Endocrine Abstracts

Background: NMDAR-Ab mediated encephalitis is a neuro-immunological disorder that presents with seizures. NMDAR-Abs cause internalisation of NMDARs while Pregnenolone sulfate (PregS) a neurosteroid upregulates NMDARs in the brain. Our previous in vitro studies have shown that PregS can reduce established ictal activity caused by NMDAR-Abs (Wright et al., 2021). Before proceeding with in vivo treatment studies, we aimed to determine baseline brain PregS levels in vivo and following subcutaneous PregS injections using an in-house modified LC-MS method.

Method: Rats were randomised and divided into three groups; Control, Cyclodextrin (CDX) and CDX+PregS treated. Rats were perfused after 8 hours and brain were extracted, immediately snap-freezed and stored in -80°C. PregS levels were determined from rat brain tissue (0.1 mg) spiked with internal standard (1ng of pregnenolone-17α,21,21,21-d4 sulfate) by adding 1 ml of ACN-ZnSO4 [89 g/l, 4:1 (v/v)], followed by alternate vortexing and sonication for 10min. PregS was enriched using two-step solid phase extraction (SPE) using a polymeric SPE column (HLB PRiME, Waters). A reverse phase LC method using 85% water/acetonitrile to methanol/acetonitrile gradient was systematically developed for optimal separation of PregS. Method validation was performed to establish linearity, sensitivity, recovery, and accuracy.

Results: The endogenous PregS levels were observed in control and CDX brain samples which were increased following PregS injection. On the other hand, there was no significant change in the cholesterol levels in the same brain samples. This assay has a linear dynamic range (R² > 0.94) of 0.02ng/ml-1ng/ml for PregS and the extraction recoveries were reproducible (>90% consistent).

Conclusion: Increased levels of PregS proves the used LC-MS method to be sensitive and accurate to identify endogenous brain neurosteroid levels. Further work will include in vivo treatment of NMDAR-Ab mediated rat models with PregS.