ATYPICAL BILE ACIDS AS CHEMICAL PROBES FOR DIAGNOSIS AND THERAPHY MONITORING OF PROGRESSIVE CHOLESTATIC LIVER DISEASE IN HSD3B7 PATIENTS

Inborn errors in bile acid synthesis are a well-recognized category of metabolic liver disease. These autosomal recessive genetic defects manifest as a broad phenotype presenting with an overlapping spectrum of variable degrees of neonatal cholestasis, fat-soluble vitamin malabsorption, and neuropathies. 3b-Hydroxy-D5-C27-steroid oxidoreductase (HSD3B7) deficiency is the most common of the 9 known defects in bile acid biosynthesis and is often the cause of idiopathic forms of late-onset chronic cholestasis in children and adolescents, and even in adult. Early diagnosis of these genetic defects is crucial to prognosis, because if undiagnosed or untreated, the liver disease, which is a progressive form of intrahepatic cholestasis, leads to fibrosis, cirrhosis, and end-stage disease. Treatment options include liver transplantation or preferably oral bile acid therapy with the primary bile acids, cholic or chenodeoxycholic acids. Diagnosis of HSD3B7 deficiency is based on the detection of increased concentrations of atypical 3-hydroxy-D5-bile acids that accumulate in urine because of the lack of enzyme activity caused by mutations in HSDB7 gene. So far, direct and accurate measurement of these conjugated 3b-hydroxy-D5-bile acids has not been possible for the lack of reference standards. We describe here the synthesis and bio-chemical characterization of a series of D5-cholenoic acid analogs, which are the signature metabolites of the HSD3B7 deficiency in humans. Using these synthesized compounds as tools to gain insights into the mechanism(s) responsible of the cholestasis and liver damage in patients with the HSD3B7 deficiency, we report their cellular hepatocytotoxicity, their affinity towards a subset of bile acid-responsive nuclear receptors and the effects on genes and cytochromes involved in bile acid homeostasis and detoxification. Moreover, the availability of these reference compounds has allowed developing an electrospray ionization (ESI) LC-MS/MS method for the accurate measurement of their concentrations in clinical diagnosis and monitoring of response to therapy in patients with HSD3B7 deficiency.