Su Hyeon Lee

Changes in steroid metabolism among girls with precocious puberty may not be associated with urinary levels of bisphenol A

Precocious puberty (PP) refers to the appearance of physical and hormonal signs of pubertal development at an abnormally early age. Urinary steroid signatures obtained from 42 patients with central PP and 40 patients with peripheral PP were assessed to compare metabolic changes. Levels of androgens such as testosterone, androstenedione, androstenediol, 16α-hydroxy-dehydroepiandrosterone, and 5α-androstenedione tended to be high in both PP groups, and the level of 17β-estradiol was higher in the central-PP group (P<0.01) than in the peripheral-PP and 32 age-matched healthy girls. Altered steroid metabolism was also associated with urinary BPA levels, and levels of testosterone, 17β-estradiol, and pregnenolone were significantly increased among individuals with high BPA levels. In particular, a correlation was observed between estrogen metabolism and BPA levels irrespective of the type of PP. These findings suggest that in girls, BPA exposure causes metabolic changes in steroidogenesis, but not the early onset of PP.

Comparative GC-MS Based In vitro Assays of 5α-Reductase Activity Using Rat Liver S9 Fraction

α-Dihydrotestosterone (DHT) is the primary active metabolite of testosterone, catalyzed by 5α-reductase (5αR) in the skin, prostate, and liver. In this study, the 5αR activity in rat liver S9 fraction in the presence of a NADPH-generating system was evaluated and compared by gas chromatography-mass spectrometry (GC-MS)-based in vitro assays. Testosterone and a 5αR inhibitor, finasteride, were added to the S9 fractions and incubated at 37 o C for 1 h. Both testosterone and DHT were quanti- tatively measured and compared with two different GC-MS-based steroid profiling techniques. DHT was not detected by con- ventional GC-MS analysis in the absence of finasteride when the concentration of testosterone in the S9 fraction was less than 0.2 µM, whereas the isotope-dilution GC-MS (GC-IDMS) system was able to evaluate the 5αR activity. Because the S9 fraction contains more reactive enzymes and is easier to collect from tissues compared with a microsomal solution, the combination of the S9 fraction and GC-IDMS technique may be a promising assay for evaluating the 5αR activity in large-scale clinical studies.

Isotope-Dilution Mass Spectrometry for Quantification of Urinary Active Androgens Separated by Gas Chromatography

Cross reacting antibodies can cause an overestimation of the results of immunoassays. Therefore, alternative methods are needed for the accurate quantification of steroids. Gas chromatography combined with isotope-dilution mass spectrometry (GC-IDMS) is developed to quantify urinary active androgens, testosterone, epitestosterone and dihydrotestosterone, which are clinically relevant androgens to both hair-loss and prostate diseases. The method devised involves enzymatic hydrolysis with β- glucuronidase, solid-phase extraction, liquid-liquid extraction using methyl tert-butyl ether and subsequent conversion to pen- tafluorophenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive and selective analysis in selected-ion monitoring mode. Flophemesyl-TMS derivatization not only eliminates matrix interference but also has a good peak resolution within a 6 min-run. A selective and sensitive GC technique with flophemesyl-TMS derivatives also allows accurate quantitative analysis of three active androgens when combined with IDMS. The limit of quantification of the three analytes was <50 pg/mL, and extraction recoveries ranged from 91.9 to 102.1%. The precision and accuracy were 1.2~6.5% and 89.0~106.7%, respec- tively. This GC-IDMS method can be useful for evaluating the drug efficacy and monitoring the biological processes responsible for male-pattern baldness and prostate diseases.

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 (150mm×2.1mm) at a flow rate of 0.3 mL/min in the negative ionization mode. The LC-MS-based analysis was found to be linear (r(2)>0.99), and all steroid conjugates had a limit-of-quantification (LOQ) of 10ng/mL, except for cholesterol sulfate and 17β-estradiol-3,17-disulfate (20ng/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.

Serum levels of cholesterol, pregnenolone, DHEA, and their sulfate conjugates based on sex and pubertal stage in adolescents.

BACKGROUND The study aim was to evaluate the correlation of sexual dimorphism and pubertal stage with steroid metabolism in adolescents. METHODS The serum levels of cholesterol, pregnenolone, and dehydroepiandrosterone (DHEA) and their sulfate conjugates were quantitatively profiled from serum samples of 199 adolescents (87 boys and 112 girls), aged 6 to 14years. RESULTS In the prepubertal stage, DHEA levels in girls were higher than those in boys; however, significantly increased DHEA levels during pubertal development in boys. Pregnenolone levels were significantly higher in girls; however, the levels of its sulfate were higher in boys. The serum levels of both cholesterol and its sulfate were higher in boys, especially in the early to mid-pubertal stages. DHEA and DHEA sulfate levels in both sexes significantly increased with pubertal development (P for trend<0.05), while pregnenolone, cholesterol, and cholesterol sulfate in both sexes were stable. The metabolic ratios, indicating sulfotransferase activity, were significantly higher in boys, and increased with pubertal development in boys, but not in girls, while CYP11A1 activity levels increased significantly in both sexes with pubertal development. CONCLUSIONS Sexual dimorphism in key enzymes of androgen biosynthesis during pubertal changes may help elucidate the normal physiology of steroidogenesis.

Electrospray-Mass Spectrometric Analysis of Plasma Pyrophosphates Separated on a Multi-Modal Liquid Chromatographic Column

Pyrophosphates are the key intermediates in the biosynthesis of isoprenoids, and their concentrations could reveal the benefits of statins in cardiovascular diseases. Quantitative analysis of five pyrophosphates, including isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP), was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative ion- ization mode. After dilution with methanol, samples were separated on a 3 µm particle multi-modal C18 column (50 ×2m m) and quantified within 10 min. The gradient elution consists of 10 mM ammonium bicarbonate and 0.5% triethylamine (TEA) in water and 0.1% TEA in 80% acetonitrile was used at the flow rate of 0.4 mL/min. Overall recoveries were 51.4-106.6%, while the limit of quantification was 0.05 µg/mL for GPP and FPP and 0.1 µg/mL for IPP, DMAPP, and GGPP. The precision (% CV) and accuracy (% bias) of the assay were 1.9-12.3% and 89.6-111.8%, respectively, in 0.05-10 µg/mL calibration ranges (R 2 > 0.993). The devised LC-MS/MS technique with the multi-modal C18 column can be used to estimate the biological activity of pyrophosphates in plasma and may be applicable to cardiovascular events with cholesterol metabolism as well as the drug efficacy of statins.

Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism

Background Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. Methods Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification. Results The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R2>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases. Conclusions Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.