Radioimmunoassay of plasma and urine 6β-hydroxycortisol: Levels in healthy adults and in hypercortisolemic states

1. steroid Biocheilt.Vol. 17,pp. 343 to 350, 1982 Printed in Great Britain. All rights reserved

0022-4731/82/~0343-08~3.~/0 Copyright 0 1982Pergamon Press Ltd

RA~IOIMMUNOASSAY OF PLASMA AND URINE BP-HYDROXYCORTISOL: LEVELS IN HEALTHY ADULTS AND IN HYPERCORTISOLEMIC STATES K. NAHOUL, J. ADELINE, F. PAYSANTand R. SCHOLLER Fondation de Recherche en Hormonologie, 67, boulevard Pasteur, 94260 Fresnes, and 26, boulevard Brune. 75014 Paris, France (Receiaed 3 September 1981) SUMMARY

A reliable radioimmuno~say for plasma and urine 6~-hydroxycortisol after chromatography on Sephadex LH 20 column has been described and evaluated. The antiserum used was raised in the rabbit injected with 6a-hydroxycortisol-3(0-carboxymethyl)oxime-bovine serum albumin. In control subjects, urine b/%hydroxycortisol levels ranged from 137 to 348 pg/24 h (mean + SD: 238 + 66) in adult males (n = 14) and from 80 to 432 pg/24 h (mean k SD: 210 f 93) in adult females (n = 15). In plasma, 6~-hydroxycortisol levels ranged from 0.53 to 3.13 ng/ml (mean & SD: 1.14 f 0.57) in adult males (n = 17) and from 0.53 to 2.69ng/ml (mean + SD: 1.22 & 0.53) in adult females (n = 19). In patients with Gushing’s syndrome high levels were found in urine as well as in plasma. Finally the higher concentrations of 6p-hydroxycortisol found in adrenal effluent in comparison with those of the peripheral vein blood has clearly demonstrated that 6fi-hydroxycortisol is secreted by the adrenal besides its extra-adrenal formation from cortisol.

INTRODUCTION

Many drugs have been shown to increase the hepatic turnover of cortisol by enhancing the activity of 68hydroxylase so that a greater proportion of cortisol is converted to 6~-hydroxy~ortisol (6/&OHF) [i-3]. In the absence of a defect in cortisol biosynthesis plasma cortisol levels remain within the normal range because of an increase in cortisol production rate [4,5]. Similarly, patients with Addison’s disease require an increase in their replacement dose of hydrocortisone [6,7]. Measurement of plasma and/or urine 6/j’-OHF appears therefore to be a reliable index for monitoring the effects of drugs acting as inducers of hepatic microsomai enzymes. Many techniques have been described for 6/3-OHF determination in urine but as the calorimetric or fluorometric reactions used were not specific enough, Cor~spondencu: K. Nahoul, Fondation de Recherche en Hormonologie, 67, boulevard Pasteur, 94260 Fresnes, France. The following trivial names have been used: Aldosterone: l&l I-Hemiacetal of 11~,2l-dihydroxy-3,2~dioxo-~ pregnen-18-a]; Allo-tetrahydrocortisol: 3a,lI~,l7,21-Tetrahydroxy-5a-pregnan-20-one; Corticosterone: llP,21-Dihydroxy-4-pregnene-3.20-dione; Cortisol: 1lg.1 7,21-Trihydroxy-4-pregnene-3,20-dione; Cortisone: 17,2l-Dihydroxy-4-pregnene-3,l I.%O-trione; Deoxycorticosterone: 21Hydroxy-4-pregnene-3,20-dione; 1I-Deoxycortisol: 17,21Dihydroxy-4-pregnene-3,20-dione; (ic+Hydroxycortisol: 61, Il~,17,21-Tetrahydroxy-4-pregnene-3,20-dione; 6fi-Hydroxycortisol: 68,l I p,l7,21-Tetrahydroxy-4-pregnene-3,20dione; Tetrahydr~ortisol: 3n,l l~,l7,21-Tetrahydroxy-5~pregnan-20-one; Tetrahydrocortisone: 3q17,21WTrihydroxy5/l-pregnane-3,20-dione. 343

extraction and multiple chromatographic steps were needed [8-20). Gas-liquid [Zl-231 and high pressure liquid chromatography [24] have also been used. Recently, specific antisera anti-6/?-OHF have been prepared [25,26] so that urine 6fi-OHF could be determined either just after [26] or even without extraction [27]. However, preliminary chromatographic purification of plasma extracts was still necessary and this was achieved by celite column chromatography [26]. This kind of chromatography has many pitfalls such as the activation of celite, the preparation and elution of the columns and more particularly the existence of frequently high water blanks. No such drawbacks are generally observed with Sephadex LH 20 [ZS] and this paper describes a rapid radioimmunoassay for plasma and urine (S/3-OHF determination including one purification step, Sephadex LH 20 column chromatography. While this work was in progress, a paper appeared using Sephadex LH 20 column chromatography before competitive protein binding assay [29].

MATERIALS

Chemicals All chemicals were of analytical grade and were used without further purification. Sephadex LH-20 was purchased from Pharmacia. The steroids used were obtained from Steraloids and tritiated 6/l-OHF with specific activity of 42 Ci/mmoI from The Radiochemical Centre, Amersham. Regular purification of this labeled compound was performed on column

K.

344 chromatography of Sephadex mixture described below.

LH-20

et al.

NAHOUL

in the solvent

Preparation of the antiserum The hapten was synthesized according to Park et a/.[251 except that the acetate group at C-21 was not removed. The 3 (0-carboxymethyl) oxime derivative was coupled to bovine serum albumin via the mixed anhydride method [30]. The antigen thus obtained was injected with Freund’s adjuvant to four rabbits (fauve de Bourgogne), with a monthly booster injection. The animals were bled once a month after the first booster injection. Blood samples were centrifuged and the plasma assayed for antibody titer and specificity at different dilutions in phosphate buffer pH 6.8 [31]. The third bleeding gave the highest antibody titer and it was used at the 1: 1,000 dilution.

Radioimmunoassay The standard curve was prepared by pipetting in test tubes duplicate aliquots of pure 6/I-OHF solution in ethanol (1 ng or 10 ng per ml) containing from 10 to 8OOpg. After evaporation to dryness, approximately 5000 c.p.m. of tritiated 6/I-OHF and 0.2 ml of diluted antiserum (l/1000) were added. The tubes were mixed, incubated for 30min in a water-bath at 37°C and then kept at 4°C for 30min. Separation of free and bound 6/J-OHF was performed by the addition of 0.5 ml of cold dextran-coated charcoal (0.5% w/v Norit A activated charcoal and 0.05% w/v dextran). Following quick mixing the tubes were incubated for 15 min in an ice water bath then centrifuged at 4°C for 20 min at 3000 rev./min; 0.5 ml of the supernatant was taken and added to 10 ml of the scintillating mixture and counted.

Preparation of Sephadex LH-20 columns Twenty grams of Sephadex LH-20 were suspended in 200 ml of the mixture methylene chloride-methanol (95:5, v/v) and left to swell overnight. Aliquots of this suspension were poured into 5 ml disposable pipettes (5 mm internal diameter; Volac) obturated with a piece of glass wool and the Sephadex LH-20 left to settle to a height of 168 mm. After prewashing the columns with 6ml of the solvent mixture they were ready for use. Dextran-coated charcoal Norit A activated charcoal 0.5 g and dextran 0.05 g were mixed with 100 ml of phosphate buffer pH 6.8. METHODS

Extraction of plasma and urine

Standard curve For each concentration of the non-radioactive standard plotted on the abscissa the percent B/B, was calculated and plotted on the ordinate scale. A representative standard curve is shown in Fig. 1. The working range was from 30 to 250 pg. The reproducibility was studied by calculating the coefficient of variation (Cv) of the results obtained on 14 different days for each amount of the standard. It was found that the C’V never exceeded 5.5%. Sensitivity and blank value The sensitivity of the assay was estimated to be 100 pg/ml and blank value was generally similar to B,, and when it could be distinguished from the BO, it never exceeded the detection limit.

To 0.5 ml of plasma or 5 or 10 ~1 of 24-h urine were added approximately 1000 c.p.m. of tritiated 6/?-OHF. Each sample was then completed to 0.5 ml with bidistilled water. Water blanks were similarly prepared with each series. Extraction was carried out by agitation with 2 x 3 ml of ethyl acetate on a vortex mixer. The tubes were then allowed to stand at room temperature for 15 min and the two phases were separated by quick freezing of the aqueous phase. The ethyl acetate extract was transferred to a clean test tube and evaporated to dryness at 40°C under a stream of nitrogen. Chromatography The dried extracts were redissolved with 2 x 0.1 ml of methylene chloride-ethanol (95: 5, v/v) and applied on the Sephadex LH-20 columns. After a 11 ml washing with the same solvent mixture, 6/?-OHF was eluted with the next 5 ml. The eluates were evaporated to dryness at 40°C under a stream of nitrogen and the residues dissolved with 2ml of ethanol. Duplicate 0.2 ml aliquots were pipetted for radioimmunoassay and 1 ml aliquot for the estimation of the recovery.

1I

10

10

25

so

100

800 200 100 6p-Hydroxycortirol (pg per tube)

Fig. 1. Standard curve. Each point represents the mean of 14 determinations run on different days and the vertical bars, the standard deviation

.

6~-Hydroxycortisol

radioimmunoassay

345

Table 1. Evaluation of the precision Inter-assay variability

Intra-assay variability n

mean

SD

900

-

700

-

-(2.47+-

) x 14.1

1

CVl%)

7

1.60

0.11

6.9

309

11.80

3.8

15

319

23.00

7.3

6-@OHF

y-(1962.7i:42.6

SD

6.8

PQ

t

mean

0.09

The mean recovery of tritiated 6/J-OHF added to plasma or urine samples was 83.7 &- 1.3% (SD) (range: 81.3-85.0; n = 12) and 85.8 + 1.0% (SD) (range: 84.8-87.7; n = 12) respectively. Accuracy was also assessed by adding increasing quantities of non-radioactive 6fi-OHF to ahquots of pooled plasma and urine obtained from dexamethasone-treated patients and containing non-detectable amounts and 3.93 J- 0.23 ng/ml respectively. For each quantity (varying from 0.5 to 4.0ng/ml for plasma and from 50 to ~ng/ml for urine) determinations were performed in triplicate. The calculated regression lines for plasma and urine were represented by: y = (0.005 f 0.030) + (0.979 + 0.014) x and y = (I .472 + 2.400) f (0.999 _t 0.012) x respectively. The slopes were not significantly different from 1 indicating no systematic error and the intercepts with the ordinate axis were not significantly different from the basal levels measured in these two pools. Besides the same study was also performed on aliquots of a charcoal-treated plasma pool where no 6/j’-OHF could be detected. The regression line was found to be represented by the equation: y = -(0.006 f 0.023) + (1.010 + 0.010) x. This experiment led to the same conclusion as that drawn from the two preceding experiments.

“O”

n

1.47

AtCWlXg

t

CV(%)

/ l

These conclusions were confirmed by the results obtained by the determination in triplicate of the concentration of 6/J-OH in aliquots of different volumes of the same plasma. The regression lines obtained for three different plasmas are shown in Fig. 2. The intercepts with the ordinate axis of these three regression lines were not significantly different from 0. Precision Intra- and interassay variability was evaluated by the coefficient of variation (Cv) of the results obtained on repeated determinations of 6fi-OHF in the same pool of plasma or urine either in the same series or different series of assays. As shown on Table 1 the method proved to be highly reproducible for plasma as well as for urine. Speci$city The specificity of the technique was achieved by the combination of column chromatography with radioimmunoassay. In fact, chromatography on Sephadex LH 20 column resulted in a good separation of 6b-OHF from other non-conjugated corticosteroids known to be present in human plasma or urine namely cortisol, corticosterone, cortisone, 1 I-deoxycortisol, deoxycorticosterone and aldosterone. The elution profile of the 6u-isomer could not, however, be studied since the tritiated derivative was not available. The specificity of the antiserum was tested with the steroids listed in Table 2. The only significant crossreaction was demonstrated with cortisol but this steroid was not liable to interfere with 6/?-OHF determination since it was not eluted in the same fraction from the Sephadex LH 20 column. Table 2. Specificity of the antiserum

tUSStl5.6)

Steroids

0

I

0.3

0.5

0.7

ml

Fig. 2. Effect of plasma dilution on the measured levels of 6/?-hydroxycortisol.

6/J-Hydroxycortisol Cortisol 11-Deoxycortisol Ailo-tetrahydrocortisol Cortisone Aldosterone Corticosterone Deoxycorticosterone Tetrahydrocortisol Tetrahydrocortisone

Cross-reactivity (%I 100 17.1 3.5 0.3 0.8 to.1
SP SP GLC BT UV RIA RIA HPLC F RIA

Poland et al.[32] Berman and Green[lB] Chamberlain[22] Smith and Rawlins[33] Hildebrandt er al.[19] Kishida and Fukushima[26] Park[27] Roots et a/.[241 Pal[20] Present me&hod

8 14 10 27 8 4 4 4 18 13 10 10* 19 6* 10 12 100 14

N

** SP: Silber and Porter reaction; UV: Ultra-violet absorption; F: Fluorometry; HPLC: High Performance Liquid Chromatography. * Sex not stated; $ t SEM.

BT uv SP SP

Detection**

Frantz et a1.[8] Katz et a!.[ 1I] Werk et ~I.[131 Berthold and Staudinger[14] Thrasher et al.[15] Yamaji et ai.[l6]

Authors

316-726 216333 143-421 289-457 137-348

41-355 4*1160 78-402 124450

309510 156378 15c-840 344779 100-800

Range

reaction: GLC: Gas-liquid

436 298 rt 286 400 + 200 525 k 123 350 & 200 346 2 115 315 + 149 430 k 135 450 + 60 185 f 17 490 rf: 240 255 245 & 108 445 * 79.9 487 & 144.8 286 + 54 273 + 21.69 377 * 60 238 + 66

Mean rt: SD

BT: Blue tetrazolium

2440 18-62 20-40

2040 2t332

23-75 20-50 1569 20-29 30-39 40-49 5&59 40.2 f 1.9 21-24

Age

Men

Table 3. Urine 6/&hydroxycortisol @g/24 h) in healthy subjects

2(r60 2&40

20-50 15-68 2&29 3&39 4@49 5&59

Age

Chromatography:

100 15

8

10 10 7 5 4 5

8

N

& 156 + 110 f 143 It 141 &-215 t 164

298-565 80-432

113-340

300-836 105420

181-760 246555

Range

RIA: Radioimmunoassay;

417 + 81 210 f 93

233 k 80

534 260 517 416 490 452

378 399 k 36%

Mean ) SD

Women

6/I-Hydroxycortisol radioimmunoassay

347

Table 4. Plasma 6/Lhydroxycortisol levels (ng/ml) in healthy subjects N

Range

Mean + SD

4* 17 M** 19F

4.01-5.40 0.53-3.13 0.53-2.69

4.69 k 0.58 1.14 & 0.57 1.22 * 0.53

Authors Kishida and Fukushima[26] Present method

* Sex not stated; M** = males; F = females. Table 5. Urine 6p-hydroxycortisol and cortisol levels (pg/24 h) in Cushing’s syndrome 6b-OHF Females (n = 6)

Mean Range

Females with o,p’-DDD

GA. BO. JA.

Males (n = 8)

Mean Range

Male with ectopic ACTHproducing tumor

Determination

of plasma and urine cortisol

Plasma or urine cortisol levels were determined by radioimmunoassay after preliminary extraction with methylene chloride and chromatography on Sephadex LH-20 column (168 x 5 mm) in the solvent system methylene chloride:methanol (98:2) [28]. The antiserum used was raised in rabbits injected with cortisol-3 (0-carboxymethyl) oxime-BSA. It cross-reacted at 65% with 1 I-deoxycortisol, 7% with 21-deoxycortisol and at 0.1% with corticosterone and deoxycorticosterone. Separation of the free and bound cortisol was carried out by extracting the free fraction with chloroform. The bound fraction in the aqueous phase was counted. Mean overall recovery of labeled cortisol was 74.9% (range: 71.9-80.3; n = 10) for plasma and 76.7% (range: 74.9-78.5; n = 10) for urine. At the level of 134 ng/ml of plasma intra- and inter-assay variability were 5.4 and 9.87; respectively.

RESULTS Normul

levels

Blood samples and 24-h urine were collected from healthy volunteer subjects ranging in age from 20 to 40 years and without any apparent endocrine, hepatic or renal disorder. The mean and the range of urine and plasma 6&OHF levels obtained in these healthy subjects are reported in Tables 3 and 4 respectively. The literature data are also shown in these tables. In these urine samples cortisol levels ranged from 22 to 49 yg/24 h with a mean of 33 + 8 pg/24 h (*SD) in men (n = 14) and from 17 to 54 pg/24 h with a mean of 35 & 12 pg/24 h in women (n = 14).

1261 + 1093 407-2987

Cortisol 207 & 85 111-338

15,540 2088 2354

1212 133 54

1068 i 674 311-2221

262 _t 148 65463

82,477

35,700

The mean ratio of 6fl-OHF to cortisol was 6.77 f 3.44 in women (range: 1.86-13.76) and 7.32 + 1.73 in men (range: 4.15-10.33). Mean plasma cortisol was 135 k 37 ng/ml in men (range: 74187; n = 17) and 139 f 43 ng/ml in women (range: 7Ck228; n = 19). No statistically significant difference between males and females could be demonstrated for either plasma or urine 6fl-OHF and cortisol levels. In addition, 24-h urine collections were obtained from 8 normal pregnant women in the course of the third trimester. The levels of 6/J-OHF ranged from 253 to 926~g/24 h with a mean of 671.8 k 214.0 pg/24 h. The difference with non-pregnant women was highly significant (P < 0.001). Levels in Cushing’s syndrome Eighteen patients with Cushing’s syndrome (9 males and 9 females) were studied. Three of the female patients were treated with op’-DDD and one male patient had an ectopic ACTH-producing tumor. The urine and plasma 6/j’-OHF and cortisol levels observed in these patients are shown on Tables 5 and 6 respectively. Comparison with the control group of the same sex demonstrated that urine levels were significantly higher at P -=I0.001 for 6/?-OHF as well as for cortisol in males and at P < 0.001 for cortisol and at 0.001 < P < 0.01 for 6/?-OHF in females. Except in one female patient, the ratio of 6/I-OHF to cortisol was generally lower than in the control group, ranging from 2.6 to 6.7 in females and from 1.8 to 7.9 in males. This difference was statistically significant in males (P < 0.01) but this was not the case in females. Concerning plasma levels, they were, except in one

K. NAHOUL era/.

348

Table 6. Plasma 6/?-hydroxycortisol and cortisol levels (ng/ml) in Cushing’s syndrome S-9a.m.

Patient 6/I-OHF Cortisoi

205

DO.(F)

6/I-OHF Cortisol

6.3 290

GA.(F)*

6/?-OHF Cortisol

280

HA. (M)

6/I-OHF Cortisol

1.2 177

NI. (M)

f$-OHF Cortisol

3.4 191

CA. (M)**

6&OHF Cortisoi

BA. (M)

2p.m.

7p.m.

1lp.m.

215

4.7 206

4.8 223

2.8 192

4.7 226

3.4 213

5.2

-

7.0

47 1287

* o,p’-DDD therapy; ing tumor.

-

6.8 1323

** With ectopic ACTH-produc-

patient, above the range observed in the control group and no circadian variations could be demonstrated. It should be noted that the patient with the ACTH-producing tumor had the highest circulating 6b-OHF level and this is consistent with what has been found in urine (Table 6). 6~-~ydroxycortisol

levels in adrenal e#?uenr

In five patients who underwent selective venous catheterization for the evaluation of their disease (Table 7) the determination of 6/I-OHF in the adrenal and in a peripheral vein blood yielded the results reported in Table 7. The ratio of the level observed in the adrenal effluent to that of the peripheral vein blood demonstrated that the adrenal secreted a sig nificant amount of this steroid.

DISCUSSION

The method described is sensitive, reliable and simple. Chromatography on Sephadex LH-20 columns has many advantages as outlined by Murphy[28]. More particularly it is easy to carry out, highly reproducible and the elution pattern not temperature dependent. Besides, columns may be reused Table 7. Concentrations of 6jShydroxycortisol (ng/ml) in adrenal (A.V.) and peripheral (P.V.) vein blood Patient (sex and age)

Diagnosis

GA. (F.22)

Hirsutism

DE. (M.20) BE. (M.34) LO. (M.44)

Hypertension Gyn~omastia Gynecomastia

* L = left; R = right;

A.V.

P.V.

48 L* 45 R 23 L 36L 14L 7.6 R

1.2

** Inferior vena cava.

2.3*” 1.8 1.4

provided they are well washed and stored thoroughly immersed in the solvent mixture at 4°C. Moreover, no special equipment is required to perform the whole technique so that it may be applied very easily in routine clinical assays. However, in spite of all the tests performed, specificity might be qu~tionable in regard to 6a-OHF interference which could not be studied as this steroid was not commercially available. This interference can however be assumed to be minimal since tic+OHF has been shown to be present in small amounts in urine at a level approximating 510% of that of 6P-OHF [P]. The urine 6P-OHF levels observed in healthy adult subjects were compared to other authors’ data (Table 3). They were found to be lower than those obtained by non-specific methods and very similar to GLC and HPLC data. Concerning radioimmunoassay data, our results agree with those of Park[27] but they are at variance with those of Kishida and Fukushima[Z6]. As no chromatography was included in these two techniques, the difference observed might be accounted for by the small number of subjects studied or by the difference in the specificity of the antiserum. Concerning plasma levels, they could only be compared with those of Kishida and Fukushima[26] and here again our data were lower than theirs. This variance does not seem, however, to be related to methodology since these authors have submitted their plasma extracts to celite column chromatography before radioimmunoassay. Sex difference in urine 6P-OHF excretion has been reported [ll, 12,161 and it was claimed that it was related to estrogens since they were shown to enhance cortisol 6/I-hydroxylation in uiuo [l l] as well as in vitro [34]. Such difference was not, however, observed by most of the authors and our results did not demonstrate any sex difference. No explanation for such discrepancy could be found. In hypercorticism, high urine 6&OHF levels has been reported [9, 10,26,35,36-J and our findings are in agreement with these observations although they are much lower than the data of Yoccia et a1.[36]. This increase in 6p-OHF output was however less im~rtant than that of cortisoi so that the ratio of 6P-OHF to cortisol was, except in one patient, lower than that of the control group. This finding is at variance with the data reported by Voccia et ai.[36] who observed an important increase of this ratio in Cushing’s syndrome. Since these authors have used the direct technique of Kishida and Fukushima[26] which yields higher values in normal subjects in comparison with either our own results obtained after Sephadex LH 20 column chromatography or those reported by Park[27] and Roots et aI.[24], it might be assumed that this discrepancy is related to the methodoIogy used. Indeed, the decrease in this ratio in hypercorticism is consistent with the results of Ghosh and Pennington[35] who demonstrated that, in spite of high 6/I-OHF excretion in Cushing’s syndrome, the percentage of urine 6/I-OHF to cortisol production rate

6B-Hydroxycortisol radioimmunoassay was diminished in comparison with normal subjects. Conversely the excretion of another metabolite namely the 20-dihydro derivative of 6/?-OHF was increased. On the other hand, in the female patients under o,p’-DDD, the ratio was elevated and this is in agreement with the fact that this drug is a hepatic microsomal enzyme-inducer [37,38]. The source of urine 6/3-OHF has appeared to be entirely extra-adrenal in view of the initial studies of Katz et al.[ll] and Werk et aI.[13]. However, following administration of labeled cortisol the specific activity of urine 6/SOHF has been shown to be lower than that of tetrahydrocortisol and tetrahydrocortisane [15,39] and this difference was no more demonstratable after total adrenalectomy [15]. Moreover, a 6P-hydroxylase activity was found in the adrenal cortex [34,40]. These findings suggested a direct adrenocortical secretion of 6&OHF in addition to its peripheral formation from cortisoi. Our results demonstrating, for the first time, higher concentrations of 6fi-OHF in adrenal effluent than in the peripheral plasma are consistent with adrenal secretion of this steroid. REFERENCES 1.

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