Combined Liquid and Solid-Phase Extraction Improves Quantification of Brain Estrogen Content

Abstract

Accuracy in quantifying brain-derived steroid hormones ("neurosteroids") has become increasingly important for understanding the modulation of neuronal activity, development, and physiology. Relative to other neuroactive compounds and classical neurotransmitters, steroids pose particular challenges with regard to isolation and analysis, owing to their lipid solubility. Consequently, anatomical studies of the distribution of neurosteroids have relied primarily on the expression of neurosteroid synthesis enzymes.To evaluate the distribution of synthesis enzymes vis-à-vis the actual steroids themselves, traditional steroid quantification assays, including radioimmunoassays, have successfully employed liquid extraction methods (e.g., ether, dichloromethane, or methanol) to isolate steroids from microdissected brain tissue. Due to their sensitivity, safety, and reliability, the use of commercial enzyme-immunoassays (EIA) for laboratory quantification of steroids in plasma and brain has become increasingly widespread. However, EIAs rely on enzymatic reactions in vitro, making them sensitive to interfering substances in brain tissue and thus producing unreliable results. Here, we evaluate the effectiveness of a protocol for combined, two-stage liquid/solid-phase extraction (SPE) as compared to conventional liquid extraction alone for the isolation of estradiol (E 2 ) from brain tissue. We employ the songbird model system, in which brain steroid production is pronounced and linked to neural mechanisms of learning and plasticity. This study outlines a combined liquid-SPE protocol that improves the performance of a commercial EIA for the quantification of brain E 2 content. We demonstrate the effectiveness of our optimized method for evaluating the region specificity of brain E 2 content, compare these results to established anatomy of the estrogen synthesis enzyme and estrogen receptor, and discuss the nature of potential EIA interfering substances.

A Quantification of the Injury‐Induced Changes in Central Aromatase, Oestrogenic Milieu and Steroid Receptor Expression in the Zebra Finch

“…These studies are ongoing in our laboratory. Using the same extraction and analysis techniques utilised in our experiment, Chao et al (32) measured approximately 10 pg/mg of E 2 in the posterior and anterior portions of the telencephalon of uninjured male zebra finches. Although this concentration is higher than our measurements for the male uninjured telencephalon of the brain, it is decreased compared to our concentrations from the male injured telencephalon (approximately 19 pg/mg).…”

“…As described previously (32,33), E 2 is readily detectable in the zebra finch brain. Statistical analyses using log-transformed data revealed that levels of E 2 were significantly increased in injured brain tissue compared to uninjured brain tissue (Figs 2 and 3) of adult male (t 4 = À2.46, P = 0.035) and female (t 4 = 2.99, P = 0.02) zebra finches 48 h after injury.…”

“…Thus, although the location of the injury was slightly different, the nature of the injury and the time between trauma and tissue collection was identical. The methods utilised to analyse brain E 2 levels have been validated in several recent songbird models, including a demonstration of the combined liquid-liquid and solid-phase extraction providing sufficient purification for an accurate analysis of low tissue levels of E 2 and, importantly, validation with results obtained by mass spectrometry (29)(30)(31)(32).…”

“…Samples were assayed in triplicates using an E 2 Enzyme Immunoassay kit (Cayman Chemical) previously validated for use with zebra finch brain tissue in accordance with the manufacturer's instructions (30)(31)(32). Prior to ether and solid-phase extraction, an additional sample was spiked with radioinert E 2 to the concentration of 256 pg/ml, processed in an identical manner, and assayed in triplicate alongside the experimental samples to estimate recovery.…”

“…Following homogenisation of tissue in 1 ml of 0.1 M PB, 300 ll of each sample was ether extracted in accordance with previously validated protocols (32,33). Two millilitres of diethyl ether (Sigma-Aldrich) was added to each sample, and tubes were vortexed on a low setting for 30 s. Samples were next centrifuged at 1000 g for 5 min at 4°C and tubes were submerged in a MeOH/dry ice bath for 1 min to freeze the mixture.…”