MSACL 2024 Abstract

Mass Spectrometry Unraveling a ‘Secondary’ AKR1D1 Deficiency and Monitoring the Successful Resolution of Neonatal Acute Liver Failure by Cholic Acid Therapy

(1) Kenneth D R Setchell, PhD, FAASLD, (2) Sarah Kemme, MD MSCS, (3) Kathy Campbell MD, (1) Wujuan Zhang PhD, (4) Amy G Feldman MD, MSCS
(1) Clinical Mass Spectrometry, Division of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, (2) D. Brent Polk Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Monroe Carrell Jr. Children’s Hospital at Vanderbilt, Nashville, TN, (3) Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, (4) Digestive Health Institute, Section of Gastroenterology, Hepatology, and Nutrition, University of Colorado Denver School of Medicine and Children’s Hospital Colorado, Aurora, CO

Kenneth Setchell, PhD (Presenter) Cincinnati Children’s Hospital Medical Center

Presenter Bio: Kenneth Setchell is Professor of Pediatrics and the Director of Clinical Mass Spectrometry in the Division of Pathology and Laboratory Medicine at the Cincinnati Children’s Hospital Medical Center and Department of Pediatrics of the University of Cincinnati College of Medicine. His expertise is in clinical and biomedical applications of mass spectrometry and allied techniques, primarily in small molecules and in the area of bile acids, steroids, sterols, lipids and small molecules with a focus on liver disease, gastroenterology and nutrition. Moving from the Medical Research Center’s Clinical Resarch Center in 1984 he established a mass spectrometry facility that provides clinical diagnostic testing, therapeutic drug monitoring and biomedical research support to investigators within CCHMC and the University of Cincinnati College of Medicine. This academic facility uniquely operates under GLP standards, is CAP/CLIA accredited, and consequently has provided analytical support for numerous Phase 1-3 clinical trials for the pharmaceutical industry, leading to 2 new drugs (Cholbam and Maralixibat) being FDA approved. He has published >430 peer-reviewed publications, is the inventor of 13 patents. He was awarded the 2004 Adolf Windaus Prize for research on bile acids following the discovery with mass spectrometry of 6 genetic defects in the cholesterol-bile acid synthetic pathway that cause liver disease in infants and children and is an internationally recognized center for the diagnosis of these defects. With the late James E. Heubi MD, they developed a successful therapy using oral cholic acid to reverse the disease in otherwise fatal forms of liver disease that was approved in 2015 by the FDA - the first drug to gain FDA approval in over 40 years for cholestatic liver disease. He is also recognized in the field of nutrition, specifically related to soy and isoflavones, having discovered the intestinally derived metabolite, S-(-)equol in human urine and making the association with soy food intake that has driven much of the interest in soy foods. He has received awards for his discoveries and in 2014 was listed by Thomson-Reuters as one of “The World’s Most Influential Scientific Minds” based on highly cited publications. He received the Distinguished Contributor Award 2016 at the Mass Spectrometry Applied to the Clinical Laboratory (MSACL) for lifetime contributions to the application of mass spectrometry to the clinical research. In 2023, named as one of the leading national and international medical researchers with 36,663 citations from 302 publications (from a total of >400 publications) with a 92 D-Index.

Relevant Financial Disclosures (within past 24 months, reported on Apr 05, 2025) Stock/Bonds Asklepion Pharmaceuticals, Aliveris srl, Italy Salary Mirum Pharmaceuticals Discussing Products? YES, presenter will present educational content related to products/services of one or more ineligible company(ies) listed above. Conflict mitigation IN PROCESS.

Case Description
Presented is a case of a patient with neonatal acute liver failure (NALF) with elevated atypical Δ4-3-oxo bile acids identified by mass spectrometry that subsequently was proven by genetic analysis and mass spectrometry to be a ‘secondary’ rather than ‘primary’ AKR1D1 deficiency. The patient was a male born in NALF with extensive negative evaluation and found on urine fast atom bombardment ionization mass spectrometry (FAB-MS) to have a Δ4-3-oxosteroid 5β-reductase (AKR1D1) deficiency. Due to clinical deterioration, cholic acid therapy was started on day of life (DOL) 8 and he was evaluated for liver transplantation on DOL 9. Rapid whole genome sequencing at the time of cholic acid initiation was pending. Cholic acid treatment led to a complete resolution of liver failure.

Background
Bile acid synthesis disorders (BASD) are now a well-established class of metabolic liver disease. Hepatotoxicity is caused by the failure to synthesize the normal primary bile acids, cholic and chenodeoxycholic acids, that promote bile flow and accumulation of atypical bile acids and intermediary metabolites. In the Δ4-3-oxosteroid 5β-reductase (AKR1D1) deficiency, the second most common BASD, accumulation of hepatotoxic/cholestatic Δ4-3-oxo bile acids that cannot be transported across the canaliculus, causes severe neonatal liver disease with rapid progression to cirrhosis. Diagnosis of the AKR1D1 deficiency is complicated by the fact that Δ4-3-oxo bile acids are often transiently elevated in the urine of healthy infants during early life and also a feature of end-stage disease, referred to as a ‘secondary’ AKR1D1 deficiency. Cholic acid therapy, approved for primary BASD has not been systematically studied as an adjunctive therapy in patients with a ‘secondary’ AKR1D1 deficiency from acute or advanced liver disease.

Methods
Mass spectrometry methodologies included the application of fast atom bombardment ionization (FAB-MS) for screening for BASD, and a newly developed electrospray ionization LC-ESI-MS/MS for targeted quantification of bile acid species at baseline and following therapy. Rapid whole genome sequencing was used to identify mutations in the AKR1D1 gene.

Results
Urinary bile acid analysis by negative ionization FAB-MS at age 5-days, revealed a significant elevation of atypical Δ4-3-oxo bile acids consistent with a diagnosis of an AKR1D1 deficiency. ESI-LC-MS/MS with stable-isotope dilution analysis confirmed high concentrations of taurine and glycine conjugated 7α-hydroxy-4-cholesten-3-one and 7α,12α-dihydroxy-4-cholesten-3-one (in the range 15-35 μmol/L). On this basis he was started on cholic acid therapy, which is approved for the treatment of BASD. Whole genome sequencing later failed to find mutations in the AKR1D1 gene leading to the conclusion that the diagnosed AKR1D1 deficiency was ‘secondary’ to liver failure. Mass spectrometry served to later confirm this was the case. Despite this, his NALF rapidly improved after initiation of cholic acid. His serum total and direct bilirubin and INR levels normalized and he was discharged on DOL 38 without a need to list for transplantation. His urine FAB-MS profile on DOL 25 confirmed compliance to cholic acid therapy and a concomitant decrease of the atypical Δ4-3-oxo bile acids. By DOL 84, his serum liver biochemistries had improved and cholic acid therapy was discontinued. Subsequent urine FAB-MS analysis at 3-months of age revealed a normal mass spectrum and targeted quantification confirmed a complete disappearance of the atypical hepatotoxic Δ4-3-oxo bile acids consistent with a complete resolution of his ‘secondary’AKR1D1 deficiency. Cholic acid treatment led to a complete resolution of liver failure.

Discussion and Conclusion
This case exemplifies how mass spectrometry was critical to the management of this case of NALF, in changing the clinical course in a life-saving manner, while circumventing the need for liver transplantation. Neonates in NALF, with ‘secondary’ AKR1D1 deficiency, for which treatments are limited and in which liver transplantation can be difficult due to a shortage of organ availability, may show a successful clinical response to cholic acid therapy. These novel findings warrant further investigation of cholic acid therapy in NALF.