Identification of multiple cardiotonic steroids in faecal material of untreated humans and rat strains

• Extraction of cardiotonic steroids from faecal material is critically dependent on the solvent. • Multiple cardiotonic steroids were identified in the faecal material of untreated rats and humans. • Steroids varied with rat strains with marinobufogenin being most abundant. • Bufalin and digitoxin were predominant in SHR and WKY rat faeces respectively. • Identified steroids isolated aligned with the phenotype of the rat strain. • The study provides preliminary data for future studies determining: • Associations between cardiotonic steroids and the disease phenotype. • The role of the microbiome in transformations and metabolism of these steroids. Endogenous cardiotonic steroid (CTS) concentrations are raised in cardiovascular diseases. CTSs undergo gastro-hepatobiliary recirculation, with the gut being an important route of elimination, yet the presence of CTSs in faecal material is seldom reported. This study investigated methods to extract and identify the presence of CTSs in faecal material of rats and humans without prior treatment. Freeze-dried faecal material from different untreated rat strains was extracted using various solvents, with separation and identification of CTSs using HPLC/MS. Preliminary results were obtained from human faecal material. Multiple CTSs were identified in faecal material, with marinobufagenin (MBG) predominant. Telocinobufagin was only detected in certain rat strains, whereas the extraction methods used did not recover ouabain. MBG and digoxin were elevated in Dahl salt sensitive rats fed supplementary salt. Bufalin was present in most spontaneously hypertensive rats (SHRs) but was not detectable in Wistar Kyoto rats (WKY). Conversely, digitoxin was detected in most WKYs but only few SHRs. Levels of digitoxin and bufalin remained relatively constant over 24 days in untreated rats. Solvent selection was critical in determining the CTSs extracted from human faecal material. Multiple CTSs were detected in faecal material of untreated rats and humans Steroids varied between rat strains and aligned with phenotype. Extraction requires further solvent optimisation and the use of tandem MS/MS is essential to reliably detect the profile of CTSs present. Analysis of CTSs present in readily available faecal material will enable studies to determine relationships between CTSs, the microbiome and disease progression.