| Endogenous steroid hormones include corticoid, estrogen, androgen and progestogen, which transform into each other by different enzymes in vivo. Steroid hormones play an important role in the nerve and tissue and influence the physiological activities by regulating the metabolic activity of the cells. The abnormal change in the concentration of the hormones is closely related to the pathogenesis of diseases. Recently, all kinds of the methods are used to determine the endogenous hormones. Through establishing high sensitivity and specificity analysis method, we can accurately measured endogenous hormones and explore the regulation function and disease pathogenesis in vivo.In this study,11endogenous steroid hormones and their metabolites were selected, including corticoid (cortisol, cortisone, tetrahydrocortisone, tetrahydrocortisol, allo-tetrahydrocortisol), estrogen (17β-estradiol), androgen (testosterone,5α-dihydrotestosterone, dehydroepiandrosterone) and progesterone (progesterone,17α-hydroxyprogesterone) as the research objects and established three analysis methods to determination these steroid hormones in human plasma and urine. These hormones are closely related to metabolic activity, human growth and development and common clinical diseases. The accurate determination of them could provide reference for clinical treatment and diagnosis. For example, progesterone has an important role in embryonic development while17β-estradiol is associated with amblyopia and osteoporosis. Cortisol and cortisone transform into each otherby11β-hydroxysteroid dehydrogenase (11β-HSD). Since11β-HSD contacts with diabetes, cognitive function and embryonic development, determination of the concentration ratio of cortisol and cortisone or their metabolites could evaluate the activity of the enzyme and thus provide references for clinical treatment and diagnosis of some diseases.In chapter1, a high-performance liquid chromatography method was developed for the determination of nine kinds of endogenous steroid hormones in the human plasma and urine. Tetrahydrocortisone, tetrahydrocortisol and allo-tetrahydrocortisol exist as conjugates, which need to be hydrolyzed by β-glucuronidase. Since no conjugated system exsit in tetrahydrocortisone, tetrahydrocortisol, allo-tetrahydrocortisol,205nm wavelength was selected where17β-estradiol has strong absorption in. Cortisol, cortisone, testosterone, progesterone and 17a-hydroxyprogesterone contain unsaturated ketone group which leading to maximum absorption at a wavelength of245nm. So,205nm and245nm wavelength were simultaneous chosen for detection of9endogenous steroid hormones. Specificity, sensitivity, linearity, precision, recovery and stability were investigated. The results showed the method was specific, but not sensitive enough to measure endogenous steroid hormones in biological samples. The measure was also affected by endogenous interferences obviously.In order to improve the specificity and sensitivity of the method, liquid chromatography-mass spectrometry method (LC-MS) was developed to analysis of seven kinds of endogenous hormones. The lowest limit of detection was shown in chapter2. Compared with previous high-performance liquid chromatography method, specificity, sensitivity and speed of analysis had significantly improved. However, during the measurement of the actual biological sample, the sensitivity still could not reach the requirement.The above methods can not be able to meet the requirements of the determination of endogenous steroid hormones. To overcome this difficulty, a new method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) developed for the simultaneous determination of nine endogenous steroid hormones, including cortisol, cortisone, tetrahydrocortisone, allo-/tetrahydrocortisol,17(3-estradiol, testosterone, progesterone and17α-hydroxyprogesterone in chapter3. Considering matrix effect, recovery and simple operation, solid phase extraction was used as pre-treatment. Positive and negative electrospray ionization modes were studied to optimize mass spectrometry. It was found that less background and signal interference and more stable ionization efficiency existed in positive mode.It was also found that0.1%formic acid in the mobile phase can enhance ion response obviously. Similarly, specificity, sensitivity, linearity, matrix effect, precision, accuracy and stability were investigated and the method was applied to detect endogenous hormones in cord plasma and urine of pregnant woman.The results showed that the concentration of17β-estradiol and progesterone were higher in pregnant woman than normal adult female while the concentration of testosterone was lower. The raito F/E and (allo-THF+THF)/THE in case group were higher than control group which suggested that low11β-HSD2level in pregnant woman may cause embryo developing slowly. Three endogenous androgens were measured for studying the mechanism of epilepsy. After determine the plasma samples, it could be found that y-amino butyric acid and glutamate levels were higher than normal in epilepsy patients while androgen level was lower. This might explain androgens could reduce the levels of excitatory amino acids, so as to achieve the role of antiepileptic. |
PDF Full Text Request