Department
of
Pharmacology, University of Heidelberg, Heidelberg, Germany
RADIOIMMUNOASSAYS OF TETRAHYDROCORTISONE AND TETRAHYDROCORTISOL IN HUMAN URINE
By H. Will, R.
Aderjan,
Th. Winkler, B. Penke and P. Vecsei
ABSTRACT
simple radioimmunoassay for the determination of tetrahydrocortisone and tetrahydrocortisol (THF) as well as their 21-glucosiduronate conjugates is described.
A
(THE)1),
White New Zealand rabbits were immunized with THF- and THE-20\x=req-\ oxime-bovine serum-albumin conjugates. The resulting antisera proved to be very specific. Both allo-THE and allo-THF gave minimal cross-reactions; however, THE- and THF-21-glucosiduronate bind with the corresponding antibodies at a level of 100 %. The following types of estimations were introduced: a) Estimation of the sum of unconjugated THF and THF-21-glucosiduronate in highly diluted unprocessed human urine. b) A similar estimation of THE and THF-21-glucosiduronate as in a). c) Estimation of THF after \g=b\-glucuronidase treatment and dilution without further procedures. d) A similar estimation for THE as in c). THF and THE values correlate significantly with known parameters that characterize the cortisol production, such as cortisol secretion rate and the urinary corticoid (C21-\g=a\-ketolic-steroid) levels.
Previously fluorometric and photometric techniques have been used for the determination of tetrahydrocortisol (THF) and tetrahydrocortisone (THE). These methods have a limited sensitivity and include several working steps Requests for reprints should be addressed to Dr. P. Vecsei der Universität, Im Neuenheimer Feld 366, Heidelberg.
Pharmakologisches
Institut
369 Acta endocr. 86, 2
24
and Chromatographie separation (Bush 8c Willoughby 1957; Karl 8c Raith 1961; Starnes et al. 1963, etc.). In clinical pathology other methods with less specific group reactions are routinely used: tetrazolium-blue-reaction (Kingsley 8c Getchell 1961), PorterSilber-reaction (Silber 8c Porter 1954) and ketogenic steroid estimation (Norymberski et al. 1953). These reactions as well as their modifications are summarized by Labhart (1974). Unfortunately, these group reactions are non-specific; they are also given by other cortisol metabolites as well as by metabolites of other steroids, and materials unrelated to steroids (Labhart 1974; Voigt 1975). We have decided to utilize the high specificity of radioimmunoassay technique and have developed a sensitive and practicable radioimmunoassay of THF and THE.
MATERIAL AND METHODS
Tetrahydrocortisol
Steroids.
and
tetrahydrocortisone
Darmstadt, W.-Germany. All other steroids -
Israel. The steroid 3-
or
were
21-glucosiduronates
purchased from Merck, Ikapharm, Ramat-Gan, kindly supplied by Dr. Vernon R. were
obtained from
were
Mattox, Rochester, Minn. Radioactive steroids. [1,2-3H]Tetrahydrocortisol (48.4 Ci/mMole) and [1,2-3H] te¬ trahydrocortisone (53.9 Ci/mMole) were purchased from NEN GmbH, Dreieichenhain, W.-Germany. -
!) List
of abbreviations
THF THE THB THS a-DHF a-DHE
/?-DHF /Î-DHE allo-THF allo-THE allo-THB allo-THS F E B S 18-OH-DOC
jg-Cortol
a-Cortolone
Steroid-3-gluc Steroid-21 -glue
3ct,ll/î,17a,21-tetrahydroxy-5^-pregnan-20-one
3a, 17a,21 -trihydroxy-5y§-pregnane-11,20-dione
3a,ll/J,21-trihydroxy-5/?-pregnan-20-one 3a, 17a,21 -trihy droxy-5/5-pregnan-20-one 11/5,17a,21 -trihydroxy-5a-pregnane-3,20-dione 17a,21-dihydroxy-5a-pregnane-3,ll,20-trione 1 ly?,17a,21-trihydroxy-5/?-pregnane-3,20-dione 17ct,21-dihydroxy-5/9-pregnane-3,ll,20-trione 3a,ll/$,17a,21-tetrahydroxy-5a-pregnan-20-one 3a, 17 a, 21 -trihy droxy-5a-pregnane-11,20-dione 3a, 11/?,21-trihy droxy-5a-pregnan-20-one 3a,17a,21-trihydroxy-5a-pregnan-20-one 11 ß, 17,21 -trihy droxy-4-pregnene-3,20-dione 17,21-dihydroxy-4-pregnene-3,l 1,20-trione 11/?,21 -dihydroxy-4-pregnene-3,20-dione 17a,21-dihydroxy-4-pregnene-3,20-dione 18,21-dihydroxy-4-pregnene-3,20-dione
5,5-pregnane-3a,ll/?,17a,20/3,21-pentol 3a,17a,20a,21-tetrahydroxy-5/5-pregnan-ll-one Steroid-3a-yl-/?,D-glucopyranosiduronate Steroid-21 -yl-/?,D-glucopyranosiduronate 370
was controlled in E2B (2) paper Bongiovanni 1955). Other chemicals. All organic solvents were purchased from Merck, Darmstadt, W.-Germany. These were of analytical grade and used without further purification. Charcoal (Norit A) was obtained from Serva (Heidelberg, W.-Germany), Dextran T 70 from Pharmacia (Uppsala, Sweden), /?-glucuronidase (Ketodase®) from Goedecke (Frei¬ burg, W.-Germany), human y-globulin (Beriglobin) from Behringwerke (Marburg, W.-Germany) and lysozyme from Serva (Heidelberg, W.-Germany). The radioactivity was measured by liquid scintillation counting. The samples were dissolved in 10 ml Bray's scintillation liquid (4 g diphenyloxazol, 200 mg dimethylPOPOP, 60 ng naphtalene, 20 ml ethylcneglycol, 100 ml methanol, made up to 1000 ml with dioxane) and measured in Packard Tri-Carb liquid scintillation spectrometers (Types 3003 and 3375). Urinary total corticoid (Cgj-a-ketolic steroids) excretion was measured using a tetrazolium-blue-method (Kingsley Se Getchell 1961) by Dr. Schmidt, Department of Clinical Chemistry in the Medical Faculty Mannheim of the University of Heidelberg. The cortisol secretion rate was estimated according to the method of Cope 8e Black (1958), slightly modified by our group (Vecsei et al. 1969). Preparation of antigen. THF and THE were incubated with O-(carboxymethyl)hydroxylamine and the resulting 20-oxime derivatives were coupled to bovine serum albumin by a carbodiimide reaction (Erlanger et al. 1957). The approximate saturation rate of the antigen measured with the DNS-C1 method (Weber 1952) was 38-40 steroids/
The purity of the radioactive Chromatographie system (Eberlein
THF and THE
Se
-
-
carrier molecule.
For the immunization 1 mg of the steroid-protein complex was dissolved in 0.5 ml 0.9 % saline solution and mixed with 0.5 ml Freund's adjuvant. Titers were read at 50 % of the radioactivity measured with undiluted antiserum.
of the assay. A calibration curve was made by incubating different THF and THE (0-5000 pg) dissolved in 0.1 ml of 0.05 m borate buffer containing 0.1 % lysozyme, 3H-labelled THF and THE (about 5000 cpm) dissolved in 0.1 ml, and 0.5 ml of the antibody diluted in the same buffer. The incubation time varied between 1 hour and 3 days with the duration of the incubation not altering the results significantly. To separate free from antibody bound radioactivity, 0.1 ml of a charcoal suspension of the following composition was used: 5 g Norit A charcoal, 600 mg Dextran T 70 in 560 ml of 0.05 m borate buffer containing 0.06 % human gamma globulin. After centrifugation the incubation tubes were decanted simultaneously into counting vials. For this purpose a special décantation equipment was used (Vecsei 1974). The urinary steroid measurements were performed in normal subjects, who were not removed from their daily profession. Procedure
amounts of
-
RESULTS
a)
Immunization-antisera
White male New Zealand rabbits were immunized with 20-oxime-BSA and THE-20-oxime-BSA. Booster injections vals of 14-30 days.
of THEmade at inter¬
complexes were
371 24*
The duration of the immunization period varied between 5 and 12 months. The maximum titers obtained in the four rabbits immunized against THE are: 65 000; 90 000; 2600 and 27 000. The same figures for the seven animals immunized against THF are: 450; 700; 3000; 4800; 5500; 10 000 and 27 000. When standard curves were made by plotting bound radioactivity against the logarithm of the steroid dose, 50 pg THE or THF showed a significant difference from zero. Cross-reaction studies show the antisera to be highly specific. The crossreactions of possible interfering steroids with THF and THE antibody are
Table 1. Per cent cross-reaction at 50 % displacement of [1,2-3H]THE or THF.
H
Antisera of rabbit THF THE THB THS a-DHF a-DHE
100.00 0.32 1.5 3.97
0.76 100.00
of
Pharmacology, University of Heidelberg, Heidelberg, Germany
RADIOIMMUNOASSAYS OF TETRAHYDROCORTISONE AND TETRAHYDROCORTISOL IN HUMAN URINE
By H. Will, R.
Aderjan,
Th. Winkler, B. Penke and P. Vecsei
ABSTRACT
simple radioimmunoassay for the determination of tetrahydrocortisone and tetrahydrocortisol (THF) as well as their 21-glucosiduronate conjugates is described.
A
(THE)1),
White New Zealand rabbits were immunized with THF- and THE-20\x=req-\ oxime-bovine serum-albumin conjugates. The resulting antisera proved to be very specific. Both allo-THE and allo-THF gave minimal cross-reactions; however, THE- and THF-21-glucosiduronate bind with the corresponding antibodies at a level of 100 %. The following types of estimations were introduced: a) Estimation of the sum of unconjugated THF and THF-21-glucosiduronate in highly diluted unprocessed human urine. b) A similar estimation of THE and THF-21-glucosiduronate as in a). c) Estimation of THF after \g=b\-glucuronidase treatment and dilution without further procedures. d) A similar estimation for THE as in c). THF and THE values correlate significantly with known parameters that characterize the cortisol production, such as cortisol secretion rate and the urinary corticoid (C21-\g=a\-ketolic-steroid) levels.
Previously fluorometric and photometric techniques have been used for the determination of tetrahydrocortisol (THF) and tetrahydrocortisone (THE). These methods have a limited sensitivity and include several working steps Requests for reprints should be addressed to Dr. P. Vecsei der Universität, Im Neuenheimer Feld 366, Heidelberg.
Pharmakologisches
Institut
369 Acta endocr. 86, 2
24
and Chromatographie separation (Bush 8c Willoughby 1957; Karl 8c Raith 1961; Starnes et al. 1963, etc.). In clinical pathology other methods with less specific group reactions are routinely used: tetrazolium-blue-reaction (Kingsley 8c Getchell 1961), PorterSilber-reaction (Silber 8c Porter 1954) and ketogenic steroid estimation (Norymberski et al. 1953). These reactions as well as their modifications are summarized by Labhart (1974). Unfortunately, these group reactions are non-specific; they are also given by other cortisol metabolites as well as by metabolites of other steroids, and materials unrelated to steroids (Labhart 1974; Voigt 1975). We have decided to utilize the high specificity of radioimmunoassay technique and have developed a sensitive and practicable radioimmunoassay of THF and THE.
MATERIAL AND METHODS
Tetrahydrocortisol
Steroids.
and
tetrahydrocortisone
Darmstadt, W.-Germany. All other steroids -
Israel. The steroid 3-
or
were
21-glucosiduronates
purchased from Merck, Ikapharm, Ramat-Gan, kindly supplied by Dr. Vernon R. were
obtained from
were
Mattox, Rochester, Minn. Radioactive steroids. [1,2-3H]Tetrahydrocortisol (48.4 Ci/mMole) and [1,2-3H] te¬ trahydrocortisone (53.9 Ci/mMole) were purchased from NEN GmbH, Dreieichenhain, W.-Germany. -
!) List
of abbreviations
THF THE THB THS a-DHF a-DHE
/?-DHF /Î-DHE allo-THF allo-THE allo-THB allo-THS F E B S 18-OH-DOC
jg-Cortol
a-Cortolone
Steroid-3-gluc Steroid-21 -glue
3ct,ll/î,17a,21-tetrahydroxy-5^-pregnan-20-one
3a, 17a,21 -trihydroxy-5y§-pregnane-11,20-dione
3a,ll/J,21-trihydroxy-5/?-pregnan-20-one 3a, 17a,21 -trihy droxy-5/5-pregnan-20-one 11/5,17a,21 -trihydroxy-5a-pregnane-3,20-dione 17a,21-dihydroxy-5a-pregnane-3,ll,20-trione 1 ly?,17a,21-trihydroxy-5/?-pregnane-3,20-dione 17ct,21-dihydroxy-5/9-pregnane-3,ll,20-trione 3a,ll/$,17a,21-tetrahydroxy-5a-pregnan-20-one 3a, 17 a, 21 -trihy droxy-5a-pregnane-11,20-dione 3a, 11/?,21-trihy droxy-5a-pregnan-20-one 3a,17a,21-trihydroxy-5a-pregnan-20-one 11 ß, 17,21 -trihy droxy-4-pregnene-3,20-dione 17,21-dihydroxy-4-pregnene-3,l 1,20-trione 11/?,21 -dihydroxy-4-pregnene-3,20-dione 17a,21-dihydroxy-4-pregnene-3,20-dione 18,21-dihydroxy-4-pregnene-3,20-dione
5,5-pregnane-3a,ll/?,17a,20/3,21-pentol 3a,17a,20a,21-tetrahydroxy-5/5-pregnan-ll-one Steroid-3a-yl-/?,D-glucopyranosiduronate Steroid-21 -yl-/?,D-glucopyranosiduronate 370
was controlled in E2B (2) paper Bongiovanni 1955). Other chemicals. All organic solvents were purchased from Merck, Darmstadt, W.-Germany. These were of analytical grade and used without further purification. Charcoal (Norit A) was obtained from Serva (Heidelberg, W.-Germany), Dextran T 70 from Pharmacia (Uppsala, Sweden), /?-glucuronidase (Ketodase®) from Goedecke (Frei¬ burg, W.-Germany), human y-globulin (Beriglobin) from Behringwerke (Marburg, W.-Germany) and lysozyme from Serva (Heidelberg, W.-Germany). The radioactivity was measured by liquid scintillation counting. The samples were dissolved in 10 ml Bray's scintillation liquid (4 g diphenyloxazol, 200 mg dimethylPOPOP, 60 ng naphtalene, 20 ml ethylcneglycol, 100 ml methanol, made up to 1000 ml with dioxane) and measured in Packard Tri-Carb liquid scintillation spectrometers (Types 3003 and 3375). Urinary total corticoid (Cgj-a-ketolic steroids) excretion was measured using a tetrazolium-blue-method (Kingsley Se Getchell 1961) by Dr. Schmidt, Department of Clinical Chemistry in the Medical Faculty Mannheim of the University of Heidelberg. The cortisol secretion rate was estimated according to the method of Cope 8e Black (1958), slightly modified by our group (Vecsei et al. 1969). Preparation of antigen. THF and THE were incubated with O-(carboxymethyl)hydroxylamine and the resulting 20-oxime derivatives were coupled to bovine serum albumin by a carbodiimide reaction (Erlanger et al. 1957). The approximate saturation rate of the antigen measured with the DNS-C1 method (Weber 1952) was 38-40 steroids/
The purity of the radioactive Chromatographie system (Eberlein
THF and THE
Se
-
-
carrier molecule.
For the immunization 1 mg of the steroid-protein complex was dissolved in 0.5 ml 0.9 % saline solution and mixed with 0.5 ml Freund's adjuvant. Titers were read at 50 % of the radioactivity measured with undiluted antiserum.
of the assay. A calibration curve was made by incubating different THF and THE (0-5000 pg) dissolved in 0.1 ml of 0.05 m borate buffer containing 0.1 % lysozyme, 3H-labelled THF and THE (about 5000 cpm) dissolved in 0.1 ml, and 0.5 ml of the antibody diluted in the same buffer. The incubation time varied between 1 hour and 3 days with the duration of the incubation not altering the results significantly. To separate free from antibody bound radioactivity, 0.1 ml of a charcoal suspension of the following composition was used: 5 g Norit A charcoal, 600 mg Dextran T 70 in 560 ml of 0.05 m borate buffer containing 0.06 % human gamma globulin. After centrifugation the incubation tubes were decanted simultaneously into counting vials. For this purpose a special décantation equipment was used (Vecsei 1974). The urinary steroid measurements were performed in normal subjects, who were not removed from their daily profession. Procedure
amounts of
-
RESULTS
a)
Immunization-antisera
White male New Zealand rabbits were immunized with 20-oxime-BSA and THE-20-oxime-BSA. Booster injections vals of 14-30 days.
of THEmade at inter¬
complexes were
371 24*
The duration of the immunization period varied between 5 and 12 months. The maximum titers obtained in the four rabbits immunized against THE are: 65 000; 90 000; 2600 and 27 000. The same figures for the seven animals immunized against THF are: 450; 700; 3000; 4800; 5500; 10 000 and 27 000. When standard curves were made by plotting bound radioactivity against the logarithm of the steroid dose, 50 pg THE or THF showed a significant difference from zero. Cross-reaction studies show the antisera to be highly specific. The crossreactions of possible interfering steroids with THF and THE antibody are
Table 1. Per cent cross-reaction at 50 % displacement of [1,2-3H]THE or THF.
H
Antisera of rabbit THF THE THB THS a-DHF a-DHE
100.00 0.32 1.5 3.97
0.76 100.00
