Synthesis of multi-labeled cortisols and cortisones with 2H and 13C for study of cortisol metabolism in humans

doi:10.1016/S0039-128X(99)00102-6

Steroids

Volume 65, Issue 4, April 2000, Pages 180-189

https://doi.org/10.1016/S0039-128X(99)00102-6 Get rights and content

Abstract

A method is described for the preparation of multi-labeled cortisol and cortisone with ¹³C and ²H via the indan synthon method, starting from chiral 11-oxoindanylpropionic acid. [1,3-¹³C₂]Acetone was used for the syntheses of [1,2,4,19-¹³C₄]cortisol (cortisol-¹³C₄) and [1,2,4,19-¹³C₄]cortisone (cortisone-¹³C₄), and [1,3-¹³C₂,1,1,1,3,3,3-²H₆]acetone was for [1,2,4,19-¹³C₄,1,1,19,19,19-²H₅]cortisol (cortisol-¹³C₄,²H₅) and [1,2,4,19-¹³C₄,1,1,19,19,19-²H₅]cortisone (cortisone-¹³C₄,²H₅). The chemical shifts for the ¹³C and ¹H NMR spectra of cortisol and cortisone were fully assigned.

Introduction

Cortisol is reversibly metabolized to biologically inactive cortisone by 11β-hydroxysteroid dehydrogenase (11β-HSD) (Fig. 1). The interconversion of cortisol to cortisone in human has recently been subjects of biochemical and clinical investigations in connection with patients with low activities of 11β-HSD, characterized by hypertension, hypokalemia, and low renin [1], [2], [3], [4], [5], [6], [7]. Stable isotope methodology has widely been accepted for investigating the pharmacokinetics and metabolism of steroid compounds in humans. One of the major advantages of the methodology coupled with mass spectrometry is that endogenous and exogenous compounds with the same basic structure can be differentiated easily by using the stable isotopically labeled compound as biologic internal standard.

In our previous paper, we have developed a method for a concise 11-step total synthesis of optically pure (+) cortisol by using the chiral hydrindan as a starting material and applied it to the introduction of five deuterium atoms at C-19 methyl and C-1 methylene of cortisol [8]. The labeled cortisol has then been used as both analytical and biologic internal standards for the pharmacokinetic studies of cortisol and cortisone in human [9], [10].

One of the important considerations in selecting stable isotopically labeled compounds for use as tracers in the in vivo study is that the exogenously administered labeled tracer behaves in an identical fashion to its endogenous counterpart through all processes of absorption, distribution, metabolism and elimination. ¹³C-Labeled compounds offer considerable advantages, in terms of minimal chemical alteration of the molecules and non-exchangeable nature of the label, over the deuterium-labeled compounds [11]. Problems associated with the isotope effect can be avoided with the ¹³C label, whereas more pronounced isotope effect may not be negligible with deuterium-label. The use of deuterium-labeled compounds as biologic internal standards then requires an in vivo study to prove whether or not the kinetic isotope effect of the deuterium-label is minor.

The present study describes an efficient synthesis of ¹³C-labeled cortisol (cortisol-¹³C₄) and cortisone (cortisone-¹³C₄) containing four ¹³C at C-1, C-2, C-4, and C-19 for use as ideal biologic internal standards. We have also synthesized multi-labeled cortisol (cortisol-¹³C₄,²H₅) and cortisone (cortisone-¹³C₄,²H₅) with five deuterium atoms at C-1 and C-19 in addition to the four ¹³C atoms for use as analytical internal standards for GC-MS. The structures of cortisol and cortisone labeled with ¹³C₄ and ²H₅ are given in Fig. 2.

Section snippets

Experimental

¹H NMR spectra were determined on Varian Gemini-300 300-MHz and Bruker DPX-400 400 MHz spectrometers for samples in CDCl₃ and CD₃OD with tetramethylsilane as internal reference. Capillary gas chromatographic-mass spectrometric (GC-MS) analysis was done on a Shimadzu QP1000EX GC-MS equipped with a data-processing system. GC-MS employed an SPB-1 fused-silica capillary column (15 m × 0.25 mm I.D.) with the stationary phase coated at a 0.25-μm film thickness (Supelco, Bellefonte, PA, USA).

Results and discussion

Zomer et al. [16], [17], [18] reported a general procedure for the synthesis of ¹³C-labeled steroid compounds such as testosterone, estradiol and cortisol. The method, however, requires 24 steps for introducing ¹³C at C-1, C-2, C-3, and C-4 of cortisol, starting from des A-17β-t-butoxy-estr-9-en-5-one as the ‘BCD’ ring precursor. The indan synthon method has provided the useful steroidal C/D ring system for the total synthesis of 11-oxigenated steroids [19], [20], [21]. An elegant construction

Acknowledgements

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

References (25)

P.M Stewart et al.
Steroid hormones and hypertensionthe cortisol-cortisone shuttle
Steroids
(1993)
F Mantero et al.
Apparent mineralocorticoid excess type II
Steroids
(1994)
P.M Stewart et al.
Type 2 11β-hydroxysteroid dehydrogenase in fetal and adult life
J Steroid Biochem Molec Biol
(1995)
J.W Funder
Apparent mineralocorticoid excess
Endocrinol Metab Clin North Am
(1995)
F Mantero et al.
Apparent mineralocorticoid excesstype I and type II
Steroids
(1996)
Y Kasuya et al.
Stable isotope methodology for kinetic studies of interconversion of cortisol and cortisone in a human subject
Steroids
(1998)
S.A Wudy
Synthetic procedures for the preparation of deuterium-labeled analogs of naturally occurring steroids
Steroids
(1990)
T Furuta et al.
Simultaneous determination of teterahydrocortisol and tetrahydrocortisone in human plasma and urine by stable isotope dilution mass spectrometry
J Chromatogr B
(1998)
G Zomer et al.
[1,2,3,4-¹³C]Testosterone and [1,2,3,4-¹³C]estradiol
Steroids
(1984)
G Zomer et al.
The synthesis of [1,2,3,4-¹³C]cortisol
Steroids
(1984)

G Zomer et al.

Synthesis of ¹³C-labeled steroid hormones

Steroids

(1990)

S Ulick et al.

Pathogenesis of the type 2 variant of the syndrome of apparent mineralocorticoid excess

J Clin Endocrinol Metab

(1990)

Cited by (25)

The evolution of methods for urinary steroid metabolomics in clinical investigations particularly in childhood
2018, Journal of Steroid Biochemistry and Molecular Biology
Citation Excerpt :
Recovery experiments of added steroid conjugates have been satisfactory [26]. Carbon 13 labelled steroids conjugates have also been prepared [94,95]. In a useful method that has survived many years free steroids and glucuronide conjugated are separated from the sulphates by elution pattern from Sephadex LH-20 with chloroform/methanol saturated with sodium chloride.
The metabolites of cortisol, and the intermediates in the pathways from cholesterol to cortisol and the adrenal sex steroids can be analysed in a single separation of steroids by gas chromatography (GC) coupled to MS to give a urinary steroid profile (USP). Steroids individually and in profile are now commonly measured in plasma by liquid chromatography (LC) coupled with MS/MS. The steroid conjugates in urine can be determined after hydrolysis and derivative formation and for the first time without hydrolysis using GC–MS, GC–MS/MS and liquid chromatography with mass spectrometry (LC–MS/MS). The evolution of the technology, practicalities and clinical applications are examined in this review. The patterns and quantities of steroids changes through childhood. Information can be obtained on production rates, from which children with steroid excess and deficiency states can be recognised when presenting with obesity, adrenarche, adrenal suppression, hypertension, adrenal tumours, intersex condition and early puberty, as examples. Genetic defects in steroid production and action can be detected by abnormalities from the GC–MS of steroids in urine. New mechanisms of steroid synthesis and metabolism have been recognised through steroid profiling. GC with tandem mass spectrometry (GC–MS/MS) has been used for the tentative identification of unknown steroids in urine from newborn infants with congenital adrenal hyperplasia. Suggestions are made as to areas for future research and for future applications of steroid profiling. As routine hospital laboratories become more familiar with the problems of chromatographic and MS analysis they can consider steroid profiling in their test repertoire although with LC–MS/MS of urinary steroids this is unlikely to become a routine test because of the availability, cost and purity of the internal standards and the complexity of data interpretation. Steroid profiling with quantitative analysis by mass spectrometry (MS) after chromatography now provides the most versatile of tests of adrenal function in childhood.
Use of endogenous cortisol 6β-hydroxylation clearance for phenotyping in vivo CYP3A activity in women after sequential administration of an oral contraceptive (OC) containing ethinylestradiol and levonorgestrel as weak CYP3A inhibitors
2014, Steroids
Citation Excerpt :
Cortisol was purchased from Sigma (St. Louis, MO, USA). Stable isotopically labeled cortisol i.e., [1,2,4,19-13C4]cortisol (cortisol-13C4) for use as analytical internal standard was synthesized in our laboratory [25]. The isotopic composition of the labeled cortisol was >97.3 atom%.
The present study was undertaken to evaluate the time courses of in vivo cytochrome P450 3A (CYP3A) inhibition in four healthy women after sequential administration of an oral contraceptive (OC) containing ethinylestradiol and levonorgestrel, using 6β-hydroxylation clearance of endogenous cortisol (CLm(6β)) as a new index for CYP3A phenotyping. The 6β-hydroxylation clearance (CLm(6β)) was followed every 2 h from 9:00 or 11:00 to 17:00 on days 0 (baseline), 1, 2, 21, and 28 during a single menstrual cycle. The serum concentrations of endogenous estradiol and progesterone were also measured. The time course data of CLm(6β) clearly demonstrated 43–64% inhibition of CYP3A activity in women taking a low daily dose of the OC for 21 days. The average CLm(6β) levels that were suppressed by the OC in four women were extremely low (0.60–1.23 mL/min) compared with the normal CLm(6β) range (1.5–3.5 mL/min) that was obtained from 49 healthy subjects in our previous study. The in vivo inhibitory potencies (43–64%) obtained in this study were stronger than expected from reported in vitro studies (∼20%). Furthermore, it would take at least seven days to return to the baseline activity of CYP3A after discontinuation of the OC. The results presented here should provide important information on the inhibitory effect of OC on the CYP3A activities in women, which are involved in the metabolism of a number of drugs with a narrow therapeutic range.
The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11β-hydroxysteroid dehydrogenase 2 activity
2012, Steroids
Citation Excerpt :
Finally, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment method for the 11β-HSD2 activities. [1,2,4,19-13C4]Cortisol (cortisol-13C4) and [1,1,19,19,19-2H5]cortisone (cortisone-2H5) were synthesized in our laboratory [12]. The isotopic compositions of the labeled compounds were >98 at.
Factors that give rise to a large variation in the urinary ratio of free cortisone to cortisol (UFE/UFF) were investigated to accurately estimate 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity in humans in vivo. A water loading test was first carried out in two healthy subjects to examine the effect of water intake or urine volume on the urinary ratio of free cortisone to cortisol (UFE/UFF). The ratio was found to increase by water loading. We also examined urinary concentrations and amounts of cortisol, cortisone, creatinine, Na⁺, K⁺, and Cl⁻, and urine volume, as possible factors affecting the urinary ratio (UFE/UFF), in 60 urine samples obtained from 15 healthy volunteers. Among these factors tested, the urinary concentration of cortisol was most highly correlated with the UFE/UFF ratio (r = −0.858), indicating that the in vivo activity of 11β-HSD2 (UFE/UFF) should fluctuate with the changes of the urinary concentration of cortisol. Based on the findings, we proposed a new estimation method of in vivo activity of 11β-HSD2 in humans, using the UFE/UFF ratio correlated with the urinary concentration of cortisol (UFE/UFF–cortisol concentration). Taking into consideration the intra-individual variabilities in the urinary concentration of cortisol, there were no significant within-day variations in 11β-HSD2 activity. The findings indicate that 11β-HSD2 activities can be accurately evaluated by simply measuring free cortisol and cortisone concentrations in spot urine samples. Furthermore, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment for the activity of 11β-HSD2.
Lipase-catalyzed regioselective preparation of fatty acid esters of hydrocortisone
2009, Steroids
A series of fatty acid derivatives of hydrocortisone has been prepared by an enzymatic methodology. Nine 21-monoacyl products and one 3,11,17-triacetyl derivative, nine of them novel compounds, were obtained in a highly regioselective way through lipase-catalyzed esterification, transesterification and alcoholysis reactions. The influence of various reaction parameters such as acylating agent: substrate ratio, enzyme: substrate ratio, solvent, temperature and nature of acylating agent and alcohol was evaluated. Among the tested lipases, Candida antarctica lipase appeared to be the most appropriate and showed a high efficient behavior especially in a one-pot transesterification. The advantages presented by this methodology, such as mild reaction conditions and low environmental impact, make the biocatalysis a convenient way to prepare acyl derivatives of hydrocortisone. These lipophilic compounds are potential products in the pharmaceutical industry.
Simultaneous determination of androstenedione, 11β-hydroxyandrostenedione, and testosterone in human plasma by stable isotope dilution mass spectrometry
2009, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
This study describes a GC–MS method for the simultaneous determination of androstenedione (AD), 11β-hydroxyandrostenedione (11β-OHAD), and testosterone (TS) in human plasma. [19,19,19-²H₃]Androstenedione (AD-²H₃), 11β-hydroxy-[1,2,4,19-¹³C₄]androstenedione (11β-OHAD-¹³C₄), and [1,16,16,17-²H₄]testosterone (TS-²H₄) were used as internal standards. Pentafluoropropionic (PFP) derivatization with good GC behavior was employed for the GC–MS analysis of the three steroids. The detection limit of the present GC–MS–SIM method was found to be 1 pg per injection for AD (S/N ratio = 4.5), 5 pg for 11β-OHAD (S/N ratio = 5.0), and 1 pg for TS (S/N ratio = 4.4), respectively. Calibration curves were linear from 0.22 to 2.80 ng/mL (r = 0.9998) for AD, from 0.56 to 3.19 ng/mL (r = 0.9996) for 11β-OHAD, and from 2.05 to 10.3 ng/mL (r = 0.9996) for TS. The intra- and inter-day assay reproducibilities in the amounts of the three androgens determined were in good agreement with the actual amounts added, the relative errors (R.E.) were −3.1 to 2.4%. The inter-assay relative standard deviation (R.S.D.) was less than 5.3%. The present method provides a sensitive and reliable technique for the simultaneous determination of AD, 11β-OHAD, and TS in plasma. The method can be applied to pharmacokinetic and metabolic studies of androgens with a particular interest in evaluating the conversion of AD to 11β-OHAD and the interconversion of AD and TS in humans.
Validation of the plasma half-life of 11α-deuterium cortisol as a sensitive index for the analysis of human 11β-HSD2 activity in vivo
2005, Steroids
This study is concerned with validating the measurement of the plasma half-life of 11α-²H cortisol in an attempt to accurately assess the in vivo activity of 11β-HSD2 in man. Oral administration of 5 mg of cortisol-¹³C₄,²H₁ to a human subject after repeated ingestions of 130 mg/day of glycyrrhetinic acid for 5 days resulted in a decrease in the rate constant of the cortisol-¹³C₄,²H₁ to cortisone-¹³C₄ conversion, a direct index reflecting 11β-HSD2 activity. The reduced 11β-HSD2 activity led to an increase in the elimination half-life of cortisol-¹³C₄,²H₁, indicating that the loss of 11α-²H is a sensitive in vivo means of assessing 11β-HSD2 activity. A simultaneous oral administration of 3 mg each of [1,2,4,19-¹³C₄,11α-²H]cortisol (cortisol-¹³C₄,²H₁) and 11α-²H cortisol to another human subject confirmed the bioequivalency of the two labeled cortisols. The information obtained from the kinetic analysis of the 11β-HSD2-catalyzed conversion of cortisol-¹³C₄,²H₁ to cortisone-¹³C₄ indicated that the elimination half-life of 11α-²H cortisol was a sensitive index of renal 11β-HSD2 activity. The use of 11α-²H cortisol as a tracer appears to offer a significant advance in evaluating human 11β-HSD2 activity in vivo.

View all citing articles on Scopus

View full text

Original ArticlesSynthesis of multi-labeled cortisols and cortisones with 2H and 13C for study of cortisol metabolism in humans

Abstract

Introduction

Section snippets

Experimental

Results and discussion

Acknowledgements

Steroids

Steroids

J Steroid Biochem Molec Biol

Endocrinol Metab Clin North Am

Steroids

Steroids

Steroids

J Chromatogr B

Steroids

Steroids

Steroids

Pathogenesis of the type 2 variant of the syndrome of apparent mineralocorticoid excess

J Clin Endocrinol Metab

Original Articles
Synthesis of multi-labeled cortisols and cortisones with ²H and ¹³C for study of cortisol metabolism in humans