Metabolite profiling of sex developmental steroid conjugates reveals an association between decreased levels of steroid sulfates and adiposity in obese girls

Free and conjugated steroids coexist in a dynamic equilibrium due to complex biosynthetic and metabolic processes. This may have clinical significance related to various physiological conditions, including sex development involving the reproductive system. Therefore, we performed quantitative profiling of 16 serum steroids conjugated with glucuronic and sulfuric acids using liquid chromatography-mass spectrometry (LC-MS). All steroid conjugates were purified by solid-phase extraction and then separated through a 3-μm particle size C18 column (150 mm × 2.1 mm) at a flow rate of 0.3 mL/min in the negative ionization mode. The LC-MS-based analysis was found to be linear (r2 > 0.99), and all steroid conjugates had a limit-of-quantification (LOQ) of 10 ng/mL, except for cholesterol sulfate and 17β-estradiol-3,17-disulfate (20 ng/mL). The extraction recoveries of all steroid conjugates ranged from 97.9% to 110.7%, while the overall precision (% CV) and accuracy (% bias) ranged from 4.8% to 10.9% and from 94.4% to 112.9% at four different concentrations, respectively. Profiling of steroid conjugates corrected by adiposity revealed decreased levels of steroid sulfates (P < 0.01) in overweight and obese girls compared to normal girls. The suggested technique can be used for evaluating metabolic changes in steroid conjugates and for understanding the pathophysiology and relative contributions of adiposity in childhood obesity.