Profiles of 21-Carbon Steroids in 21-hydroxylase Deficiency

Turcu, Adina F.; Rege, Juilee; Chomic, Robert; Liu, Jiayan; Nishimoto, Hiromi; Else, Tobias; Moraitis, Andreas; Palapattu, Ganesh S.; Rainey, William E.; Auchus, Richard J.

doi:10.1210/jc.2015-1023

Cited by 71 publications

(43 citation statements)

References 40 publications

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“…Steroids produced by the H295R and H295RA cells at baseline and after ACTH stimulation are summarized in Table 2. Similar to our previous study of H295R steroid profiles (Xing et al 2011, Rege et al 2015, the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, androstenedione, and cortisol, while the mass of aldosterone was low under basal conditions. After ACTH stimulation for 24 h, H295RA cells showed a significant increase in 17a-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, pregnenolone sulfate, and 17a-hydroxypregnenolone sulfate as well as aldosterone and cortisol (P!0.05; Table 2).…”

Section: Steroidogenic Ability Of the H295ra Cell Linesupporting

confidence: 87%

“…Preparation and measurement of D 4 steroids were performed as previously described (Turcu et al 2015). Steroid sulfates were extracted with 1 ml of 1:1 chloroform:2-butanol from a 50 ml serum aliquot after mixing with 200 ml 1 M ammonium sulfate.…”

Section: Steroid Quantitation By Liquid Chromatography-tandem Mass Spmentioning

confidence: 99%

“…The dried extracts were reconstituted with 200 ml of methanol/deionized water (1:1) and transferred to a 0.25 ml vial insert. Steroid quantitation was performed as previously described (Turcu et al 2015). Supplementary Table 1 gives retention times and precursor/product ion pairs for the targeted steroids not previously published.…”

Section: Steroid Quantitation By Liquid Chromatography-tandem Mass Spmentioning

confidence: 99%

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H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

Nanba¹,

Chen²,

Turcu³

et al. 2015

Journal of Molecular Endocrinology

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The H295R adrenocortical cell line is widely used for molecular analysis of adrenal functions but is known to have only modest ACTH responsiveness. The lack of ACTH response was linked to a low expression of its receptor, melanocortin 2 receptor (MC2R). We hypothesized that increasing the MC2R accessory protein (MRAP), which is required to traffic MC2R from the endoplasmic reticulum to the cell surface, would increase ACTH responsiveness. Lentiviral particles containing human MRAP-open reading frame were generated and transduced in H295R cells. Using antibiotic resistance, 18 clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, H295RA, was used for further experiments. Successful induction of MRAP and increased expression of MC2R in H295RA cells was confirmed by quantitative real-time RT-PCR and protein analysis. Treatment with ACTH significantly increased aldosterone, cortisol, and dehydroepiandrosterone production in H295RA cells. ACTH also significantly increased transcript levels for all of the steroidogenic enzymes required to produce aldosterone, cortisol, and dehydroepiandrosterone, as well as MC2R mRNA. Using liquid chromatography/tandem mass spectrometry, we further revealed that the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, cortisol, and androstenedione. Treatment of H295RA cells with ACTH also acutely increased cAMP production and cellular protein levels for total and phosphorylated steroidogenic acute regulatory protein. In summary, through genetic manipulation, we have developed an ACTH-responsive human adrenocortical cell line. The cell line will provide a powerful in vitro tool for molecular analysis of physiologic and pathologic conditions involving the hypothalamic-pituitary-adrenal axis.

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Section: Steroidogenic Ability Of the H295ra Cell Linesupporting

confidence: 87%

Section: Steroid Quantitation By Liquid Chromatography-tandem Mass Spmentioning

confidence: 99%

Section: Steroid Quantitation By Liquid Chromatography-tandem Mass Spmentioning

confidence: 99%

See 1 more Smart Citation

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

Nanba¹,

Chen²,

Turcu³

et al. 2015

Journal of Molecular Endocrinology

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“…In keeping with best practices in the measurement of adrenal steroids [17] , we measured serum cortisol and cortisone using liquid chromatography-tandem mass spectrometry as described previously [18] . Serum (0.2 ml) was mixed with deuteriumlabeled internal standards and deproteinated with methanol and acetonitrile.…”

Section: Cortisol and Cortisone Assaysmentioning

confidence: 99%

Serum Cortisol-to-Cortisone Ratio and Blood Pressure in Severe Obesity before and after Weight Loss

et al. 2015

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Background/Aims: The pathogenesis of obesity-associated hypertension is poorly understood. Serum cortisol-to-cortisone ratio (F/E ratio) is a marker of cortisol metabolism. Our objective was to determine whether the serum F/E ratio is associated with blood pressure (BP) in patients after significant weight loss ( ≥ 15% from baseline weight). Methods: Sera from 43 nondiabetic, severely obese males participating in a weight management program were assayed for F and E by mass spectrometry. We assessed whether changes in the F/E ratio accompanying weight loss correlate with changes in the systolic (SBP) and diastolic BP (DBP). Linear regression was used to evaluate change in the F/E ratio as a predictor of change in BP. Results: The body mass index decreased from 40.8 ± 5.6 to 33.7 ± 4.8 (p < 0.001); also, SBP (133.2 ± 13.8 vs. 124.1 ± 14.3 mm Hg; p < 0.001) and DBP (69.8 ± 8.0 vs. 66.6 ± 9.4 mm Hg; p = 0.026) decreased during the study. The baseline F/E ratio tended to associate with baseline DBP (Spearman's r = -0.29, p = 0.06), and change in the serum F/E ratio correlated with change in DBP (Spearman's r = -0.32, p = 0.036). Change in the F/E ratio also tended to associate with change in SBP (Spearman's r = -0.27, p = 0.08). A multiple linear regression model adjusted for change in the F/E ratio and age explained 22% of the variance in SBP change (R 2 = 0.22, p = 0.007). Change in the F/E ratio independently predicted change in SBP (p = 0.036). Conclusion: In our sample of nondiabetic, severely obese males, change in the serum F/E ratio was associated with change in BP after weight loss.

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“…Finally, if the urine metabolites are characteristically elevated, the corresponding steroids produced from the tumors themselves might be easy targets for serum measurements. Furthermore, LC-MS/MS methods to profile serum steroids are well developed with much quicker turn-around times than GC/MS profiling [9].…”

mentioning

confidence: 99%

Exploiting the Unusual to Find the Rare: Novel Steroid Metabolites Pinpoint Adrenal Cancer Patients

2015

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Profiles of 21-Carbon Steroids in 21-hydroxylase Deficiency

Abstract: Measurement of 16OHP and 11OHP along with 17OHP and 21dF by liquid chromatography/tandem mass spectrometry might comprise a biomarker panel to accurately diagnose all forms of 21OHD.

Cited by 71 publications

References 40 publications

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

Serum Cortisol-to-Cortisone Ratio and Blood Pressure in Severe Obesity before and after Weight Loss

Exploiting the Unusual to Find the Rare: Novel Steroid Metabolites Pinpoint Adrenal Cancer Patients

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