Ultrasensitive Quantification of Multiple Estrogens in Songbird Blood and Microdissected Brain by LC-MS/MS

Abstract

Neuroestrogens are synthesized within the brain and regulate social behavior, learning and memory, and cognition. In song sparrows, Melospiza melodia, 17β-estradiol (17β-E 2 ) promotes aggressive behavior, including during the non-breeding season when circulating steroid levels are low. Estrogens are challenging to measure because they are present at very low levels, and current techniques often lack the sensitivity required.Furthermore, current methods often focus on 17β-E 2 and disregard other estrogens.Here, we developed and validated a method to measure four estrogens (estrone, 17β-E 2 , 17α-estradiol, estriol) simultaneously in microdissected songbird brain, with high specificity, sensitivity, accuracy, and precision. We used liquid chromatography tandem mass spectrometry (LC-MS/MS), and to improve sensitivity, we derivatized estrogens using 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). The straightforward protocol improved sensitivity by 10-fold for some analytes. There is substantial regional variation in neuroestrogen levels in brain areas that regulate social behavior in male song sparrows. For example, the auditory area NCM, which has high aromatase levels, has the highest estrone and 17β-E 2 levels. In contrast, estrogen levels in blood are very low. Estrogen levels in both brain and circulation are lower in the non-breeding season than in the breeding season. This technique will be useful for estrogen measurement in songbirds and potentially other animal models.

Brain and circulating steroids in an electric fish: Relevance for non-breeding aggression

“…A value was considered non-detectable if it was below the lowest standard on the calibration curve. When the percentage of detectable samples was � 20% of the total values in a group, the non-detectable values were estimated via quantile regression imputation of left-censored missing data using MetImp web tool [14,16,[46][47][48][49]. When the percentage of detectable samples in a group was < 20% of the total values in the group, the non-detectable values were set to 0 to perform statistical analysis.…”

“…This study showed seasonal patterns in different regions of the social behavior network that differ greatly from those in the blood [ 14 ]. The use of ultrasensitive LC-MS/MS assays that can measure multiple steroids at low analyte levels can further our understanding of local steroidogenesis, the precursors involved, and the balance between local and systemic steroid signaling [ 14 – 16 ].…”

Brain and circulating steroids in an electric fish: relevance for non-breeding aggression

“…A value was considered non-detectable if it was below the lowest standard on the calibration curve. When detectable samples in a group were ≥ 20%, missing values were estimated via quantile regression imputation of left-censored missing data using MetImp web tool 13,[38][39][40][41][42] . When detectable samples in a group were < 20%, non-detectable values were set to 0 to perform statistical analysis.…”

Sex-dependent effects of multiple acute concurrent stresses on memory: a role for hippocampal estrogens

“…We probably failed to detect estradiol with our current mass spectrometry approach because estradiol quantities were too low or extraction procedures insufficient. While large quantities of tissue estradiol have been quantified without derivatization (McNamara et al, 2010 ), other groups have successfully detected small quantities through estradiol-specific derivatization (Kato et al, 2013 ; Jalabert et al, 2022 ) or signal-enhancing additives (Lozan et al, 2017 ).…”