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Clinical Chemistry 43: 786-793, 1997;
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(Clinical Chemistry. 1997;43:786-793.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Convenient chromatographic prepurification step before measurement of urinary cortisol by radioimmunoassay

Gilles Morineau1,a, James Gosling2, Marie-Claude Patricot3, Hany Soliman1, Philippe Boudou1, Akram Al Halnak1, Gaëlle Le Brun1, Jean-Louis Brérault1, René Julien1, Jean-Marie Villette1 and Jean Fiet1,4

1 Laboratoire de Biologie Hormonale, Hôpital Saint-Louis, 75010 Paris, France.

2 Department of Biochemistry and National Diagnostics Centre, University College, Galway, Ireland.

3 Laboratoire d'Hormonologie, bâtiment 3B, Centre hospitalier Lyon-Sud, 69495 Pierre-Benite cedex, France.

4 Laboratoire de Biochimie, Faculté de pharmacie, 75006 Paris, France.
a Address correspondence to this author at: Laboratoire de Biologie Hormonale, Hôpital Saint-Louis, 1 ave. Claude Vellefaux, 75475 Paris cedex 10, France. Fax Int + 33 1 42 49 42 80; e-mail bio.horm.fiet{at}chu-stlouis.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We applied various prepurification protocols (extraction with different solvents, liquid/solid separation on bonded silica media, Celite, and Sephadex LH20 chromatography) with a range of commercially available RIA kits to measure cortisol in urine samples. We then compared the results with the concentrations measured by a HPLC method validated with reference to isotope dilution gas chromatography–mass spectrometry. We conclude that chromatography on a commercial, prepacked diol minicolumn (WatersTM Sep-Pak Vac RC) in combination with dichloromethane extraction is a convenient and very effective purification step before RIA of urinary cortisol in patients not receiving corticoid medication. We tested numerous steroids for interference and found that free polar cortisol derivatives (hydroxylated or hydrogenated) could only partially account for the overestimations routinely encountered when free urinary cortisol concentrations are measured by direct RIA.


Key Words: indexing terms: chromatography • Celite • Sephadex LH 20 • HPLC • ID GC-MS • diol minicolumns.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The concentration of cortisol in 24-h urine samples is useful because it reflects the free and active fraction of the steroid in serum compensated for diurnal fluctuations. Nevertheless, accurate measurements are difficult because of the presence of interfering substances. The most accurate method for cortisol measurement is gas chromatography with isotope dilution mass spectrometry (ID GC-MS) (1), but this requires very expensive apparatus and a highly trained operator.1 However, some simpler methods, when implemented carefully, are capable of approaching the accuracy of ID GC-MS and are useful as secondary reference methods in the validation of new methodology. HPLC with UV detection (2)(3)(4) or RIA after HPLC (5)(6) or after Sephadex LH20 chromatography (3) can be useful secondary reference methods, whereas standard immunoassay procedures such as RIA, even when performed after a solvent extraction step, are not so (3)(7). In fact, such methods often perform so poorly in external quality-assurance programs that the suitability of some of them for routine use is questionable.

The reaction of organosilanes with a range of functional groups with activated silica gives rise to a range of bonded silicas with surface groups of varying hydrophobicity and hydrophilicity linked through stable silyl ether linkages. Bonded silicas can be equilibrated rapidly with different solvents because they do not shrink or swell and they are widely used for HPLC. Bonded silicas in prepackaged cartridges or minicolumns and useful ancillary equipment are almost always used for sample clean-up before HPLC, and they are widely commercially available for such purposes (8). Our demonstration that a commercial, prepacked, bonded silica diol minicolumn may be used to selectively separate cortisol from most of the other steroids that interfere in the measurement of cortisol in urine expands the range of potential applications for these convenient devices.

The objective of the present study was to identify an accurate method based on a commercial immunoassay kit for cortisol measurement in urine that is simpler and more convenient than the secondary reference methods cited above. We found that an extraction step and fractionation on a commercial prepacked diol minicolumn combined with a standard immunoassay kit resulted in a procedure suitable for the routine assay of cortisol in urine. Finally, to identify the substances responsible for the overestimations encountered, we also tested numerous steroids for interference.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
subjects and samples
Aliquots of 24-h urines from hospitalized patients of different adrenal status [8 hypocortisolics (<20 nmol/day), 23 hypercortisolics (>220 nmol/day), and 47 normocortisolics (20–200 nmol/day)] or from 48 healthy volunteers (28 women, 20 men) were stored at -20 °C until assayed. All investigations conformed with the ethical standards laid down by the Helsinki Declaration (1975) as revised at Tokyo (1983).

reagents and materials
All steroids, dimethoxypropane, and hexamethyldisilizane were obtained from Sigma Chemical Co. (St Quentin Fallavier, France), except 5{alpha}-tetrahydrodehydrocorticosterone (5{alpha}-THA), 6ß-hydroxycortisol (6ß-OHF), and cortisol-21-glucuronide, which were from Steraloids (Wilton, NH). Methoxyamine hydrochloride (MOX reagent) and N-trimethylsilylimidazole were from Pierce (Rockford, IL). All solvents were analytical grade (for extraction) or HPLC grade. Tritiated steroids [1,2,6,7-3H]cortisol and [1,2-3H]11-deoxycortisol were from NEN Products-France (Les Ulis, France), and scintillation liquid was purchased from Amersham France (Les Ulis, France).

Phosphate gelatin buffer (PGB) was 0.04 mol/L Na2HPO4/NaH2PO4, 1 g/L gelatin, pH 7.4.

The columns for Celite and Sephadex chromatography were 5-mL glass pipettes (5 mm i. d.) (Kimble, Vineland, NJ), siliconized and stoppered at the bottom with small glass beads. Celite (Celite Corp., Touzart et Matignon, Vitry/Seine, France) was washed in cyclohexane and heated for 16–18 h at 800 °C, then kept dry at 100 °C until use. Sephadex LH20 (Pharmacia, St Quentin en Yvelines, France) was prepared by suspending 50 g in 350 mL of dichloromethane (DCM) and leaving it to swell overnight. Lipidex®-5000, a predominantly C15 hydroxyalkoxypropyl Sephadex, was from Packard Instruments (Rungis, France).

The prepacked commercial columns (500 mg) used for prepurification of urine samples before RIA had sorbent beds consisting of native silica unfunctionalized (Si) or silica coated with the following functional groups: ethyl (C2), octyl (C8), octadecyl (C18), phenyl (PH), aminopropyl (NH2), or dipropyl ether-1,2-diol covalently linked via the 3' carbon [i.e., -(CH2)3-O-CH2CH(OH)-CH2OH, abbreviated to "diol"]. The columns were from three suppliers: Amersham [Amprep RPN C2, C8, C18, 2OH (i.e., diol), NH2, PH, Si], Waters-Millipore Corp. (St Quentin en Yvelines, France; Sep-Pak Vac RC diol), and Baker Corp. (Noisy le Sec, France; Bakerbond spe diol). A special manifold made in house allowed simultaneous operation of up to 36 of any of the above columns.

Immunoassay kits.
Six "coated-tube" RIA kits for the measurement of cortisol in serum and urine were chosen. Five had polyclonal anti-cortisol antibodies including those from CIS bio International (CORT-CT® and CORT-CT2®; Gif sur Yvette, France), Behring Diagnostic (Coat-a-count® cortisol; Rueil Malmaison, France), Kallestad Diagnostics (Quanticoat® cortisol; Chaska, MN), ERIA Diagnostics Pasteur (Marnes la Coquette, France), Incstar Corp. (Gamma coatTM cortisol; Stillwater, MN), Sorin Biomedica (CA 1549; Antony, France); one had a monoclonal antibody, Immunotech (Cortisol ref.1114; Marseille, France).

The radioactivity counters used were a LKB Gammamaster (Pharmacia-LKB, St Quentin en Yvelines, France) and a Wallac 1409 beta counter (Pharmacia-LKB).

Gas chromatograph–mass spectrometer.
A Girdel serial 32 gas–liquid chromatograph apparatus (Girdel, Suresnes, France) with R.10.10 quadrupole mass spectrometer (Nermag, Rueil-malmaison, France) coupled to a PDP8-based data system (Digital Equipment, Maynard, MA) was used to measure cortisol with correction for losses by isotope dilution analysis of added deuterated cortisol.

methods
ID GC-MS.
The method used has been previously published (9)(10).

HPLC.
The method used has been described previously (3)(4). This method gave similar results to HPLC followed by RIA (5).

RIA.
With each of the six RIA kits we analyzed up to 78 urine samples containing 10–650 nmol/L cortisol: (a) according to the manufacturer's prescriptions without an extraction step; (b) as above but including the manufacturer's protocol for extraction except that a small amount of tritiated cortisol (500 cpm) was added to the samples to allow monitoring of methodological losses, as in the chromatographic purifications.

For the solvent extraction study and for preliminary studies on chromatographic purification of samples, we used only one kit, the Kallestad Quanticoat kit, because it gave generally lower values than the others without an extraction step. It also appeared to be quite specific (Table 1 ).


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  		Table 1. Cross-reactivities of a wide range of natural steroids and drugs in RIA kits used in this investigation.1

Starting with 400-µL samples of urine in 6-mL glass test tubes, four procedures of solvent extraction were tested: (a) 2 mL of ethyl acetate; (b) 2 mL of cyclohexane:ethyl acetate (50:50 by vol) (CH:EA); (c) preliminary extraction with 3 mL of carbon tetrachloride followed by extraction with 2 mL of CH:EA; and (d) 2 mL of DCM. For each tube with urine and solvent, vortex for 60 s, centrifuge for 5 min at 1800g, and separate the phases by transferring the organic solvent to another tube after removal of the aqueous phase by aspiration when the upper phase is aqueous, or by freezing and decanting.

chromatographic purification before ria
Extraction and Celite chromatography
was carried out as described previously (11)(12)(13)(14), with DCM and Celite/ethylene glycol column.

Extraction and Sephadex LH20 chromatography
was carried out as described (3)(15)(16), with DCM and a Sephadex LH20 column.

Extraction and chromatography on diol-bonded silicas.
Efficiency in separating tritiated analogs of cortisol and 11-deoxycortisol was the criterion used when we chose the solid-phase diols and optimized the elution procedure (Fig. 1 ). Diol columns were better than Si and NH2 and all of these were better still than C18, C8, C2, and PH. We preferred Waters Vac RC cartridges over minicolumns from Amersham because of their integrated reservoirs. Baker diol columns gave less efficient separation.



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  		Figure 1. Chromatographic elution profile: separation of [3H]cortisol from [3H]11-deoxycortisol upon chromatography on Amersham diol minicolumns.

To perform diol chromatography on urine samples, extract 0.4 mL of urine with 2 mL of DCM, add the extract directly to the previously conditioned column (5 mL of ethanol followed by 5 mL of CH), and elute at 1–2 drop/s according to a procedure slightly modified depending on the brand (Waters or Amersham) and the batch. For example, with an Amersham column, add and discard 12 mL of CH:EA (65:35 by vol), then add and collect 10 mL of CH:EA (40:60 by vol); with a Waters column add and discard 12 mL of CH:EA (80:20 by vol), add and collect 12 mL of CH:EA (70:30 by vol), and evaporate this. We used each column up to three times, flushing them with 6 mL of ethanol and 8 mL of CH before each use.

We redissolved the final residue from each purification in PGB and analyzed 100-µL aliquots by RIA after addition of 20 µL of steroid-free plasma.

cross-reactions
We estimated cross-reactivities (17) of natural steroids and selected therapeutic steroids prepared in PGB without extraction (Table 1Up ). For the CORT-CT2 kit only, we relied on the data supplied by the manufacturer.

To test interferences from endogenous steroids by direct assay, we added them to cortisol-free urine and assayed for cortisol-like reactivity (Table 2 ). To test for interferences from exogenous steroids in RIA after chromatography, we proceeded in the same way but with increasing concentrations of steroid, and measured the cortisol-like reactivity according to the Celite, Sephadex, diol, or HPLC procedures above (Table 5 ).


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  		Table 2. Estimates of actual potentials for interference of endogenous corticoids determined by directly measuring apparent cortisol concentrations in steroid-free urine supplemented with concentrations likely to be present when unconjugated cortisol is 100 nmol/L.


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  		Table 5. Apparent cortisol concentrations due to therapeutic corticoids seen by Kallestad kit after DCM extraction and diol purification.

statistics
Intra- (n = 10) and interassay (n = 10) CVs were determined at three concentrations (60, 120, and 600 nmol/L). Comparison of measurements with each method under investigation and measurements by HPLC (as independant variable) were made by constructing scattergraphs around lines of agreement, and by fitting the points to a straight line by the least-squares method. The significance of slopes and intercepts was determined by Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
characteristics of the methods
HPLC.
The HPLC detection limit was 1.7 pmol per injection or 5 nmol/L of urine. Intraassay variability was 6.41% at 268 nmol/L (n = 10) and interassay variability was 8.18% and 14.84% at 1160 nmol/L (n = 26) and 51.5 nmol/L (n = 42) respectively. Recovery of added cortisol to urine was 90.3% ± 4.94% (mean ± SD). When cortisol between 100 and 600 nmol/L (n = 8) was added (x) to aliquots of a single charcoal-treated urine sample and measured (y), the fitted correlation was y = 0.89x + 4.52, r = 0.991.

When the HPLC method (y) was compared with ID GC-MS (x) for nine urine samples containing 20–4000 nmol/L cortisol (each one in duplicate), the regression equation for the scatterplot was y = 0.98x - 0.36, r = 0.999.

Recoveries in procedures preliminary to RIA.
With ethyl acetate, extraction was nearly complete [99.0% ± 1.9%, 95–103% (mean ± SD, range); n = 78] but was lower with DCM (93.0% ± 1.5%, 90–96%; n = 78). For the Celite, Sephadex, and diol chromatographic separations after DCM extraction, the overall recoveries were 84% ± 3.9% (range 73–92%), 76% ± 3.1% (70–94%), and 74% ± 2.9% (69–86%), respectively (n = 78 each).

RIA kits
. Unlabeled cortisol was added to charcoal-treated serum at three concentrations (100, 400, and 850 nmol/L) and measured without extraction with each RIA kit. Mean results are not significantly different from those expected (P = 0.05) for any kit; they were (mean ± SD) 96 ± 5.5, 408 ± 13.6, and 830 ± 32.8 nmol/L, respectively.

Measurements of control urine samples at regular intervals allowed the estimation of assay variability with the Kallestad kit and different preliminary steps. Intraassay CVs (n = 10) for preliminary extraction and chromatography on Celite, Sephadex LH20, and diol were respectively 7.2%, 8.5%, and 9.6% at 60 nmol/L, 7.7%, 7.1%, and 4.8% at 120 nmol/L, and 8.4%, 7.4%, and 5.5% at 600 nmol/L. Interassay CVs (n = 10) were 7.8%, 8.8%, and 9.8% at 60 nmol/L, 9.0%, 7.5%, and 4.9% at 120 nmol/L, and 9.0%, 7.8%, and 5.6% at 600 nmol/L, respectively.

method comparisons
Specificity.
We have no information on the nature of immunogens used to raise the antibodies used in the different kits, except for the Kallestad kit, in which the immunogen was cortisol-3-(O-carboxymethyl)-oxime–bovine serum albumin. The susceptibilities of the six kits to interfere with a wide range of natural steroids, their metabolites, and steroidal drugs were assessed experimentally (Table 1Up ). The most pronounced and widely interfering compound was the drug prednisolone; it cross-reacted 40–80% in all kits except one, Immunotech, for which it was 10%. Of the "natural" steroids and metabolites, 5{alpha}-dihydrocortisol (5{alpha}-DHF) was the most interfering, giving 25–85% in four of the kits. None of the kits was clearly superior overall. The Immunotech kit, which has a monoclonal antibody, was best with prednisolone (10%) and 5{alpha}-DHF (2.8%) but worst with methylprednisolone (50%), 11-deoxycortisol (S) (16%), 11-deoxycorticosterone (DOC) (27%), and triamcinolone (13%).

Among 10 natural steroids, only 6ß-OHF and 20{alpha}-dihydrocortisol (20{alpha}-DHF) showed significant interference at physiological concentration (Table 2Up ).

Cortisol measurements without and with extraction.
Cortisol was measured in urine samples by HPLC and by RIA without or with extraction steps performed according to the instructions of the respective kit manufacturers (Table 3 ). The results represent mainly gross overestimates of the actual cortisol present, particularly at lower concentrations and when no preliminary extraction step was performed. At 300 nmol/L, for Kallestad and CORT-CT2 kits, the apparently higher results after extraction represent, in fact, the low recovery of high concentrations of cortisol by direct assay in urine (7). To make feasible further investigations, one kit (Kallestad) was chosen for combination with each of a range of extraction and preliminary chromatographic procedures.


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  		Table 3. Correlations between apparent cortisol concentrations (nmol/L) measured by RIA without and with extraction and HPLC (n = 78; range 10–650 nmol/L).

Extraction and chromatographic procedures.
Of the four extraction procedures tested in combination with the Kallestad kit, a simple extraction with DCM was the most satisfactory (Table 4 , upper section). We then combined the same kit with an extraction step and three different column chromatography steps (Table 4 , lower part). The concentrations measured by DCM extraction and all three column methods were in quite good accordance with HPLC results. The correlation coefficients (r) were all >0.92, the slope of no regression line differed significantly from unity, and the intersection of no line differed from zero (P >0.05). The combination of CH:EA extraction and separation on a diol column was much less efficient at removing interfering substances.


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  		Table 4. Correlations between apparent cortisol concentrations (nmol/L) measured by Kallestad RIA kit with different extractions or extraction plus chromatography prepurification methods, and HPLC (n = 78; range 10–650 nmol/L).

The combination of extraction with DCM and purification on a diol minicolumn, rather than either of the other chromatographic methods, was chosen for further studies because of the convenience of the prepacked minicolumns.

Interference of exogenous corticoids.
Sephadex or Celite chromatography prevented interference in most common therapeutic situations, but at high concentrations (>=300 nmol/L prednisolone) interference did occur, whereas HPLC (up to 300 000 nmol/L prednisolone) still allowed quantification of cortisol without interference (data not shown). Extraction with DCM and diol separation was clearly imperfect and did not allow cortisol quantification in these cases (Table 5Up ).

concentrations in volunteers
With the above diol procedure, urinary free cortisol on 24-h diuresis ranged from 38 to 248 nmol/24 h (119 ± 58.8, mean ± SD) for 48 volunteers between 20 and 40 years old (28 women, 20 men). Cortisol was significantly lower among women (94.1 ± 46.5) than men (143.9 ± 62.5) (Mann–Whitney U-test, P = 0.003).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Plasma cortisol measurements by immunoassay are relatively precise and accurate (18). Urinary cortisol measurements pose much greater difficulties because of matrix effects and the high concentrations of cross-reacting compounds (1)(3)(7). Specific methods such as HPLC with UV detection or RIA after HPLC require heavy investments and (or) much technical time and skill.

Manufacturers offer immunoassays that either are used directly with urine samples or involve only a simple extraction step. In the present study, we looked for sample pretreatment methods that could offer the best compromise between simplicity and accuracy.

The basic problem is easily demonstrated: Direct, nonextraction measurements by RIA give concentrations that are two to six times greater than the actual concentrations of cortisol present at about 20 nmol/L cortisol, depending on the kit used (Table 3Up ). When the extraction procedures recommended by three of the manufacturers were used, the results obtained were generally better. Nonetheless, no kit, even with the recommended extraction step, gave results that agreed, or even nearly agreed, with HPLC results over a wide range of cortisol concentrations.

As found above, inaccuracy with cortisol immunoassays is generally overestimation, suggesting that the problem is due to interfering compounds detected in addition to cortisol. A range of compounds more polar than cortisol have been suggested as the principal cross-reactants (27), and extraction procedures designed to selectively remove these do have some effect. An ideal solvent would efficiently extract cortisol and none of the cross-reactants. DCM appears to have about the optimum degree of polarity for selective cortisol extraction (3)(28), giving the smallest degree of overestimation of the solvents tested (Table 4Up ).

Knowledge of the actual identities of the interfering substances would be important to the design of cross-reactant-resistant procedures, particularly if a small number of compounds turned out to be responsible for most of the problem. Among the endogenous corticoids, the conjugates (glucuronides or sulfates) are usually present in too low concentrations to interfere, except in some cases such as newborn infants, in whom cortisol and cortisone sulfates may be highly increased (29), or in Cushing syndrome (30), in which cortisol sulfate is high. However, conjugates can be expected to be efficiently removed by an extraction step. Many unconjugated steroids are possible candidates for causing interference in the kits included in this study. For example, 6ß-OHF and 20{alpha}-DHF may interfere, but probably not in all assays; the Kallestad kit is particularly susceptible to 20{alpha}-DHF and the Behring kit to 6ß-OHF (Tables 1Up and 2Up ). In routine practice such specific interferences may be highly significant, particularly in samples from patients with conditions in which the concentrations of unconjugated corticosteroids are greatly increased, such as 6ß-OHF in Cushing syndrome (16)(25), in newborn infants (31), and in pregnant women (32), and 5{alpha}-DHF in apparent mineralocorticoid excess (22). Some other steroids found to markedly cross-react, such as 5{alpha}-tetrahydrocortisol (5{alpha}-THF) and 5ß-tetrahydrocortisol (5ß-THF), may be less important, since they are almost entirely conjugated in urine.

In addition, kits that appear to be comparatively robust in a cross-reactivity study may be more subject to overestimations when compared with a reference method (24)(27). The role of nonsteroids and of other "nonspecifically" interfering substances may be crucially important and is little understood. Therefore, particularly when extraction is not used, cortisol RIA of urine samples give concentration estimates that, in addition to cortisol, include the concentrations of specific and nonspecific interferences, and these results would be better referred to as cortisol-like immunoreactivity, or some such term rather than "cortisol" (24), and the method used must be constant if comparisons are to be made.

Most authors agree that selective extraction followed by chromatographic purification are necessary for an accurate urinary cortisol assay (2)(3)(6)(24)(27). Chromatographic pretreatement of samples for immunoassay has been used since the earliest steroid RIA, but has been relatively neglected for reasons that can be justified on analytical grounds (it increases imprecision, and the range of media and methods available were limited) or because it requires highly skilled operators, increases costs, and slows the production of results. A variety of additional chromatographic media that are commercially prepared as minicolumns or cartridges have become widely used for "cleaning" samples before HPLC. Prepacked in minicolumns, diol and other media of this type are almost always used for the global extraction of analytes (e.g., "all" steroids) from the general biological matrix of samples, but we report here a finer separation of compounds within the same general group (e.g., "cortisol" from interfering corticoids), as other authors did for the isolation of vitamin D3(33).

Under common physiological and pathological conditions, the proposed method with DCM extraction, diol minicolumn fractionation, and the Kallestad kit gave cortisol concentrations matching those of the secondary reference method. The main advantages of the diol columns (over Celite and Sephadex LH20 columns) are their commercial availability to all potential users and their ease of use for >20 samples with a vacuum extractor. The principal limitation of the method is that it is not suitable for samples from patients undergoing therapy with prednisone, prednisolone, or 6{alpha}-methylprednisolone. In these cases, we recommend HPLC, which allows visualization (and quantification) of the drug while accurately measuring cortisol.


   Acknowledgments
 
We are grateful to E. Rolle, C. Crouin, and Ph. Aubin for their technical support and to C. Courtois for typing the manuscript.


   Footnotes
 
1 Nonstandard abbreviations: ID GC-MS, isotope dilution gas chromatography–mass spectrometry; 5{alpha}-THA, 5{alpha}-tetrahydrodehydrocorticosterone; 6ß-OHF, 6ß-hydroxycortisol; PGB, phosphate gelatin buffer; DCM, dichloromethane; PH, phenyl; CH:EA, cyclohexane:ethyl acetate; 5{alpha}- and 5ß-DHF, 5{alpha}- and 5ß-dihydrocortisol; S, 11-deoxycortisol; DOC, 11-deoxycorticosterone; 20{alpha}- and 20ß-DHF, 20{alpha}- and 20ß-dihydrocortisol; 5{alpha}- and 5ß-THF, 5{alpha}- and 5ß-tetrahydrocortisol; 5ß-THE, 5ß-tetrahydrocortisone; and 5{alpha}- and 5ß-THS, 5{alpha}- and 5ß-tetrahydro-11-deoxycortisol.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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