The human colon harbors one of the most densely populated bacterial ecosystems known (>1011 bacteria/g feces). Cholic acid (CA) and chenodeoxycholic acid (CDCA) are primary bile acids synthesized from cholesterol in the liver. Bile acids undergo enterohepatic circulation several times each day. Primary bile acids entering the colon are deconjugated and free bile acids are biotransformed to more than 20 different metabolites by gut bacteria. The major biotransformation is 7?-dehydroxylation (7?- DeOH) of CA and CDCA yielding the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. Hydrophobic secondary bile acids are highly toxic to mammalian cells and can accumulate to high levels (>70%) in the bile acid pool of some individuals as the human liver cannot 7?-hydroxylate secondary bile acids. High levels of DCA in blood, bile, and feces have been correlated with an increase risk of colon cancer and cholesterol gallstone disease, in some patients. The formation of secondary bile acids is carried out by a few species of the genus Clostridium and the level of these bacteria in feces is correlated with DCA levels in bile. The biochemical pathway of bile acid 7?-DeOH has been elucidated by our group. However, many of the genes encoding enzymes in this pathway have not been isolated and characterized nor have the 3D structure of key enzymes in this pathway determined. Some strains of 7?- dehydroxylating bacteria can convert glucocorticoids into C-19 androgenic compounds and the genes encoding these enzymes have not been isolated. Isolation of these genes is crucial for the development of molecular techniques (RT-PCR) to easily monitor intestinal bile acid and glucocorticoid metabolism and pharmacological or dietary ways to decrease formation of secondary bile acids and C-19 androgenic compounds.
Sub aim 1 A. Identify CA, allo-CA, and glucocorticoid inducible genes from the human bile acid 7?-dehydroxylating bacterium, Clostridium scindens ATCC 35704, via a wholetranscriptome approach (RNAseq).
Sub aim 1 B. Develop quantitative PCR assays for key genes involved in allo-DCA and C-19 androgen formation.
Aim 2. Characterize the allo-CA and glucocorticoid inducible enzymes from Clostridium scindens ATCC 35704 catalyzing the formation of allo-DCA acid and C-19 androgens, respectively.
Aim 3 : Determine the 3D structure of bile acid 7? and 7?-dehydratases, the rate limiting enzymes in bile acid 7?/7?-DeOH, from Clostridium scindens. The results of these studies will give us the tools to carry out clinical studies to determine the most effective ways to decrease DCA in the bile acid pool with the aim of decreasing the risk of colon cancer and cholesterol gallstones, in some patients.

Public Health Relevance

The primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) are converted to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, by a small population of gut bacteria belonging to the genus Clostridium. Secondary bile acids are more hydrophobic and toxic to mammalian cells than primary bile acids. Increased levels of DCA in blood, bile and feces have been correlated with an increase incidence of colon cancer and cholesterol gallstone disease, in some patients. The normal level of DCA in human bile is around 20% of the total bile acid pool. However, in a preliminary study of patients (92) at the McGuire VA Hospital in Richmond, Virginia, we found that approximately 50% of these patients had DCA between 30% and 70%, which is considered to be high levels of DCA. The studies proposed in this application are aimed at determining why this population of patients has high levels of DCA in bile and developing ways to monitor bile acid 7?-dehydroxylating gut bacteria in fecal samples.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX001328-02
Application #
8536579
Study Section
Gastroenterology (GAST)
Project Start
2012-07-01
Project End
2016-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
2
Fiscal Year
2013
Total Cost
Indirect Cost
Name
VA Veterans Administration Hospital
Department
Type
DUNS #
146678115
City
Richmond
State
VA
Country
United States
Zip Code
23249