This presentation will highlight how mass spectrometry was successfully applied to define new genetic defects in the cholesterol-bile acid biosynthetic pathway as a specific class of metabolic liver disease. Bile acid synthesis disorders due to single enzyme defects generally present in infancy or early childhood with a progressive cholestatic hepatitis that, unchecked, lead to cirrhosis, liver failure, and death. Prior to the seminal work of Setchell and colleagues in identifying 6 genetic diseases as discrete entities, and conceiving of an effective therapy, children with these autosomal recessive diseases either underwent liver transplantation, or more commonly, were given supportive care until they died of liver failure of unknown origin. To be described are the combined use an untargeted and targeted approach with FAB-MS, GC-MS and ESI-LC-MS/MS that led to the elucidation of the biochemical basis of these diseases, the development of an international screening program, and the evaluation of the therapeutic responses that served to ultimately gain regulatory approval from the FDA for a life-saving therapy based on oral administration of cholic acid. This application of mass spectrometry to clinical chemistry has been a game-changer that has led to a radical change in the evaluation and treatment of patients with idiopathic progressive familial intrahepatic cholestasis syndromes.1
In 1919 Adolf Windaus made the critical discovery that coprostane could be oxidized to cholanic acid and thereafter recognized the close relationship between cholesterol and bile acids, and later, steroids and vitamin D. The pathway for bile acid synthesis was defined >60 years ago and the importance of bile acids in facilitating lipid absorption and liver function realized. The formation of bile acids in the liver involves a complex multi-stepped sequence of reactions in which the neutral sterol cholesterol is converted to the acidic steroids, cholic and chenodeoxycholic acids, referred to as primary bile acids1. The reactions in this pathway are catalyzed by at least 17 hepatic enzymes, but only in the last two-plus decades have disorders in bile acid synthesis become a recognizable cause of progressive familial intrahepatic cholestasis (PFIC), fatal forms of liver disease, thus defining a new category of ‘metabolic liver disease’. These discoveries were exclusively the result of the application of mass spectrometric techniques involving the use of the soft ionization technique of FAB-MS complemented by GC-MS, and later LC-MS/MS approaches to the metabolic profiling of a ‘drop of urine’ – it can be considered to represent an early successful example of ‘metabolomics’ long predating the coining of the ‘omics’ terminology. Rapid analysis of bile acids directly in urine samples with minimal sample preparation led to the discovery of the first 6 autosomal recessive single-enzyme genetic defects in the bile acid synthetic pathway. The biochemical basis of each defect was characterized, and these defects have accounted for approximately 2% of patients with idiopathic cholestasis screened in an international diagnostic screening program established in 1985 at Cincinnati Children’s Hospital Medical Center. Biochemically, all share the common feature of an absence of hepatic synthesis of the normal primary bile acids, cholic and chenodeoxycholic acids, that are essential for the promotion of bile flow, with concomitant elevated levels of atypical bile acids that are cholestatic and hepatotoxic. The clinical manifestation includes varying degrees of progressive intrahepatic cholestasis, fat-soluble vitamin malabsorption, and neurological disease. The poor prognosis for many of these patients prompted our development of a highly successful therapy based on oral administration of cholic acid. The hypothesis being that the toxic, hydrophobic bile acid intermediates that accumulate to abnormally high levels could through a feedback mechanism be suppressed with the expectation of a profound therapeutic effect in normalizing liver function. This hypothesis was confirmed by monitoring the disappearance of atypical bile acids with FAB-MS and ESI tandem mass spectrometry. Long-term survival and impressive clinical improvements including normalization in serum liver enzymes, and improvement in growth and liver histology is now the norm. Based on this successful therapeutic strategy, cholic acid therapy was recently approved by the European Medicines Agency and by FDA. This research program spanning some 30+ years highlights how mass spectrometry has been successfully applied to investigations of idiopathic liver disease in infants and children to define biochemical defects in cholesterol-bile acid biosynthetic pathway as the underlying cause, understanding the mechanism of liver injury, and developing a life-saving therapy.
References & Acknowledgements:
1. Russell DW, Setchell KDR. Bile Acid Biosynthesis. Biochemistry 1992; 31:4737-4749.
2. Clayton PT, Leonard JV, Lawson AM, Setchell KDR, Andersson S, Egestad B, Sjövall J. Familial giant cell hepatitis associated with synthesis of 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholenoic acids. J. Clin. Invest. 1987;79:1031-1038.
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