The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4628-4633
Copyright © 2004 by The Endocrine Society
Studies on the Origin of Circulating 18-Hydroxycortisol and 18-Oxocortisol in Normal Human Subjects
E. Marie Freel,
Loai A. Shakerdi,
Elaine C. Friel,
A. Michael Wallace,
Eleanor Davies,
Robert Fraser and
John M. C. Connell
Medical Research Council Blood Pressure Group (E.M.F., L.A.S., E.C.F., E.D., R.F., J.M.C.C.), University of Glasgow, Division of Cardiovascular and Medical Sciences, Western Infirmary, Glasgow G11 6NT, Scotland, United Kingdom; and Department of Clinical Biochemistry (A.M.W.), Macewen Building, Royal Infirmary, Glasgow G4 0SF, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Dr. Robert Fraser, Medical Research Council Blood Pressure Group, Western Infirmary, Glasgow G11 6NT, Scotland, United Kingdom. E-mail: rfraser{at}clinmed.gla.ac.uk.
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Abstract
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18-Hydroxycortisol (18-OHF) and 18-oxocortisol (18-oxoF) are derivatives of cortisol found in primary aldosteronism but whose origin and regulation in normal subjects are uncertain. 18-OHF can be synthesized by zona fasciculata 11-ß hydroxylase; 18-oxoF can only be produced by zona glomerulosa aldosterone synthase (AS).
Stably transfected cell lines expressing either CYP11B1 (11ß-hydroxylase) or CYP11B2 (AS) were incubated with cortisol and other substrates over a range of concentrations. Both enzymes could synthesize 18-OHF from cortisol, but only AS could synthesize 18-oxoF. AS was more efficient than 11ß-hydroxylase at 18-hydroxylation. The apparent Michaelis-Menten constant (Km) of AS for cortisol was estimated to be 2.6 µM.
In five patients with adrenal insufficiency maintained on hydrocortisone, urinary free cortisol and cortisone levels were high; 18-oxoF was detectable in all patients and 18-OHF in three. It is likely that the 18-oxygenated steroids were synthesized from circulating cortisol, either in the zona glomerulosa or at extraadrenal sites. In eight male volunteers, dexamethasone treatment decreased urinary excretion rates of free cortisol, cortisone, 18-OHF, and 18-oxoF, confirming dependence of 18-oxygenated steroid levels on cortisol availability. In both groups, hydrocortisone administration resulted in detectable levels of 18-OHF and raised levels of 18-oxoF. There was close correlation between 18-oxoF and cortisol excretion during hydrocortisone administration in normal subjects (r = 0.86; P < 0.001).
These data show, for the first time, that 18-OHF and 18-oxoF can be synthesized from circulating cortisol. The close correlation between 18-oxoF and cortisol suggests that 18-oxoF is normally produced by the action of AS using circulating cortisol as a substrate. Although 18OHF can be synthesized using circulating cortisol as substrate, our data suggest this is normally produced in the zona fasciculata by 11ß-hydroxylase from locally available cortisol.
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Introduction
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THERE ARE TWO principal active end products of corticosteroidogenesis in man. Aldosterone from the zona glomerulosa (ZG) is formed from 11-deoxycorticosterone (DOC) by aldosterone synthase (AS), and cortisol from the zona fasciculata (ZF) is formed from 11-deoxycortisol by 11ß-hydroxylase. This latter enzyme also converts DOC to corticosterone. Importantly, 11ß-hydroxylase is probably also the source of many circulating 18-hydroxysteroids, for example, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and 18-hydroxyprogesterone, in normal subjects.
In 1982, Chu and Ulick (1) isolated a novel compound from the urine of patients with primary hyperaldosteronism, which they identified as the 20,18-hemiketal form of 11ß, 17
, 18,21-tetrahydroxy-4-pregnene-3, 20-dione [18-hydroxycortisol (18-OHF),
17-hydroxyaldosterone]. In 1983, they isolated another C-18-oxygenated steroid after incubating cortisol with bullfrog interrenal gland; this was identified as 11ß, 17, 21-trihydroxy-3, 20-diketo-4-pregnene-18-al [18-oxocortisol (18-oxoF)] (2). Subsequently, Lifton and Pascoe coined the term "hybrid corticosteroids" for these compounds with both ZG (i.e. 18-oxygenation) and ZF (i.e. cortisol-like, hydroxylation at C-17) characteristics (3, 4, 5). High plasma concentrations in patients with aldosterone-producing tumors (Conn’s syndrome) was attributed to abnormal access of AS to cortisol (6, 7). AS is abnormally expressed in the ZF of patients with glucocorticoid-remediable aldosteronism; they also have high levels of these compounds, presumably also due to high local cortisol levels (3).
However, both compounds have now been identified in normal subjects (8, 9). In the normal human adrenal cortex, 18-OHF may be produced in the ZF from cortisol by the action of 11ß-hydroxylase (10). In vitro studies suggest that 18-OHF originates from the action of 11ß-hydroxylase. Apparent control of its levels by ACTH is further evidence of this (6, 11). However, 18-oxoF synthesis requires the additional 18-oxidation step, the capacity for which is restricted to the normal ZG. Cortisol is not made in this zone and, because blood flow is centripetal, it is not likely to perfuse from the ZF. What, therefore, is the origin of 18-oxoF in the normal circulation? One possibility is that cortisol enters the adrenal cortex in the peripheral circulation and is converted during passage through the ZG. Although the Michaelis-Menten constant (Km) for the action of AS on cortisol is not known, plasma cortisol is largely bound to corticosteroid binding globulin, and the concentration of free cortisol is low.
In the following study, we investigated the origins of 18- OHF and 18-oxoF. We evaluated the potential of 11ß-hydroxylase and AS to metabolize cortisol in vitro and obtained an estimate of Km for the latter reaction. We then tested the effect of increasing cortisol levels in vivo on 18-OHF and 18-oxoF excretion rates.
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Patients and Methods
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Cell culture and transfection
V79 Chinese hamster lung cells stably transfected with either CYP11B1 (11ß-hydroxylase) or CYP11B2 (AS) were a gift from Dr. Rita Bernhardt (Universitat des Saarlandes, Saarbrucken, Germany). They were grown to confluence in medium (DMEM supplemented with L-glutamine, 5% fetal calf serum, penicillin G, streptomycin, and amphotericin B). The medium was removed, and the cells washed in PBS followed by trypsin-EDTA solution and incubated at room temperature for 2 min. The cells were isolated by centrifugation, resuspended in incubation medium, and aliquots were transferred to six-well plates. These were incubated at 37 C for 24 h before use. CYP11B1-transfected cells were incubated with cortisol or 11-deoxycortisol. CYP11B2-transfected cells were incubated with cortisol, DOC, or 18-OHF. Incubation was for 24 h at 37 C. Medium was then removed and stored at –20 C. Where necessary, protein was measured by the Bio-Rad procedure.
Patients and volunteers
Local ethical committee approval for the study was obtained from the North Glasgow Hospitals University National Health Service Trust (Glasgow, Scotland, UK). Informed, written consent was obtained from all subjects.
Patients.
Twenty-four-hour urine collections were obtained from six patients with primary adrenocortical insufficiency. Five had autoimmune adrenal failure (Addison’s disease), and one had undergone bilateral adrenalectomy for phaeochromocytoma. Five of the subjects were taking hydrocortisone (15–30 mg/d) as replacement, and one was on dexamethasone (0.75 mg/d).
Volunteers.
Eight healthy male volunteers were recruited by advertisement. They were aged between 27–34 yr, and their body mass index was between 20–25 kg/m2. None had a history of hypertension (i.e. above the normal limits for age), excessive alcohol intake (>21 U/wk), or obesity (body mass index > 33 kg/m2). The study was subdivided into three stages, 1 mo apart. In stage one, 24-h urine collections were obtained without treatment. In stage two, 24-h urine collections were obtained after treatment with dexamethasone only [1 mg twice daily (bd) for 3 d]. In stage three, 24-h urine collections were obtained after treatment with dexamethasone (1 mg bd for 3 d) and hydrocortisone (20 mg bd for 1 d only). A period of 1 mo separated each phase.
Steroid analysis
Urinary steroid excretion rates were measured by gas chromatography-mass spectrometry using the methods of Shackleton (12) and Palermo et al. (13) with minor modifications. The internal standard was allo tetrahydrodeoxycorticosterone. A DB5 MS column (J&W Scientific, Folsom, CA), 30 m (0.25-mm inner diameter, 0.25-µm stationary phase) was used. The temperature program was as follows: 50 C for 3 min, 27 C/min up to 220 C, 1.7 degrees/min up to 300 C. Separate Sep-pak cartridges were used for extraction of conjugates and steroids released by hydrolysis (dictated by change in specification), and the source of the ß-glucuronidase was different (Biosepra, Cergy-Saint-Christophe, France). A Varian Saturn GCMS was used. Results were compared by one-way ANOVA.
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Results
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In vitro cell incubations
CYP11B1-transfected cells converted 11-deoxycortisol to cortisol (
42%) in a dose-dependent manner (Fig. 1
) Cortisol was converted to 18-OHF (0.5%) but not to 18-oxoF (Fig. 2). Small quantities of 18-OH-11-deoxycortisol, identified by mass spectrum only (results not shown), were also found. It was not possible to measure conversion rate to this steroid because no reference standard was available.
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FIG. 1. Cortisol (F) production by CYP11B1-transfected V79 Chinese hamster lung cells incubated for 24 h with 11-deoxycortisol (S).
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FIG. 2. 18-OHF production by CYP11B1-transfected Chinese hamster lung cells incubated for 24 h with cortisol.
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CYP11B2-transfected cells converted DOC to corticosterone (1.8%), 18-hydroxycorticosterone (1.7%), and aldosterone (3%), again in a dose-dependent manner (Fig. 3). Cortisol was converted to 18-OHF (4.3%) and to 18-oxoF (1.1%) (Fig. 4).
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FIG. 3. Corticosteroid production by CYP11B2-transfected V79 Chinese hamster lung cells incubated for 24 h with DOC. 18-OH-B, 18-Hydroxycorticosterone.
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FIG. 4. 18-OxoF and 18-OHF production by CYP11B2-transfected Chinese hamster lung cells incubated for 24 h with cortisol (F).
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Ratios of steroids synthesized by CYP11B1- and CYP11B2-transfected cells to their substrates are presented in Fig. 5. 11ß-Hydroxylase converted 11-deoxycortisol into cortisol with high efficiency, but efficiency to convert cortisol to 18-OHF was much lower. AS converted DOC into aldosterone less efficiently than 11ß-hydroxylase converted 11-deoxycortisol into cortisol. The ability of AS to convert cortisol to 18-OHF was higher than that of 11ß-hydroxylase.
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FIG. 5. Precursor to product ratios of steroid synthesis by CYP11B1- and CYP11B2-transfected Chinese hamster lung cells. F, Cortisol; S, 11-deoxycortisol.
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The apparent Km of AS for cortisol (see discussion) was estimated as 2.6 µM. No steroids apart from 18-OHF were detected when CYP11B2-transfected cells were incubated with 18-OHF.
Patients
Cortisol and cortisone were found in high concentration in the urine of the five patients taking hydrocortisone replacement therapy (Fig. 6). Low concentrations of 18-OHF, at the limit of detection, were also found in the urine of three patients who were taking hydrocortisone. 18-OxoF was also found in all patients who were taking cortisol. No endogenous steroids were detected in the patient receiving dexamethasone.
Volunteers
Average 24-h urinary excretion rates of cortisol, cortisone, 18-OHF, and 18-oxoF as well as comparisons between urinary excretion rates of these steroids are presented in Table 1. Urinary excretion rates of cortisol, cortisone, and 18-oxoF were significantly decreased in volunteers who were receiving dexamethasone compared with no treatment. They were significantly increased when the volunteers were taking cortisol and dexamethasone together compared with dexamethasone alone. Using the sum of cortisol and cortisone excretion rates as an index of cortisol status, 18-oxoF excretion rate correlated closely with this (r = 0.86; P < 0.01) whereas that of 18-OHF did not (r = 0.12; P > 0.05).
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TABLE 1. Urinary steroid metabolites excretion rates (nmol/24 h; mean ± SEM) in eight normal male subjects on no treatment, under dexamethasone suppression, and on dexamethasone plus cortisol
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Discussion
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The cell incubation experiments confirm that 11ß-hydroxylase is capable of converting cortisol to 18-OHF but not to 18-oxoF. AS was capable of both conversions. All conversions were dose dependent. The substrate conversion rates provide useful information on individual enzyme reactions. In CYP11B1-transfected cells, 11ß-hydroxylation by 11ß-hydroxylase is clearly more efficient than 18-hydroxylation. Cozza et al. (14) incubated slices of rat adrenals with 11-deoxycortisol. Both ZG and ZF were able to produce 18-hydroxy-11-deoxycortisol. Mulatero et al. (15) made a tentative identification of this compound in transfected COS-1 cells. In the current study, when CYP11B1-transfected cells were incubated with 11-deoxycortisol, they were also able to produce 18-hydroxy-11-deoxycortisol, confirming the ubiquitous ability of 11ß-hydroxylase to 18-hydroxylate steroids. In CYP11B2-transfected cells, 18-hydroxylation by AS is more efficient than in the CYP11B1-transfected cells. This may be related to the orientation of the substrate molecule at the catalytic site; for AS, this is perhaps more likely to favor interaction with the angular methyl group. Mulatero et al. (15) studied this problem in some detail and demonstrated that amino acid residues 288 and 320 are crucial to 18-hydroxylation and subsequent oxidation. That AS was unable to use 18-OHF as a precursor for 18-oxoF in the current study may again be due to difficulties in accessing the catalytic site or because 18-hydroxysteroids assume a thermodynamically hindered form in aqueous solution (16). The overall activity of CYP11B1-transfected cells (11ß-hydroxylase) in converting 11-deoxycortisol into cortisol was higher than the activity of CYP11B2-transfected cells (AS) in converting DOC into aldosterone. This may reflect functional differences between the two enzymes; the AS reaction is more complex, and released intermediates (18-hydroxycorticosterone and corticosterone) were not taken into account in calculating efficiency. In addition, transfection efficiency of the two enzymes in vitro may have been different.
In the patients with primary adrenal insufficiency, traces of 18-oxoF and 18-OHF were found in the urine in those on hydrocortisone treatment, but none was detectable in the patient on dexamethasone treatment. Because endogenous adrenal cortisol production is absent, circulating cortisol from replacement therapy must have been the substrate for the production of these steroids. In the one adrenalectomized patient, it is likely that they were synthesized from cortisol at extra-adrenal sites (for review, see Ref. 17); in the other four patients, a proportion might also have arisen from perfusion by plasma of residual functional ZG tissue.
In the volunteers, suppressing the adrenal source of cortisol by dexamethasone reduced 18-OHF and 18-oxoF excretion rates. Subsequent administration of hydrocortisone to the volunteers simultaneously with dexamethasone increased excretion rates of both compounds. In this hydrocortisone + dexamethasone phase, 18-oxoF excretion rate exceeded control (untreated) levels, but that of 18-OHF did not achieve control levels. The cortisol status achieved, of which the sum of cortisol and cortisone excretion rates is a convenient index, was higher during cortisol + dexamethasone than during the control (untreated) period. This suggests that, in this extreme situation, the ZG used extra-adrenal cortisol to make 18-oxoF and 18-OHF. 11ß-Hydroxylase production of 18-OHF is probably less important here because extra-adrenal cortisol is less accessible to the ZF than to the ZG. Moreover, 11ß-hydroxylase is less efficient than AS at 18-hydroxylation (see above).
Although the conversion efficiency of cortisol to 18-OHF was low in the transfected cells in vitro, the estimate of Km suggests that, in vivo, high plasma levels of cortisol could contribute to the maintenance of normal levels of 18-OHF. However, two reservations must be emphasized. Firstly, true apparent Km of AS for cortisol cannot be calculated accurately because of the complex nature of the reaction and because no pure enzyme protein is available. The estimate given here is an approximation. Secondly, in plasma, most cortisol is specifically bound to corticosteroid-binding globulin, and only a small fraction is free; availability to AS (and 11ß-hydroxylase) will depend on competing affinities of enzyme and plasma protein.
In human subjects, AS is probably the only source of 18-oxoF. Its excretion rate was significantly increased by exogenous cortisol treatment, suggesting that cortisol reperfusing the ZG is a significant source of this steroid. However, AS and 11ß-hydroxylase are expressed in organs other than the adrenal glands (18, 19, 20, 21), albeit at much lower rates; extra-adrenal production cannot be ruled out. The close correlation between urinary cortisol metabolites and 18-oxoF levels in the volunteer study supports the conclusion that conversion of circulating cortisol is a major source of this steroid. Other studies also support the dependence of 18-oxoF and 18-OHF on cortisol availability. The plasma levels of both compounds correlate with that of cortisol and not aldosterone (9), and, although the renin-angiotensin system may have some influence (22), the major control factor is ACTH (22, 23). Dexamethasone suppressed the levels of both compounds in the current study and in others (24).
In normal subjects, cortisol for 18-OHF or 18-oxoF synthesis may derive either by reperfusion from the circulation or from local tissue production. Cortisol is produced in the ZF, but AS is essential at least to 18-oxoF synthesis. Direct penetration of ZF-synthesized cortisol to the ZG is unlikely because the blood supply of the adrenal gland is centripetal. There are, however, plausible alternative explanations. A transitional anatomical zone was identified between the ZF and ZG in rats (25, 26) but not so far humans. In the human adrenal gland, ZG tissue may penetrate the ZF along the surfaces of small blood vessels, providing a potentially extensive interface for sufficient for cortisol to enter ZG cells. Finally, aldosterone might be converted to 18-oxoF by 17-hydroxylase in the ZF. Early experiments (14) showed that 18-OHF and 18-oxoF are produced in beef adrenal outer slices. The authors suggested that AS and 17-hydroxylase coexist in these outer slices, allowing synthesis of these two steroids, and inferred that 17-hydroxylase could convert aldosterone into 18-oxoF and 18-hydroxycorticosterone into 18-OHF. Interpretation is difficult because in vitro incubation of mixed cell slices destroys the strict compartmentalization of enzymes and substrates maintained in vivo. There is as yet no similar evidence in human tissue.
Thus, cortisol can be converted to 18-OHF in both the ZF and ZG by 11ß-hydroxylase and AS, respectively. Because cortisol synthesis is restricted to the ZF in vivo, it is likely that most 18-OHF in normal subjects is produced by 11ß-hydroxylase. 18-OxoF is a unique product of AS. The source of cortisol in normal subjects may be circulating cortisol or ZF cortisol diffusing across the interphase between ZG and ZF.
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Footnotes
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This work was supported by the Medical Research Council (Program Grant G9317119 to E.D., R.F., and J.M.C.C.) and by the Wellcome Trust (Clinical Training Fellowship to E.M.F.).
Abbreviations: AS, Aldosterone synthase; bd, twice daily; DOC, 11-deoxycorticosterone; 18-OHF, 18-hydroxycortisol; 18-oxoF, 18-oxocortisol; ZF, zona fasciculata; ZG, zona glomerulosa.
Received March 5, 2004.
Accepted May 25, 2004.
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References
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Abstract
Introduction
