Study On The Correlationship Between Steroid Hormone Levels In Patients And The Duration, Recurrence Of Primary Liver Cancer

Primary liver cancer (PLC) is one of the most common malignant tumors in theworld, which jeopardized people’s health seriously. The pathogenesis of PLC is stillobscure. The neoplasm is predominant in the male gender, proportion of male to femalepatients is6.3:1domestic and11:1aboard, besides male gender is characterized by aworse prognosis than females. Recent researches indicated the occurrence anddevelopment of liver cancer were closely linked to steroid hormones, especially sexhormones and hormone levels in serum of patients changed greatly. We developed asensitive HPLC-ESI-MS/MS method for measurement of glucocorticoid and sex hormonesin vivo to evaluate the different hormone levels between patients and healthy people. Ourintention was to investigate the correlationship between hormonal imbalance anddevelopment of primary liver cancer in order to aid tumor diagnose and get a betterknowledge of disease changes.Firstly， we developed a high performance liquid chromatography-electrosprayionization-tandem quadrupole mass spectrometry (HPLC-ESI-MS/MS) for simultaneousdetermination of three estrogens including estriol, estradiol and estrone in vivo by usingp-Phenylphenol as internal standard. Serum samples were processed by liquid-liquidextraction. After extraction with ethyl acetate and derivatized with dansyl chloride in pH10.5, the samples were then injected into instrument for analysis. LC separation wasachieved by using an Agilent EC-C18column and acetonitrile-water (0.1%formic acid)as a mobile phase, the MS detection was positive ion mass spectrometry using electrosprayionization and MRM mode. The method showed a high sensitivity by limit of quantitativewith20pg/mL and no other endogenous components were found affecting the analysis ofestrogens. The results of methodology showed three kinds of estrogens possessed goodlinear relationship in the corresponding linear range; intra-day and inter-day precisionswere less than15%; the matrix effect of estrone is about60-70%, and the recoveries andmatrix effects of estriol and estradiol were ranged from85%-118%; samples remainedstable at room temperature, auto-injector, three freeze-thaw cycles and long-term storage.The established method is suitable for the analysis and detection of estrogen levels inprimary liver cancer patients and healthy volunteers.Secondly, a high performance liquid chromatography-electrospray ionization-tandemquadrupole mass spectrometry (HPLC-ESI-MS/MS) was established in the positive ion mode for measurement of hydrocortisone (glucocorticoid), testosterone andandrostenedione (androgens) and progesterone in human serum using betamethasone asinternal standard. Methyltert-butyl ether (MTBE) was taken as extraction solvent, whichshowed high extraction efficiency and low matrix effect. The mobile phase was acetonitrileand0.1%formic acid in water for gradient elution, separation was performed in theEC-C18column same as above, and the MS detection was Dynamic MRM mode. TICfigure showed that four kinds of steroids have sufficient resolution, the background noisewas small and no signal interferes had been detected. Methodology results demonstatedthat the method is simple, sensitive and accurate. Four kinds of steroid hormones possessedgood linear relationship in the corresponding linear range; the intra-day and inter-dayprecisions, matrix effects, recoveries, and stabilities (under four conditions) all met therequirements of biological sample analysis.Serum samples were collected from180patients with primary liver cancer whoreceived treatment in our hospital from February2012to August2012and61cases ofhealthy volunteers were from outpatient clinics. Concentrations of steroid hormones in vivowere determinated by the established HPLC-MS/MS methods above. Statistical data wereanalyzed by SPSS18.0and difference of hormone levels was found between healthyvolunteers and patients. Glucocorticoids and androgens reduced in primary liver patients ’serum, estrogen level was elevated and E2/T was significantly higher than normal ones.With the development of liver cancer, E2/T was rising gradually and a notable differenceexsited between Ⅰ、Ⅱ、Ⅲ stage of the disease. Through the comparison of hormonelevels in serum samples of6patients before and after surgery, all of them showed adecreased E2/T value after tumor resection. Follow-up of30patients of liver cancer aftersurgical resection, we found elevated E2/T in recurrence patients, compared with those whodid not relapse, which can be preliminarily ascertained as a high risk factor of liver cancerrecurrence.The results of our study demonstrated that there was a relationship between sexhormone imbalance, the occurrence and development of liver cancer as well as recurrenceafter surgery, thus sex hormone levels could be considered as a valid reference index forclinical diagnose and recurrence prediction of primary liver cancer.