Application Research Of Urine Steroid Hormones Assayed By ICMA In Prepubertal Children

Background: Serum estriol(E3), hydrocortisone(HC), 17β-estradiol(17β-E2),testosterone(T), progesterone(P) are steroid hormones, which are usually regarded as the detection indicators in clinic for diagnosis and treatment of many diseases such as endocrine diseases. Urine steroid hormones can reflect the average levels of those in serum during a period of time. Concentrations of urine steroid hormones are often higher than those in serum.Instead of serum collection, urine collection is convenient and noninvasive and it can improve the compliance of patients in clinic or population in epidemiological research. However, there are only a few studies of total steroid hormones(including free and conjugate hormones)assayed by immunochemiluminometric assay(ICMA) in urine, and no report of five kinds of urine steroid hormones as shown in above assayed by ICMA simultaneously.Objective: An optimization method of enzymatic hydrolysis for five kinds of urine steroid hormones(E3、HC、17β-E2、T and P) was explored. Detection results of steroid hormones by ICMA were compared with ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method for the agreement. And the relationship between urine free and total steroid hormones assayed by ICMA in prepubertal children was analyzed.Method: An enzymatic hydrolysis method of five kinds of urine steroid hormones was optimized(the amount of enzyme, temperature and time), which was used in UPLC-MS/MS method and ICMA. And the two methods were compared with each other for the agreement of the results. Urine steroid hormones were assayed by ICMA in prepubertal healthy children,and 33 boys with the average age of(5.49±0.89) years and 33 girls of(5.53±0.81) years were included. The relationship between free and total steroid hormones was analyzed.Result:(1) Enzymatic hydrolysis optimization method: 0.5 m L of urinary supernatant was diluted with 1 m L of 0.2 M sodium acetate buffer and 2000 U β-glucuronidase, and incubated at 55 °C for 3 h under gentle agitation.(2) The results by UPLC-MS/MS showed that five kinds of steroid hormones displayed good linearity with correlation coefficients from0.9964 to 0.9994 in the corresponding concentration range. The limits of detections were from0.1 ng/m L to 1.0 ng/m L and the limits of quantitations were from 0.3 ng/m L to 3.0 ng/m L.The average recoveries were varied from 84.7% to 106.0% with relative standard deviations from 4.5% to 9.8%.(3) There was high correlation between ICMA and UPLC-MS/MS method in the determination of five kinds of urine steroid hormones. The linear correlation between two methods were from 0.905 to 0.971, and intraclass correlation coefficient were from 0.893 to 0.964.(4) Urine free and total steroid hormones(E3、HC、17β-E2、T and P) were assayed by ICMA in prepubertal children. The correlations for the concentrations of five kinds of steroid hormones between free and total steroid hormones in above were 0.759, 0.961,0.261, 0.655 and 0.820, respectively; and for the concentrations of steroid hormones to creatinine 0.742, 0.976, 0.753, 0.494 and 0.828, respectively; and for the content of steroid hormones 0.772, 0.967, 0.678, 0.582 and 0.749, respectively.Conclusion:(1) The optimization method was that 0.5 m L of urinary supernatant was enzymatic hydrolysised by 2000 U β-glucuronidase and incubated at 55 °C for 3 h under gentle agitation. Urine steroid hormones(E3, HC, 17β-E2, T and P) could simultaneously be determined by UPLC-MS/MS method or ICMA with the characteristic of rapid and accurate,which provided preliminary data for the development of integrative machine of enzymolysis and ICMA.(2) There was highly significant correlation ship between urine free E3(concentration, concentration to creatitine ratio, content) and its total level assayed by ICMA in prepubertal children, as well as HC or P. The ratio of free 17β-E2 concentration to creatitine was also highly significantly correlated with its ratio of total concentration to creatitine. There were significant correlations between urine free T(concentration, concentration to creatitine ratio, content) and their total levels, as well as the content of urine free 17β-estradiol and its total content. But there was poor correlation of concentration between free 17β-estradiol and its total 17β-estradiol.