MSACL 2022 Abstract

Application of a Novel Steroidomic LC-MS/MS Method for Investigating Metabolic Alterations in Patients with Non-Alcoholic Fatty Liver Disease

Federico Ponzetto (1,2), Mirko Parasiliti-Caprino (1,2), Chiara Rosso (1), Gian Paolo Caviglia (1), Fabio Settanni (3), Elisabetta Bugianesi (1,4), Mauro Maccario (1,2), Giulio Mengozzi (1,3)
(1) Department of Medical Sciences, University of Turin, Turin, Italy (2) Endocrinology, Diabetes and Metabolism, City of Health and Science University Hospital, Turin, Italy (3) Clinical Biochemistry Laboratory, City of Health and Science University Hospital, Turin, Italy (4) Unit of Gastroenterology, City of Health and Science University Hospital, Turin, Italy

Federico Ponzetto, PhD (Presenter) University of Turin

Presenter Bio: Federico Ponzetto, PhD is a Tenure Track Researcher in Clinical Biochemistry and Clinical Molecular Biology at the Department of Medical Sciences, University of Turin, Italy. His research activity is mainly focused on steroid analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS), with particular expertise in targeted steroidomics, endogenous steroid profiling, and the development of high-throughput analytical workflows for both clinical endocrinology and anti-doping applications. He obtained his PhD at the Swiss Laboratory for Doping Analyses, University of Lausanne (Switzerland), where he developed blood steroid profiling strategies for the detection of testosterone misuse within the Athlete Biological Passport framework.
Dr. Ponzetto has more than 10 years of experience in mass spectrometry-based steroid analysis across multiple biological matrices, including serum, plasma, urine, saliva, and microsampling devices. His work has contributed to the implementation of innovative UHPLC-MS/MS methods for the quantification of endogenous steroids, phase II metabolites, glucocorticoids, and biomarkers relevant to endocrine disorders and doping control. He has authored over 30 peer-reviewed publications in the fields of clinical mass spectrometry, steroidomics, and endocrine laboratory medicine, and has served as principal investigator of international research projects funded by the World Anti-Doping Agency and Partnership for Clean Competition. His current research interests include advanced LC-MS/MS steroidomic platforms, adrenal and metabolic disorders, and automation of mass spectrometry workflows in routine laboratory medicine.

Relevant Financial Disclosures (within past 24 months, reported on May 26, 2026) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION
The onset and progression of liver damage in non-alcoholic fatty liver disease (NAFLD) is tightly associated with metabolic derangements. Steroid hormones and their metabolites may affect lipid metabolism but their alterations in the setting of NAFLD remain to be fully explored.

OBJECTIVES
The objectives of this study were: i) the development and validation of a LC-MS method for measuring serum/plasma concentrations of an extended panel of steroid hormones and their phase II metabolites; ii) the application of the developed method to samples coming from a cohort of NAFLD patients to investigate steroidal perturbations linked to severe hepatic fibrosis.

METHODS
A novel UHPLC-MS/MS method for the quantification of major circulating steroid hormones (including glucocorticoids, mineralocorticoids, androgens and progestogens) together with an extended panel of androgens’ glucuro- and sulpho-conjugated phase 2 metabolites was developed and validated. Chromatographic set up was optimized comparing the performance of three different C18 analytical columns and accurately selecting the mobile phases with the aim of separating all the 26 target steroids, including numerous isomeric isobaric compounds. MS parameters were finely tuned to obtain the sensitivity needed for measuring the concentrations of target analytes, ranging from low pg/mL to μg/mL. Finally, sample preparation protocol was developed for efficiently extracting steroid hormones from 200µL of plasma/serum and its performances were evaluated in terms of extraction recovery and matrix effect. Plasma samples of 121 patients with biopsy-proven NAFLD and 108 controls (CT) were analyzed with the developed method and severe hepatic fibrosis (F) was defined by F≥3.

RESULTS
Compared to CT, NAFLD patients were older (median age 51 vs 43, p<0.001) and were characterized by a higher rate of Metabolic Syndrome (47% vs 2%, p<0.001). More than a half of target steroids were deregulated in patients compared to CT. At liver histology, the prevalence of absent/mild, moderate and severe fibrosis was 50.4%, 10.8% and 38.8%, respectively. Circulating levels of 16 compounds showed a significant stepwise decrease according to the degree of hepatic fibrosis. At univariate analysis, testosterone and its derivatives, androsterone metabolites, etiocholanolone metabolites and glicoandrogens metabolites were differentially expressed in patients with severe fibrosis compared to those with absent/moderate fibrosis. After multivariable logistic regression analysis adjusted for age, sex and type 2 diabetes, epitestosterone sulphate, 5α-androstan-3α,17β-diol 3-glucuronide and androsterone sulphate levels were significantly associated with F≥3. The diagnostic accuracy of the model for the identification of F≥3 was 0.91 with a sensitivity and specificity of 87% and 85 %, respectively, and with a positive and negative predictive value of 78% and 91%, respectively.

CONCLUSIONS
In NAFLD patients, alterations in androgens and their glucuro- and sulpho-conjugated metabolites levels are expression of compromised steroid homeostasis regulation by the liver and are associated with severe fibrosis.