Direct measurement of free glucocorticoids in small volumes of mouse and rat serum using ultrafiltration and liquid chromatography-tandem mass spectrometry

Glucocorticoids are critical steroid hormones secreted from the adrenal glands. In mice and rats, over 90% of circulating corticosterone is bound to proteins such as corticosteroid-binding globulin and albumin, and the rest is unbound (free). Only free glucocorticoids can enter cells and bind receptors, so it is crucial to measure free glucocorticoids. Some studies have estimated free glucocorticoid levels, but such estimates might be inaccurate as they do not take temperature and protein binding by competing steroids into account. Far fewer studies have directly measured free glucocorticoid levels in serum, and current methods to do so are time-consuming and require sample volumes (200 + µl) that are difficult to obtain from mice. Here, we developed a method to directly measure free glucocorticoids in a small volume of rodent serum using ultrafiltration and liquid chromatography-tandem mass spectrometry. We validated this method to measure free 11-deoxycorticosterone (corticosterone precursor), corticosterone, and 11-dehydrocorticosterone (corticosterone metabolite) in as little as 30 µl of mouse and rat serum. Ultrafiltration produces results that are qualitatively similar to those from equilibrium dialysis, an established method that requires a much larger sample volume (200 + µl). We then applied our novel method to examine the effects of lipopolysaccharide (LPS), an immune stressor that is known to increase total corticosterone levels, free corticosterone levels, and percent free corticosterone. We administered saline vehicle or LPS to adult male and female mice, collected blood 4 hr later, and measured total and free glucocorticoid levels in serum from individual mice. As expected, LPS increased total and free corticosterone levels and percent free corticosterone. This simple, robust, and rapid method allows direct measurement of free corticosterone and other steroids in 30 µl of rodent serum.