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A simple competitive protein binding assay for plasma cortisol
147
CIinica Chimica Acta, 55 (1974) 147-154 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 6463
A SIMPLE COMPETITIVE CORTISOL
PROTEIN
BINDING
ASSAY
FOR PLASMA
C.K. BAUMa, R. TUDORb and J. LANDON’
‘~e~artrnent of ~hern~ca~Fa#ko~o~, St. Bartko~~me~~s hospital, London, E.C.1. and bThe Radi~kem~ea~ Centre Ltd, Amersham, Buakinghamshire, HP7 9LL (U.K.) (Received April 29, 1974)
The availability of cortisol labelled with ” Se has permitted the development of a simple and rapid competitive protein binding assay for cortisol. Plasma samples are diluted with water and heated at 70” to destroy endogenous tr~sco~in, and an aliquot is then incubated with ’ 5 Se-labelled cortisol and ch~co~-treated rabbit serum. Separation of the bound and free fractions is achieved by a Sephadex equ~ibration technique. Results compare closely with those obtained using a specific, automated fluorimetric assay. The assay is more specific and technically simpler than manual fluorimetric methods.
Introduction The clinical value of assays to determine circulating cortisol levels is now well established. A calorimetric assay, based upon the reaction of phenylhydrazine with the steroid d~ydroxya~etone side chain [1,2], is unfo~una~ly not specific and has a limited throughput. Currently, most methods depend on the relatively specific fluorescence exhibited when cortisol is dissolved in suli phuric acid and activated with light at 470 nm [3,4]. Although easy to perform, such methods lack precision, unless automated [5], and employ dangerous chemicals. More recently, competitive protein binding (CPB) assays have been introduced [6,7]. These also have disadvantages including the necessity for solvent extraction to denature endogenous binding proteins (transcortin and albumin) and the time-dependence of the separation step when charcoal is used, which necessitates adherence to a strict time schedule. Finally, most CPB assays employ tritium-labelled cortisol and, therefore, require liquid scintillation counting. The present paper describes the development and assessment of a new CPB assay for cortisol designed to overcome these problems.
148
Materials and Methods Labelled cort~so~. A ’ 5selenium-lab~lled derivative of cortisol ( [ 7 ’ Se] cortisol) at a specific activity of approximately 25 Ci/nmole was supplied by The Radiochemical Centre Ltd, Amersham. Cortisol standards. Serum standards were prepared with the desired cortisol concentrations (approximately 50, 150, 400 and 1400 nmoles/l) by mixing appropriate volumes of serum from normal subjects with serum from dex~eth~one suppressed subjects (in whom circulating cortisol was virtually absent) with and without addition of cortisol. One-ml aliquots of the serum standards were freeze-dried and stored at 4” until required, when they were reconstituted with 1 ml of distilled water. Binding protein. This was prepared by treating a pool of rabbit serum with charcoal to remove endogenous cortisol and, thereby, to free the binding sites of transcortin (and albumin). Preparation of samples for assay. Two procedures were employed to denature endogenous binding proteins. Initially duplicate 500~~1 aliquots were shaken vigorously with 3 ml methylene chloride, in stoppered tubes, for 10 min, the tubes were then centrifuged, the aqueous layer aspirated and 2 ml of the organic phase transferred to glass tubes and evaporated to dryness at 40” under a stream of air. Finally, the residue was dissolved in 600 ~1 distilled water and 500 ~1 transferred into an assay vial, This procedure was later replaced by one in which 500-1.11aliquots were diluted, in glass tubes, with 1 ml distilled water, vortex mixed and heated for 10 min at 70”. The tubes were finally cooled and duplicate 500-&l aliquots of their contents transferred to assay vials. CPB assay u~u~s.These were provided by the Radiochemic~ Centre and comprised 6 ml phosphate buffer (0.05 M, pH 7.0) containing 0.1% sodium azide; 1.0 + 0.03 g Sephadex G-25 (coarse); 50 ~1 of charcoal treated rabbit serum and 12 pmoles [’ 5 Se] cortisol. CPB assay. After addition of the prepared samples and standards, the assay vials were capped, placed on a slowly rotating mixer for 20 to 30 min and then left to stand, when the Sephadex settled quickly. One ml of the supernatant from each vial was counted in a manual gamma counter (Panax) or in an automatic counter (Wallac GTL-500). The counts obtained were plotted versus the logarithm of the concentration of cortisol in the standards to provide a standard curve from which the cortisol concentration in the unknown samples was read. ~~~orirnetr~c ussuys. These were performed by the manual method described by Mattingly [ 41 and by a fully automated modification [ 51. Samples. Plasma or serum samples were obtained from normal subjects and from patients with a wide range of endocrine and non-endocrine disorders. A total of 213 samples were assayed both by the CPB and by the manual fluo~metric technique and 165 by the CPB and automated ~uo~metric technique, by staff unaware of the results obtained by the alternative analytical technique.
149
Results (A) Counting of ’ ‘Se 7 5 Se has a half-life of 120 days and decays by electron capture, emitting gamma rays with energies of 0.096, 0.12, 0.14, 0.27, 0.28 and 0.4 MeV. There is minimal overlap with the emitting energies (0.035 and 0.027 MeV) of l2 5 I and the two isotopes can, therefore, be counted simultaneously. Counting conditions were selected for 7 5 Se alone, using a Panax manual counter, and for 75 Se and ’ 25 I together (with the latter at an optimum efficiency) on a Wallac GTL-500 automatic counter. Counter settings and efficiency data are summarised in Table I. The gamma rays emitted by 7 5 Se are not significantly attenuated by quartz glass. (B) Assessment of the Sephadex equilibration technique Preliminary studies were designed to investigate the nature of the separation of protein-bound and free cortisol and to assess the suitability of Sephadex equilibration for the assay. When 6 pmoles of [ 7 5 Se] cortisol were added to 6 ml of buffer, 1 ml generated 2343 counts/l0 s and this count rate was unaffected by the presence of binding protein and/or unlabelled cortisol. When the same mass of labelled cortisol was added to 6 ml of buffer containing 1 g of Sephadex, only 1441 counts/l0 s (61.5% of the expected counts) were generated by 1 ml of the supernatant - indicating that the Sephadex was acting as an adsorbent as well as a “molecular sieve”. Adsorption of [ 7 ’ Se] cortisol was unaffected by addition of a large excess of unlabelled cortisol but decreased if more than 15 pmoles of labelled cortisol was added (Fig. 1). Addition of binding protein (50 ~1 of charcoal-treated rabbit serum), which is excluded by the Sephadex, bound some [ 7 5 Se] cortisol and resulted in an increase in the counts generated by 1 ml of supernatant from 1441 to 2901 counts/l0 s. Addition of an excess of unlabelled cortisol (35 nmoles per vial) competed for available binding sites so that most labelled cortisol was in the free fraction and again permiated the gel - as evidenced by a decrease in the counts obtained to 1515 counts/l0 s. TABLE
I
COUNTER
SETTINGS
AND EFFICIENCY
DATA
Type
Panax
High voltage (V) Attenuation Window settings (MeV) lower upper Efficiency (%) Figure of merit* *
FOR
75Se
of counter Wallac GTL-500*
650 8
810 8
60 420 46 14.2
260 1010 38 90.3
* High voltage setting optimised for ‘*‘I Efficiency’ ** Figure of merit = Background counts in 10 s.
150
,o a
y40-
b’
A
A
.
.
. P
8
.
.
A
77 0” L30B
d
s J : 4 20Q
1 100 1000 Mass cortisol added to vial (pmoles)
10
10000
Fig. 1. Adsorption of [75Se]cortisol to Sephadex. in the presence of increasing amounts of labelied cortisol (a) and of unlabelled cortisol in the presence of a constant amount (40 pmoles) of [75Se] cortisol (0).
Further studies were performed to determine the effect of adding different amounts of binding protein and of unlabelled cortisol on the binding of [ 7 5 Se] cortisol, as evidenced by supernatant counts. A direct relationship was found between the volume of rabbit serum (over the range 1 to 100 ~1) added and the number of counts generated by 1 ml of supernatant (Fig. 2). Conversely, an inverse relationship was found between the mass of unlabelled cortisol (over the range 5.5 to 180 pmoles) added to a series of vials containing 6 pmoles [’ ’ Se] cortisol, 50 1.11charcoal-treated rabbit serum, 6 ml buffer and 1 g Sephadex, and the number of counts in the supematant (Fig. 2).
0 Y
_
6
In
E
a v
&
L
xx)
50
25
12.5
6.2
Volume serum per vial
3.1
1.6
4
0.6
(1 I) CI 1
2.5
5
10
20
40
60
xx)
200
Mass unlabelled Cortisol per Vial (PmOles) c--d
Fig. 2. Serum dilution
curve (0 -0)
and cortisol standard curve (L - - - - A).
151
(C) Effect of heat denaturation A series of investigations were performed to determine whether heating samples to 70”) to denature transcortin, might damage cortisol. [ 3 H] Cortisol was added to 5OO+d aliquots of normal plasma, of haemolysed plasma and of plasma obtained from a subject receiving an oestrogen. Each sample was then diluted with 1 ml of water and heated at 70” for 45 min. After cooling, the samples were extracted with methylene chloride and the extracts chromatographed on 2 cm strips of Whatman No. 2 paper for 4 h in the Bush C system [8]. The paper was then cut into 0.5-cm pieces and the distribution of radioactivity determined by liquid scintillation counting. For all samples only a single zone of activity, greater than 0.6% of the total counts added, was found which ran in the same position as standard cortisol. Aliquots of aqueous cortisol standards, over the range 55 to 1770 nmoles/l, were heated for 45 min and compared in an assay with similar concentrations of unheated cortisol standards. No significant differences were found. Finally, 10 plasma samples were heated at 70” for 10 or 45 min and assayed. The results obtained were similar. (0) Op timisation of assays Conditions were studied such that the assay would operate with optimum precision over the clinically important range of circulating cortisol levels, namely 55 to 1770 nmoles/l. (i) Volume of binding protein. Standard curves were set up containing varying volumes of charcoal-treated rabbit serum. Addition of 50 ~1 provided a suitable assay range. (ii) Mass of 1” Se] cortisol. Standard curves were set up with varying amounts of labelled cortisol over the range 1.27 to 163 pmoles. The effect on the percentage fall in the number of counts obtained compared with the “zero” tubes (containing no unlabelled cortisol) and with those containing excess unlabelled cortisol is shown in Fig. 3. Up to 30 pmoles of label per vial could be employed without loss of assay precision. (iii) Temperature of incubation. Standard curves were incubated at a varie-
,,,, .
(
1
IO
100
Mass [‘“Se] cortisol per assuy vial ( p moles)
Fig. 3. Effect of mass ot label on the maximum (B,,) and minimum (B,& counts obtained range of the standard curve. Fall in counts over standard curve: 100 X (Bmax - B,&/B,,.
over the
152
250
500
750
Manual fluorimetry
1000 (nmoles/
1250
I)
Fig. 4. Comparison of the CPB assay with a manual flu&metric
method.
ty of temperatures. Between 4” and 25” the precision and range were independent of temperature, but above 30” there was loss of sensitivity and flattening of the standard curve leading to reduced precision. (iv) Duration of incubation. Standard curves were superimposable and precision identical provided that assay vials were incubated, with continuous rotation, for more than 10 min and less than 2 h. (v) Volume of sample and order of additions. Optimum precision over the required range was obtained by addition of 500 ~1 of 1:2 diluted sample or standard, 50 /.11of binding protein and 10 to 15 pmoles of label. The order in which the reactants were added made no significant difference to the results. .
. .
Automated fluorlmetry
. .
.
.
(n moles/l 1
Fig. 5. Correlation of CPB assay with a specific, automated
fluorimetric method.
153 TABLE II COMPARISON
OF THE CPB ASSAY
Assay method
Manual fluorlmetry Automated
[41
fluorimetry [ 61
WITH TWO FLUORIMETRIC
ASSAYS
Number of samples
Correlation coefficient
Slope
213
0.948
0.11
165
0.956
1.17
Intercept
0.7 -1.2
Thus vials containing labelled cortisol and binding protein may be prepared in advance and stored at 4” when they were found to remain stable for more than 3 months. Once equilibrium had been attained after the addition of sample and standard, the vials could be left at 4” for at least 24 h before removal of an aliquot of the supernatant for counting, without affecting the results. (E) Validation of the assay The sensitivity of the assay, defined as the minimum concentration of cortisol which could be reliably distinguished from zero, ranged from 40 to 60 nmoles/l. Precision was determined by the assay of 20 samples in duplicate on the same and on separate days. The “within batch” coefficient of variation between duplicates assayed blind by a single operator was 7.5%, while the “between batch” coefficient of variation with two assayists was 11.0%. Accuracy was assessed by determining the recovery of cortisol added to plasma from “dexamethasone-suppressed” subjects over the range 200 to 1800 nmoles/l and gave a mean value of 97.8%. The results obtained were compared with those determined by the manual fluorimetric technique. The correlation coefficient for 12 samples from subject8 to which cortisol had been added was “dexamethasone-suppressed” 0.999 and for 33 samples from subjects receiving cortisol by mouth was 0.974. The correlation obtained with 213 consecutive samples previously assayed by manual fluorimetry in the routine steroid laboratory is shown in Fig. 4, and that with 165 consecutive samples that had been assayed by an automated fluorimetric method in Fig. 5. The correlations were excellent (Table II). Maximum assay throughput and batch size depended upon the availability of automatic pipettes and an automatic gamma-counter, when 200 or more samples could be processed by a single technician in a normal working day. In their absence this was reduced; a batch of 20 samples could be assayed within 70 min using a manual gamma counter and 100 or more samples could be processed in one day. Discussion Initial solvent extraction is included in all calorimetric and fluorimetric assays for cortisol to improve their specificity and in the more specific CPB methods to denature endogenous binding proteins. Dilution with distilled water followed by heating at 70” avoids precipitate formation whilst completely
154
denaturing transcortin without significant evaporation or damage to the steroid. Most CPB assays employ cortisol labelled with the beta-emitting isotope 3 H. However, assays based on a gamma-emitting isotope are simpler and cheaper because tubes can be counted directly, without addition of scintillant; there is no need to correct for quenching and the counting time is reduced, making possible the use of relatively inexpensive manual counters. The advantages of labelling steroids with 7 5 Se rather than with ’ *‘I have not yet been fully explored, but the smaller molecular weight, longer half-life (120 days) and smaller atomic radius (1.16 as compared with 1.36 A) of the former, are of potential benefit. The Sephadex equilibration technique has been employed for a number of assays including those for thyroxine, tri-iodothyronine and digoxin, as well as for cortisol. It was introduced by Pearlman and Crepy [9] and offers some advantages over other separation procedures. Thus it is generally applicable to the saturation analysis of small ligands; is virtually time- and temperatureindependent and is simple and rapid. Precision and accuracy are satisfactory and the assay covers the clinically important range. Results correlate closely with those of an automated fluorimetric method but less well with those obtained by a manual fluorimetric technique, probably due to the lack of specificity of the latter [lo]. Nonetheless the correlation with both methods shows a regular pattern and is in agreement with published data [ 111. Finally, the ease of the present assay makes it ideally suited to a busy routine laboratory. Acknowledgements We wish to thank Professor V.H.T. James, St. Mary’s Hospital London, W.2., and the staff of the Medical Professorial Unit, St. Bartholomew’s Hospital, London, EC 1 for the plasma samples and Miss S.T.C. Williams, D. Borthwick and E. Ogunyemi for the fluorimetric assays. References 1 2 3 4 5 6 7 8 9 10 11
D.H. Nelson and L.T. Samuels. J. Clin. Endocrinol., 12 (1952) 519 R.E. Petersen, A. Karrer and S.L. Guerra, Analyt. Chem., 29 (1957) 144 P. de Moor, 0. Steeno, M. Raskin and A. Hendrik. Acta EndocrinoL (Copenhagen), D. Mattingly. J. Clin. Pathol.. 15 (1962) 374 J. Townsend and V.H.T. James, Steroids, 11 (1968) 497 B.E.P. Murphy, J. Clin. Endocrinol.. 27 (1967) 973 C.A. Nugent and D.M. Mayes, J. Clin. Endocrinol., 26 (1966) 1116 I.E. Bush. Chromatography of Steroids, Pergamon Press, London, 1961 W.H. Pearlman and 0. Crew. J. Biol. Chem.. 242 (1967) 182 V.H.T. James, J. Townsend and R. Fraser, J. Endocrinol., 37 (1967) xxviii J. Bruton, T-K. Li and G.D. Smith, Clin. Chem., 19 (1973) 748
33 (1960)
297
CIinica Chimica Acta, 55 (1974) 147-154 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 6463
A SIMPLE COMPETITIVE CORTISOL
PROTEIN
BINDING
ASSAY
FOR PLASMA
C.K. BAUMa, R. TUDORb and J. LANDON’
‘~e~artrnent of ~hern~ca~Fa#ko~o~, St. Bartko~~me~~s hospital, London, E.C.1. and bThe Radi~kem~ea~ Centre Ltd, Amersham, Buakinghamshire, HP7 9LL (U.K.) (Received April 29, 1974)
The availability of cortisol labelled with ” Se has permitted the development of a simple and rapid competitive protein binding assay for cortisol. Plasma samples are diluted with water and heated at 70” to destroy endogenous tr~sco~in, and an aliquot is then incubated with ’ 5 Se-labelled cortisol and ch~co~-treated rabbit serum. Separation of the bound and free fractions is achieved by a Sephadex equ~ibration technique. Results compare closely with those obtained using a specific, automated fluorimetric assay. The assay is more specific and technically simpler than manual fluorimetric methods.
Introduction The clinical value of assays to determine circulating cortisol levels is now well established. A calorimetric assay, based upon the reaction of phenylhydrazine with the steroid d~ydroxya~etone side chain [1,2], is unfo~una~ly not specific and has a limited throughput. Currently, most methods depend on the relatively specific fluorescence exhibited when cortisol is dissolved in suli phuric acid and activated with light at 470 nm [3,4]. Although easy to perform, such methods lack precision, unless automated [5], and employ dangerous chemicals. More recently, competitive protein binding (CPB) assays have been introduced [6,7]. These also have disadvantages including the necessity for solvent extraction to denature endogenous binding proteins (transcortin and albumin) and the time-dependence of the separation step when charcoal is used, which necessitates adherence to a strict time schedule. Finally, most CPB assays employ tritium-labelled cortisol and, therefore, require liquid scintillation counting. The present paper describes the development and assessment of a new CPB assay for cortisol designed to overcome these problems.
148
Materials and Methods Labelled cort~so~. A ’ 5selenium-lab~lled derivative of cortisol ( [ 7 ’ Se] cortisol) at a specific activity of approximately 25 Ci/nmole was supplied by The Radiochemical Centre Ltd, Amersham. Cortisol standards. Serum standards were prepared with the desired cortisol concentrations (approximately 50, 150, 400 and 1400 nmoles/l) by mixing appropriate volumes of serum from normal subjects with serum from dex~eth~one suppressed subjects (in whom circulating cortisol was virtually absent) with and without addition of cortisol. One-ml aliquots of the serum standards were freeze-dried and stored at 4” until required, when they were reconstituted with 1 ml of distilled water. Binding protein. This was prepared by treating a pool of rabbit serum with charcoal to remove endogenous cortisol and, thereby, to free the binding sites of transcortin (and albumin). Preparation of samples for assay. Two procedures were employed to denature endogenous binding proteins. Initially duplicate 500~~1 aliquots were shaken vigorously with 3 ml methylene chloride, in stoppered tubes, for 10 min, the tubes were then centrifuged, the aqueous layer aspirated and 2 ml of the organic phase transferred to glass tubes and evaporated to dryness at 40” under a stream of air. Finally, the residue was dissolved in 600 ~1 distilled water and 500 ~1 transferred into an assay vial, This procedure was later replaced by one in which 500-1.11aliquots were diluted, in glass tubes, with 1 ml distilled water, vortex mixed and heated for 10 min at 70”. The tubes were finally cooled and duplicate 500-&l aliquots of their contents transferred to assay vials. CPB assay u~u~s.These were provided by the Radiochemic~ Centre and comprised 6 ml phosphate buffer (0.05 M, pH 7.0) containing 0.1% sodium azide; 1.0 + 0.03 g Sephadex G-25 (coarse); 50 ~1 of charcoal treated rabbit serum and 12 pmoles [’ 5 Se] cortisol. CPB assay. After addition of the prepared samples and standards, the assay vials were capped, placed on a slowly rotating mixer for 20 to 30 min and then left to stand, when the Sephadex settled quickly. One ml of the supernatant from each vial was counted in a manual gamma counter (Panax) or in an automatic counter (Wallac GTL-500). The counts obtained were plotted versus the logarithm of the concentration of cortisol in the standards to provide a standard curve from which the cortisol concentration in the unknown samples was read. ~~~orirnetr~c ussuys. These were performed by the manual method described by Mattingly [ 41 and by a fully automated modification [ 51. Samples. Plasma or serum samples were obtained from normal subjects and from patients with a wide range of endocrine and non-endocrine disorders. A total of 213 samples were assayed both by the CPB and by the manual fluo~metric technique and 165 by the CPB and automated ~uo~metric technique, by staff unaware of the results obtained by the alternative analytical technique.
149
Results (A) Counting of ’ ‘Se 7 5 Se has a half-life of 120 days and decays by electron capture, emitting gamma rays with energies of 0.096, 0.12, 0.14, 0.27, 0.28 and 0.4 MeV. There is minimal overlap with the emitting energies (0.035 and 0.027 MeV) of l2 5 I and the two isotopes can, therefore, be counted simultaneously. Counting conditions were selected for 7 5 Se alone, using a Panax manual counter, and for 75 Se and ’ 25 I together (with the latter at an optimum efficiency) on a Wallac GTL-500 automatic counter. Counter settings and efficiency data are summarised in Table I. The gamma rays emitted by 7 5 Se are not significantly attenuated by quartz glass. (B) Assessment of the Sephadex equilibration technique Preliminary studies were designed to investigate the nature of the separation of protein-bound and free cortisol and to assess the suitability of Sephadex equilibration for the assay. When 6 pmoles of [ 7 5 Se] cortisol were added to 6 ml of buffer, 1 ml generated 2343 counts/l0 s and this count rate was unaffected by the presence of binding protein and/or unlabelled cortisol. When the same mass of labelled cortisol was added to 6 ml of buffer containing 1 g of Sephadex, only 1441 counts/l0 s (61.5% of the expected counts) were generated by 1 ml of the supernatant - indicating that the Sephadex was acting as an adsorbent as well as a “molecular sieve”. Adsorption of [ 7 ’ Se] cortisol was unaffected by addition of a large excess of unlabelled cortisol but decreased if more than 15 pmoles of labelled cortisol was added (Fig. 1). Addition of binding protein (50 ~1 of charcoal-treated rabbit serum), which is excluded by the Sephadex, bound some [ 7 5 Se] cortisol and resulted in an increase in the counts generated by 1 ml of supernatant from 1441 to 2901 counts/l0 s. Addition of an excess of unlabelled cortisol (35 nmoles per vial) competed for available binding sites so that most labelled cortisol was in the free fraction and again permiated the gel - as evidenced by a decrease in the counts obtained to 1515 counts/l0 s. TABLE
I
COUNTER
SETTINGS
AND EFFICIENCY
DATA
Type
Panax
High voltage (V) Attenuation Window settings (MeV) lower upper Efficiency (%) Figure of merit* *
FOR
75Se
of counter Wallac GTL-500*
650 8
810 8
60 420 46 14.2
260 1010 38 90.3
* High voltage setting optimised for ‘*‘I Efficiency’ ** Figure of merit = Background counts in 10 s.
150
,o a
y40-
b’
A
A
.
.
. P
8
.
.
A
77 0” L30B
d
s J : 4 20Q
1 100 1000 Mass cortisol added to vial (pmoles)
10
10000
Fig. 1. Adsorption of [75Se]cortisol to Sephadex. in the presence of increasing amounts of labelied cortisol (a) and of unlabelled cortisol in the presence of a constant amount (40 pmoles) of [75Se] cortisol (0).
Further studies were performed to determine the effect of adding different amounts of binding protein and of unlabelled cortisol on the binding of [ 7 5 Se] cortisol, as evidenced by supernatant counts. A direct relationship was found between the volume of rabbit serum (over the range 1 to 100 ~1) added and the number of counts generated by 1 ml of supernatant (Fig. 2). Conversely, an inverse relationship was found between the mass of unlabelled cortisol (over the range 5.5 to 180 pmoles) added to a series of vials containing 6 pmoles [’ ’ Se] cortisol, 50 1.11charcoal-treated rabbit serum, 6 ml buffer and 1 g Sephadex, and the number of counts in the supematant (Fig. 2).
0 Y
_
6
In
E
a v
&
L
xx)
50
25
12.5
6.2
Volume serum per vial
3.1
1.6
4
0.6
(1 I) CI 1
2.5
5
10
20
40
60
xx)
200
Mass unlabelled Cortisol per Vial (PmOles) c--d
Fig. 2. Serum dilution
curve (0 -0)
and cortisol standard curve (L - - - - A).
151
(C) Effect of heat denaturation A series of investigations were performed to determine whether heating samples to 70”) to denature transcortin, might damage cortisol. [ 3 H] Cortisol was added to 5OO+d aliquots of normal plasma, of haemolysed plasma and of plasma obtained from a subject receiving an oestrogen. Each sample was then diluted with 1 ml of water and heated at 70” for 45 min. After cooling, the samples were extracted with methylene chloride and the extracts chromatographed on 2 cm strips of Whatman No. 2 paper for 4 h in the Bush C system [8]. The paper was then cut into 0.5-cm pieces and the distribution of radioactivity determined by liquid scintillation counting. For all samples only a single zone of activity, greater than 0.6% of the total counts added, was found which ran in the same position as standard cortisol. Aliquots of aqueous cortisol standards, over the range 55 to 1770 nmoles/l, were heated for 45 min and compared in an assay with similar concentrations of unheated cortisol standards. No significant differences were found. Finally, 10 plasma samples were heated at 70” for 10 or 45 min and assayed. The results obtained were similar. (0) Op timisation of assays Conditions were studied such that the assay would operate with optimum precision over the clinically important range of circulating cortisol levels, namely 55 to 1770 nmoles/l. (i) Volume of binding protein. Standard curves were set up containing varying volumes of charcoal-treated rabbit serum. Addition of 50 ~1 provided a suitable assay range. (ii) Mass of 1” Se] cortisol. Standard curves were set up with varying amounts of labelled cortisol over the range 1.27 to 163 pmoles. The effect on the percentage fall in the number of counts obtained compared with the “zero” tubes (containing no unlabelled cortisol) and with those containing excess unlabelled cortisol is shown in Fig. 3. Up to 30 pmoles of label per vial could be employed without loss of assay precision. (iii) Temperature of incubation. Standard curves were incubated at a varie-
,,,, .
(
1
IO
100
Mass [‘“Se] cortisol per assuy vial ( p moles)
Fig. 3. Effect of mass ot label on the maximum (B,,) and minimum (B,& counts obtained range of the standard curve. Fall in counts over standard curve: 100 X (Bmax - B,&/B,,.
over the
152
250
500
750
Manual fluorimetry
1000 (nmoles/
1250
I)
Fig. 4. Comparison of the CPB assay with a manual flu&metric
method.
ty of temperatures. Between 4” and 25” the precision and range were independent of temperature, but above 30” there was loss of sensitivity and flattening of the standard curve leading to reduced precision. (iv) Duration of incubation. Standard curves were superimposable and precision identical provided that assay vials were incubated, with continuous rotation, for more than 10 min and less than 2 h. (v) Volume of sample and order of additions. Optimum precision over the required range was obtained by addition of 500 ~1 of 1:2 diluted sample or standard, 50 /.11of binding protein and 10 to 15 pmoles of label. The order in which the reactants were added made no significant difference to the results. .
. .
Automated fluorlmetry
. .
.
.
(n moles/l 1
Fig. 5. Correlation of CPB assay with a specific, automated
fluorimetric method.
153 TABLE II COMPARISON
OF THE CPB ASSAY
Assay method
Manual fluorlmetry Automated
[41
fluorimetry [ 61
WITH TWO FLUORIMETRIC
ASSAYS
Number of samples
Correlation coefficient
Slope
213
0.948
0.11
165
0.956
1.17
Intercept
0.7 -1.2
Thus vials containing labelled cortisol and binding protein may be prepared in advance and stored at 4” when they were found to remain stable for more than 3 months. Once equilibrium had been attained after the addition of sample and standard, the vials could be left at 4” for at least 24 h before removal of an aliquot of the supernatant for counting, without affecting the results. (E) Validation of the assay The sensitivity of the assay, defined as the minimum concentration of cortisol which could be reliably distinguished from zero, ranged from 40 to 60 nmoles/l. Precision was determined by the assay of 20 samples in duplicate on the same and on separate days. The “within batch” coefficient of variation between duplicates assayed blind by a single operator was 7.5%, while the “between batch” coefficient of variation with two assayists was 11.0%. Accuracy was assessed by determining the recovery of cortisol added to plasma from “dexamethasone-suppressed” subjects over the range 200 to 1800 nmoles/l and gave a mean value of 97.8%. The results obtained were compared with those determined by the manual fluorimetric technique. The correlation coefficient for 12 samples from subject8 to which cortisol had been added was “dexamethasone-suppressed” 0.999 and for 33 samples from subjects receiving cortisol by mouth was 0.974. The correlation obtained with 213 consecutive samples previously assayed by manual fluorimetry in the routine steroid laboratory is shown in Fig. 4, and that with 165 consecutive samples that had been assayed by an automated fluorimetric method in Fig. 5. The correlations were excellent (Table II). Maximum assay throughput and batch size depended upon the availability of automatic pipettes and an automatic gamma-counter, when 200 or more samples could be processed by a single technician in a normal working day. In their absence this was reduced; a batch of 20 samples could be assayed within 70 min using a manual gamma counter and 100 or more samples could be processed in one day. Discussion Initial solvent extraction is included in all calorimetric and fluorimetric assays for cortisol to improve their specificity and in the more specific CPB methods to denature endogenous binding proteins. Dilution with distilled water followed by heating at 70” avoids precipitate formation whilst completely
154
denaturing transcortin without significant evaporation or damage to the steroid. Most CPB assays employ cortisol labelled with the beta-emitting isotope 3 H. However, assays based on a gamma-emitting isotope are simpler and cheaper because tubes can be counted directly, without addition of scintillant; there is no need to correct for quenching and the counting time is reduced, making possible the use of relatively inexpensive manual counters. The advantages of labelling steroids with 7 5 Se rather than with ’ *‘I have not yet been fully explored, but the smaller molecular weight, longer half-life (120 days) and smaller atomic radius (1.16 as compared with 1.36 A) of the former, are of potential benefit. The Sephadex equilibration technique has been employed for a number of assays including those for thyroxine, tri-iodothyronine and digoxin, as well as for cortisol. It was introduced by Pearlman and Crepy [9] and offers some advantages over other separation procedures. Thus it is generally applicable to the saturation analysis of small ligands; is virtually time- and temperatureindependent and is simple and rapid. Precision and accuracy are satisfactory and the assay covers the clinically important range. Results correlate closely with those of an automated fluorimetric method but less well with those obtained by a manual fluorimetric technique, probably due to the lack of specificity of the latter [lo]. Nonetheless the correlation with both methods shows a regular pattern and is in agreement with published data [ 111. Finally, the ease of the present assay makes it ideally suited to a busy routine laboratory. Acknowledgements We wish to thank Professor V.H.T. James, St. Mary’s Hospital London, W.2., and the staff of the Medical Professorial Unit, St. Bartholomew’s Hospital, London, EC 1 for the plasma samples and Miss S.T.C. Williams, D. Borthwick and E. Ogunyemi for the fluorimetric assays. References 1 2 3 4 5 6 7 8 9 10 11
D.H. Nelson and L.T. Samuels. J. Clin. Endocrinol., 12 (1952) 519 R.E. Petersen, A. Karrer and S.L. Guerra, Analyt. Chem., 29 (1957) 144 P. de Moor, 0. Steeno, M. Raskin and A. Hendrik. Acta EndocrinoL (Copenhagen), D. Mattingly. J. Clin. Pathol.. 15 (1962) 374 J. Townsend and V.H.T. James, Steroids, 11 (1968) 497 B.E.P. Murphy, J. Clin. Endocrinol.. 27 (1967) 973 C.A. Nugent and D.M. Mayes, J. Clin. Endocrinol., 26 (1966) 1116 I.E. Bush. Chromatography of Steroids, Pergamon Press, London, 1961 W.H. Pearlman and 0. Crew. J. Biol. Chem.. 242 (1967) 182 V.H.T. James, J. Townsend and R. Fraser, J. Endocrinol., 37 (1967) xxviii J. Bruton, T-K. Li and G.D. Smith, Clin. Chem., 19 (1973) 748
33 (1960)
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