Abstract
Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.
Highlights
Progesterone is a steroid hormone that is synthesized mainly in the ovaries, placenta and adrenal glands
We demonstrate the utility of this approach for the analysis of progesterone metabolism in an epithelial endometrial cell line (HES cells)
Individual diols were subsequently isolated by reverse-phase separation of the mixture, using flash chromatography followed by preparative high pressure liquid chromatography (HPLC)
Summary
Progesterone is a steroid hormone that is synthesized mainly in the ovaries, placenta and adrenal glands. Progesterone has a number of important functions. It is known to be involved in differentiation of endometrium [1], proliferative changes of breast glandular tissue that occur during the menstrual cycle [2], and maintenance of pregnancy and lactation [1], while showing neuroprotective effects [3]. Elevated levels of progesterone during gestation provide protection against oxidative stress and immune reactions to the fetus, and have a role in the normal development of neurons [4]. Progesterone appears to modulate bone remodeling, and to protect against bone loss [1]. While the role of progesterone in human is well known, the possible actions and implications of progesterone metabolites are still to be determined
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