RESUMEN
Steroids that contain a 3-hydroxyl group (3-OH steroids) are widely distributed in nature. During analysis with ESI-MS, they easily become dehydrated while in the protonated form, resulting in the production of several precursor ions and leading to low sensitivity of detection. To address this analytical challenge, here, we developed a method for the quantitation of 3-OH steroids by LC-MS/MS coupled with post-column addition of lithium (Li) ions to the mobile phase. The Li ion has a high affinity for the keto group of steroids, stabilizing their structures during ionization and permitting detection of analytes exclusively as the lithiated form. This not only improved the intensities of the precursor ions, but also promoted the formation of typical lithiated fragment ions. This improvement made the quantitation by multiple reaction monitoring more sensitive and reliable, as evidenced by 1.53-188 times enhanced detection sensitivity of 13 steroids that contained at least one keto and two hydroxyl groups or one keto and one 5-olefinic double bond, among 16 different 3-OH steroids. We deployed our newly developed method for profiling steroids in mouse brain tissue and identified six steroids in one tissue sample. Among these, 16-hydroxyestrone, tetrahydrocorticosterone, and 17α-hydroxypregnenolone were detected for the first time in the mouse brain. In summary, the method described here enables the detection of lithiated steroids by LC-MS/MS, including three 3-OH steroids not previously reported in the mouse brain. We anticipate that this new method may allow the determination of 3-OH steroids in different brain regions.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Litio/química , Esteroides/análisis , Esteroides/química , Espectrometría de Masas en Tándem/métodos , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
Neuroactive steroids, termed neurosteroids, are synthesized locally in the brain and influence biological functions including cognition and behavior. These neurosteroids are synthesized from cholesterol by a series of cytochrome P450 enzymes, among which a member of P450 hydroxylase, cytochrome P450-7b1 (CYP7B1), catalyzes the formation of 7α-hydroxylated neurosteroids, 7α-hydroxypregnenolone (7α-OH-Preg) and 7α-hydroxydehydroepiandrosterone (7α-OH-DHEA). Here we demonstrated the occurrence of these neurosteroids in the mouse hippocampus after spatial-learning tasks. Cyp7b1 deficiency impaired remote spatial memory with recent memory mostly unaffected. The hippocampal dendritic spine densities were reduced in Cyp7b1-deficient mice, and they were no more increased by the training. Furthermore, chronic intracerebroventricular administration of a mixture of 7α-OH-Preg and 7α-OH-DHEA rescued the deteriorated remote memory performance in Cyp7b1-deficient mice. It is concluded that the 7α-hydroxylated neurosteroids are required for long-term maintenance of spatial memory, and we suggest that these neurosteroids may induce synaptic remodeling to maintain the hippocampal function.
RESUMEN
An in situ coupled oxidation cycle that allows catalytic oxidation of a substrate with catalytic amounts of o-chloranil and novel reusable polymer-immobilized platinum nanocluster catalysts using molecular oxygen as the terminal oxidant was developed.