Hiromi Shibasaki

Simultaneous determination of endogenous and 13C-labelled cortisols and cortisones in human plasma by stable isotope dilution mass spectrometry.

This study describes a capillary GC-MS method for the simultaneous determination of endogenous cortisol and cortisone and their 13C-labelled analogues, [1,2,4,19-13C4]cortisol (cortisol-13C4) and [1,2,4,19-13C4]cortisone (cortisone-13C4), in human plasma. [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) were used as analytical internal standards. A double derivatization (bismethylenedioxy-pentafluoropropionate, BMD-PFP) with good GC behavior was employed for the GC-MS analysis of cortisol and cortisone. Quantitation was carried out by selected-ion monitoring of the molecular ions ([M]+*) of the BMD-PFP derivatives of cortisol and cortisone. The sensitivity limit of the present GC-MS-SIM method was found to be 150 pg per injection for cortisol (s/n=5.0) and 50 pg for cortisone (s/n=8.1). The within-day reproducibility in which the amounts of unlabelled and labelled cortisols and cortisones determined were in good agreement with the actual amounts added, the relative errors being less than 3.07%. The inter-assay coefficients of variation (C.V.) were less than 1.80% for unlabelled and labelled cortisols and cortisones.

Hydrolysis of conjugated steroids by the combined use of β-glucuronidase preparations from helix pomatia and ampullaria: determination of urinary cortisol and its metabolites

This study describes the enzymatic hydrolysis of urinary conjugates of cortisol, cortisone, tetrahydrocortisol, allotetrahydrocortisol, and tetrahydrocortisone with beta-glucuronidase preparations from Helix pomatia and Ampullaria. The objective of the present studies was to find optimal hydrolysis conditions for these conjugated steroids. Assay of the isolated steroids was carried out by GC-MS using deuterium-labeled compounds as internal standards. The allotetrahydrocortisol conjugate was clearly the hardest to hydrolyze with enzyme from Helix pomatia and required increased enzyme concentration and prolonged incubation. Hydrolysis of a urine sample for 2.0 h with the simultaneous use of 3400 units/ml Ampullaria and 5400 units/ml Helix pomatia enzymes in 0.5 M acetate buffer at 55 degrees C achieved more complete cleavage of the urinary conjugates of the five steroids examined. It is thus advantageous to use the Ampullaria and Helix pomatia enzymes in combination to obtain the highest yield in the urinary corticosteroid assay.

The use of deuterium-labeled cortisol for in vivo evaluation of renal 11β-HSD activity in man: urinary excretion of cortisol, cortisone and their A-ring reduced metabolites

This study describes a new approach using stable isotope methodology in evaluating 11beta-HSD activities in vivo based on urinary excretion of cortisol, cortisone, and their A-ring reduced metabolites. The method involved the measurement of deuterium-labeled cortisol and its deuterium-labeled metabolites by GC/MS simultaneously with endogenous cortisol, cortisone, and their A-ring reduced metabolites after oral administration of deuterium-labeled cortisol to normal human subjects. This stable isotope approach offered unique advantages in assessing the appropriateness of measuring unconjugated and total (unconjugated + conjugated) cortisol, cortisone, and their A-ring reduced metabolites in urine as indices of renal 11beta-HSD2 activity in man. Our results strongly support that the measurement of urinary unconjugated cortisol and cortisone is a significant advance in assessing 11beta-HSD2 activity.

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

A method is described for the preparation of multi-labeled cortisol and cortisone with (13)C and (2)H via the indan synthon method, starting from chiral 11-oxoindanylpropionic acid. [1, 3-(13)C(2)]Acetone was used for the syntheses of [1,2,4, 19-(13)C(4)]cortisol (cortisol-(13)C(4)) and [1,2,4, 19-(13)C(4)]cortisone (cortisone-(13)C(4)), and [1,3-(13)C(2),1,1,1, 3,3,3-(2)H(6)]acetone was for [1,2,4,19-(13)C(4),1,1,19,19, 19-(2)H(5)]cortisol (cortisol-(13)C(4),(2)H(5)) and [1,2,4, 19-(13)C(4),1,1,19,19,19-(2)H(5)]cortisone (cortisone-(13)C(4), (2)H(5)). The chemical shifts for the (13)C and (1)H NMR spectra of cortisol and cortisone were fully assigned.

Simultaneous determination of 6β- and 6α-hydroxycortisols and 6β-hydroxycortisone in human urine by stable isotope dilution mass spectrometry

Abstract A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of 6β-hydroxycortisol (6β-OHF, 6β,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione), 6α-hydroxycortisol (6α-OHF, 6α,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione) and 6β-hydroxycortisone (6β-OHE, 6β,17α,21-trihydroxypregn-4-ene-3,11,20-trione) in human urine is described. Deuterium-labelled compounds, 6β-[1,1,19,19,19-2H5]OHF (6β-OHF-d5), 6α-[1,1,19,19,19-2H5]OHF (6α-OHF-d5) and 6β-[1,1,19,19,19-2H5]OHE (6β-OHE-d5) were used as internal standards. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M-31]+) of the methoxime–trimethylsilyl (MO–TMS) derivatives of 6β-OHF, 6α-OHF and 6β-OHE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring 6β-OHF, 6α-OHF and 6β-OHE in human urine.

Simultaneous determination of tetrahydrocortisol and tetrahydrocortisone in human plasma and urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of tetrahydrocortisol (THF, 3alpha,11beta,17alpha,21-tetrahydroxy-5beta-preg nane-20-one), allo-tetrahydrocortisol (allo-THF, 3alpha,11beta,17alpha,21-tetrahydroxy-5alpha-pre gnane-20-one) and tetrahydrocortisone (THE, 3alpha,17alpha,21-trihydroxy-5beta-pregnane-11,20-dion e) in human plasma and urine is described. [1,2,3,4,5-2H5]THF (THF-d5), allo-[1,2,3,4,5-2H5]THF (allo-THF-d5) and [1,2,3,4,5-2H5]THE (THE-d5) were used as internal standards. A double derivatization (bismethylenedioxypentafluoropropionate, BMD-PFP) made possible the separation of the three tetrahydrocorticoids with good gas chromatographic behavior. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M-30]+) of the BMD-PFP derivatives of THF, allo-THF and THE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring low concentrations of THF, allo-THF and THE in human plasma and urine.

Stable Isotope Methodology for Kinetic Studies of Interconversion of Cortisol and Cortisone in a Human Subject

Oral administration of 5 mg each of deuterium-labeled cortisol ([1,1,19,19,19-2H5]cortisol, cortisol-d5) and cortisone-d5 ([1,1,19,19,19-2H5]cortisone) to a human subject on two different occasions four weeks apart provided a useful means of characterizing the kinetics of the interconversion of cortisol and cortisone. From the data on plasma concentration measurements of cortisol-d5, cortisone-d5, cortisone-5, endogenous cortisol and endogenous cortisone by gas chromatography-mass spectrometry, it was demonstrated that (1) the plasma concentration ratio of cortisone-d5 to cortisol-d5 approached a plateau 4-5 h following either the cortisol-d5 or cortisone-d5 administration and the plateau values for the cortisone-d5 and cortisol-d5 administration were almost identical (about 0.43) and (2) dosing with only 5 mg of the deuterium-labeled steroids suppressed the plasma concentrations of endogenous cortisol and cortisone.

Measurement of cortisol secretion rate by stable isotope methodology following oral administration of 13C-labeled cortisol to a human subject.

Plasma concentration measurements of 13C-labeled cortisol ([1,2,4,19-13C(4)]cortisol, cortisol-13C(4)) and its metabolite cortisone-13C(4) were made simultaneously with measurements of endogenous cortisol and cortisone by gas chromatography-mass spectrometry (GC-MS). After administering a small amount (3mg) of cortisol-13C(4) to a human subject, changes in cortisol secretion rates were estimated by deconvolution techniques from the measured plasma cortisol and cortisone levels and the rates of elimination and interconversion of cortisol and cortisone were obtained from the plasma concentration-time data of cortisol-13C(4) and cortisone-13C(4). The objective of this study was to look for a novel approach to quantitate rates of minute-to-minute cortisol secretion in man by taking into account the interconversion of cortisol and cortisone by 11beta-hydroxysteroid dehydrogenase (11beta-HSD).

Syntheses of stable isotope-labeled 6β-hydroxycortisol, 6β-hydroxycortisone, and 6β-hydroxytestosterone

Abstract A method is described for the preparation of two types of multi-labeled 6β-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13 C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19- 2 H 5 ]cortisone and [1,2,4,19- 13 C 4 ,1,1,19,19,19- 2 H 5 ]cortisone (cortisone- 2 H 5 -BMD and cortisone- 13 C 4 , 2 H 5 -BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3- 2 H 5 ]- or [1,3- 13 C 2 ,1,1,3,3,3- 2 H 5 ]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3- 2 H 6 ]- or [1,3- 13 C 2 ,1,1,1,3,3,3- 2 H 6 ]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6β-hydroxy-[1,1,19,19,19- 2 H 5 ]cortisone-BMD and 6β-hydroxy-[1,2,4,19- 13 C 4 ,1,1,19,19,19- 2 H 5 ]cortisone-BMD, respectively, as a mixture of 6β- and 6α-epimers in a ratio of 4:1. Separation of 6β- and 6α-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6β-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6β-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH4 and subsequent removal of the C-3 and C-17 protecting groups gave 6β-hydroxy-[1,1,19,19,19- 2 H 5 ]cortisol (6β-hydroxycortisol- 2 H 5 ) and 6β-hydroxy-[1,2,4,19- 13 C 4 ,1,1,19,19,19- 2 H 5 ]cortisol (6β-hydroxycortisol- 13 C 4 , 2 H 5 ), respectively, as a mixture of 6β- and 6α-epimers (6β:6α=4.4:1). The isotopic compositions of 6β-hydroxycortisol- 2 H 5 and 6β-hydroxycortisol- 13 C 4 , 2 H 5 were 90.9 and 92.1 at.%, respectively. Furthermore, 6β-hydroxy-[1α,16,16,17α- 2 H 4 ]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1α,16,16,17α- 2 H 4 ]testosterone) obtained by using catalytic deuteration and hydrogen–deuterium exchange reactions.

Evaluation of 11β-HSD activities in vivo following oral administration of cortisol-13C4,2H1 to a human subject

This study is concerned with an oral administration of 5mg of [1,2,4,19-13C(4),11alpha-2H]cortisol (cortisol-13C(4),2H(1)) to a human subject to reliably evaluate the individual activities of two isozymes of 11beta-HSD. The use of a GC-MS method allowed the simultaneous measurement of the plasma concentrations of cortisol-13C(4),2H(1), cortisone-13C(4), and cortisol-13C(4) together with endogenous cortisol and cortisone. The loss of 11alpha-2H during the conversion of cortisol-13C(4),2H(1) to cortisone-13C(4) by 11beta-HSD2 and the regenerated cortisol-13C(4) from cortisone-13C(4) by 11beta-HSD1 provided a direct and accurate means of distinguishing the activities of the two isozymes. The kinetic analysis associated with the metabolism of orally administered cortisol-13C(4),2H(1) was of great importance in assessing the 11beta-HSD activities. From a viewpoint of the chemical stability and much less pronounced kinetic isotope effect of the 13C-label and the 2H-labeling in the 11alpha-position, cortisol-13C(4),2H(1) used in this study served as an appropriate tracer for elucidating the kinetics of the interconversion of cortisol to cortisone in man.