283
PLASMA 17~ HYDROXYPROGESTERONE: AN IMPROVED RADIOASSAY METHOD S.Y. TAN and B.E.P. MURPHY Department of Internal Medicine, Yale University, New Haven, Conn. and Dept. of Experimental Medicine, McGill University, Montreal, Canada Received: 11/11/74 Most of the published methods for the measurement of 17~ hydroxyprogesterone (17-OHP) in human plasma are relatively time-consuming and insensitive (1-4).
This paper describes a rapid (8 hours) and sen-
sitive (capable of detecting 50 pg 17-OHP) competitive-protein-binding radioassay method using dog transcortin and Sephadex LH-20, and is modified after the original report of Murphy (5). MATERIALS AND METHODS 3H-labelled steroids (specific activity 50-85 Ci/mMole, New England Nuclear Corp.) were purified on a Sephadex LH-20 column before use. Unlabelled 17-OHP was purchased from Sigma Chemical Company and Sephadex LH-20 from Pharmacia Fine Chemicals. Plasma from dexamethasone treated dogs (4 mg intramuscularly 1-2 days prior to venesection) was used as the source of the binding protein, transcortin. The protein-tracer solution (PTS) was made up by adding i ml dog plasma to i00 ml 0.i M phosphate buffer, pH 7.4, containing 0.5 ~Ci of 3H-corticosterone. Dextran-coated charcoal suspension was prepared by adding 25 mg dextran T-70 and 250 mg Norit-A charcoal to i00 ml phosphate buffer. All solvents were distilled before use. Plasma samples to be assayed (containing 2,000 cpm 3H-17-OHP for recovery) were extracted with 2 x 3 volumes ethyl acetate, and the dried organic extract chromatographed on a 39 x 0.9 cm Sephadex LH-20 column using the solvent system n-heptane : chloroform, 50:50. 17-OHP elutes between 34-51 ml. The assay procedure consisted of adding 0.5 ml PTS to 17-OHP standards (0, 50, 100, 200, 300 and 400 pg) in duplicates and to the unknown samples. The assay tubes (12 x 75 mm, Becton-Dickinson, Rutherford, New Jersey) were vortexed, incubated at 45°C for I0 minutes, followed by incubation at 4°C for 1/2 hour. Dextran-coated charcoal suspension, 0.5 ml, was then added, followed by inmlediate centrifugation for i0 minutes at 2,000 rpm at 4°C. The supernatant was then completely decanted into counting vials containing i0 ml Aquasol. RESULTS AND DISCUSSION In repeated assay curves, the binding when 50 pg 17-OHP was added was always outside 2 S.D. of the binding at zero, so that the assay is considered to have a sensitivity of less than 50 pg.
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2
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Februaru,
1975
284
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zero averaged 60-65%, and a 50% change in binding occurred in the presence of 200 pg 17-OHP. The intra and interassay coefficients of variation were 5.2% (mean 1040 pg, S.D. 55pg) and 17.7% (mean 970pg, S.D.170 pg) respectively. Water and solvent blanks, and plasma(l ml) from a patient with panhypopituitarism on cortisone and testosterone replacement gave negligible readings for 17-OHP. Competing steroids were removed by the chromatigraphy step as described elsewhere (6). The mean recovery of 17-OHP was 79~i0% (S.D.). Accuracy was determined by the addition of 500 and lO00pg to a plasma pool. After correction for recovery and endogenous 17-OHP concentration (830pg/ml), the values obtained were 430~15 and i080~15 pg (S.E.) respectively. Plasma samples from 29 human volunteers gave readings ranging from 250 to 2880 pg/ml. Six patients with congenital adrenal hyperplasia had markedly elevated 17-OHP ranging from 99 to 337 ng/ml with a mean of 194~40 ng/ml. The method described above is rapid, sensitive and reliable. Only O.01 to 0.5 ml plasma and a single rapid chromatography step are required. The use of dog plasma as an easily available source of binding protein obviates the need for the preparation of specific anti-17-OHP antibodies required in a radioimmunoassay. Values obtained using this method agree well with those published by others (1-4), further validating the procedure. The expert technical assistance of M. Radcliffe and A.Tan is acknowledged. REFERENCES 1. Strott, C.A. and Lipsett, M.B. J. Clin. Endocr. 28:1426, 1968. 2. Stone, S. et al. Steroids 18:161, 1971. 3. Barnes, N.D. and Atherden, S.A. Arch. Dis. Child. 47:62, 1972. 4. Abraham, G.E. et al. J. Clin. Endocr. 33:42, 1971. 5. Murphy B.E.P. J. Clin. Endocr. 27:973, 1971. 6. Tan, S.Y. and Mulrow, P.J. Steroids, in press, 1974.
PLASMA 17~ HYDROXYPROGESTERONE: AN IMPROVED RADIOASSAY METHOD S.Y. TAN and B.E.P. MURPHY Department of Internal Medicine, Yale University, New Haven, Conn. and Dept. of Experimental Medicine, McGill University, Montreal, Canada Received: 11/11/74 Most of the published methods for the measurement of 17~ hydroxyprogesterone (17-OHP) in human plasma are relatively time-consuming and insensitive (1-4).
This paper describes a rapid (8 hours) and sen-
sitive (capable of detecting 50 pg 17-OHP) competitive-protein-binding radioassay method using dog transcortin and Sephadex LH-20, and is modified after the original report of Murphy (5). MATERIALS AND METHODS 3H-labelled steroids (specific activity 50-85 Ci/mMole, New England Nuclear Corp.) were purified on a Sephadex LH-20 column before use. Unlabelled 17-OHP was purchased from Sigma Chemical Company and Sephadex LH-20 from Pharmacia Fine Chemicals. Plasma from dexamethasone treated dogs (4 mg intramuscularly 1-2 days prior to venesection) was used as the source of the binding protein, transcortin. The protein-tracer solution (PTS) was made up by adding i ml dog plasma to i00 ml 0.i M phosphate buffer, pH 7.4, containing 0.5 ~Ci of 3H-corticosterone. Dextran-coated charcoal suspension was prepared by adding 25 mg dextran T-70 and 250 mg Norit-A charcoal to i00 ml phosphate buffer. All solvents were distilled before use. Plasma samples to be assayed (containing 2,000 cpm 3H-17-OHP for recovery) were extracted with 2 x 3 volumes ethyl acetate, and the dried organic extract chromatographed on a 39 x 0.9 cm Sephadex LH-20 column using the solvent system n-heptane : chloroform, 50:50. 17-OHP elutes between 34-51 ml. The assay procedure consisted of adding 0.5 ml PTS to 17-OHP standards (0, 50, 100, 200, 300 and 400 pg) in duplicates and to the unknown samples. The assay tubes (12 x 75 mm, Becton-Dickinson, Rutherford, New Jersey) were vortexed, incubated at 45°C for I0 minutes, followed by incubation at 4°C for 1/2 hour. Dextran-coated charcoal suspension, 0.5 ml, was then added, followed by inmlediate centrifugation for i0 minutes at 2,000 rpm at 4°C. The supernatant was then completely decanted into counting vials containing i0 ml Aquasol. RESULTS AND DISCUSSION In repeated assay curves, the binding when 50 pg 17-OHP was added was always outside 2 S.D. of the binding at zero, so that the assay is considered to have a sensitivity of less than 50 pg.
Volume
25, N u m b e r
2
S
T
~
~
o
i D
~
Binding at
Februaru,
1975
284
~
"x" ~ '
]B' o
x
I~
~m
zero averaged 60-65%, and a 50% change in binding occurred in the presence of 200 pg 17-OHP. The intra and interassay coefficients of variation were 5.2% (mean 1040 pg, S.D. 55pg) and 17.7% (mean 970pg, S.D.170 pg) respectively. Water and solvent blanks, and plasma(l ml) from a patient with panhypopituitarism on cortisone and testosterone replacement gave negligible readings for 17-OHP. Competing steroids were removed by the chromatigraphy step as described elsewhere (6). The mean recovery of 17-OHP was 79~i0% (S.D.). Accuracy was determined by the addition of 500 and lO00pg to a plasma pool. After correction for recovery and endogenous 17-OHP concentration (830pg/ml), the values obtained were 430~15 and i080~15 pg (S.E.) respectively. Plasma samples from 29 human volunteers gave readings ranging from 250 to 2880 pg/ml. Six patients with congenital adrenal hyperplasia had markedly elevated 17-OHP ranging from 99 to 337 ng/ml with a mean of 194~40 ng/ml. The method described above is rapid, sensitive and reliable. Only O.01 to 0.5 ml plasma and a single rapid chromatography step are required. The use of dog plasma as an easily available source of binding protein obviates the need for the preparation of specific anti-17-OHP antibodies required in a radioimmunoassay. Values obtained using this method agree well with those published by others (1-4), further validating the procedure. The expert technical assistance of M. Radcliffe and A.Tan is acknowledged. REFERENCES 1. Strott, C.A. and Lipsett, M.B. J. Clin. Endocr. 28:1426, 1968. 2. Stone, S. et al. Steroids 18:161, 1971. 3. Barnes, N.D. and Atherden, S.A. Arch. Dis. Child. 47:62, 1972. 4. Abraham, G.E. et al. J. Clin. Endocr. 33:42, 1971. 5. Murphy B.E.P. J. Clin. Endocr. 27:973, 1971. 6. Tan, S.Y. and Mulrow, P.J. Steroids, in press, 1974.
