well received by faculty and peers from the College of Science. As with life in general, there were good days when we got great data, and bad days when we had contaminated cells; but overall it is a very invigorating experience!