MSACL 2022 Abstract

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.