Free Cortisol Index: A Rapid and Simple Estimation of Free Cortisol in Human Plasma1 GERHARD BAUMANN,2 GEORGES RAPPAPORT, THERESE LEMARCHAND-BERAUD, AND JEAN-PIERRE FELBER Department of Medicine, Hopital Cantonal, University of Lausanne Medical School, Lausanne, Switzerland and free cortisol concentration (FF) was determined by equilibrium dialysis at 37 C. 169 plasma samples with an F range of 0.8-61.2 /xg/100 ml from 18 normal, 10 ACTH-stimulated, 17 estrogen-treated and 12 pregnant subjects were analyzed. A highly significant correlation between FFI and FF was found (r = 0.952). Inter-assay coefficients of variation were 7% for total plasma F and 3.5% for CFU. The method allows the rapid processing of large numbers of samples and can he performed on 0.2 nil of plasma (J Clin Endocrinot Metah 40: 462, 1975)
ABSTRACT. A rapid and simple method for the estimation of free (nonprotein-bound) cortisol in human plasma is described. Total plasma cortisol (F) was determined by a competitive protein binding assay. An estimate of the saturation of Corticosteroid Binding Globulin was obtained by the use of "charcoal cortisol uptake" (CFU). "Charcoal cortisol uptake ratio" (CFUR) was defined as the ratio of the CFU of the unknown plasma to that of a reference serum pool. Free cortisol index (FFI) was calculated as (total plasma F) x (CFUR). Percent free cortisol
M
ETHODS currently employed in the clinical laboratory for the determination of cortisol (F) in plasma measure total (free and protein bound) unconjugated F. Approximately 90% of circulating F is bound to plasma proteins in man, mainly to Corticosteroid Binding Globulin (CBG) or Transcortin and to albumin (1-4). It is, however, widely accepted that the unbound (free) rather than the protein-bound moiety is biologically active (5-7). Total plasma F is influenced by the concentration of binding proteins, particularly CBG, while free F is less dependent on such factors, being controlled within narrow limits by pituitary ACTH secretion. A variety of drugs and diseases are known to alter CBG and albumin content of plasma, notably estrogens, which have been shown to increase CBG and F concentration threefold. With a large portion of the population taking oral contraReceived September 12, 1974. Address reprint requests to Dr. J. P. Felber. 1 A preliminary report of this work has appeared in the program of the 56th Annual Meeting of the Endocrine Society, No. 316, 1974. 2 Present address: Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014.
ceptives, this often renders interpretation of plasma F values difficult. For these reasons, the measurement of free F would be preferable, but methods heretofore used, such as dialysis, ultrafiltration or gel chromatography are time consuming, cumbersome and generally not suitable for routine laboratory use. This report describes a simple and rapid method for the estimation of plasma free F by means of analysis of CBG saturation in conjunction with total F determination. A similar method for the estimation of free thyroxine ("free thyroxine index") has found wide application as a routine clinical test. Materials and Methods 1) Reagents 1,2-3H-Cortisol (51.7 Ci/mmol) was purchased from New England Nuclear Company and examined for purity by ascending paper chromatography. It was found to be > 95% pure. Unlabeled cortisol was obtained from Merck, Darmstadt, Germany. The reference serum pool was obtained in lyophilized form from Hyland Laboratories, Costa Mesa, California. All other chemicals were reagent grade. Dextran-coated charcoal suspension was prepared as follows: 150 ml borate buffer 0.05M, pH 7.8, 500 mg Norit A (Fisher), 50 mg dextran
462
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FREE CORTISOL INDEX IN HUMAN PLASMA T 70 (Pharmacia). The suspension was found to be stable for at least 4 weeks at 4 C. CBG-tracer solution: 150 ml borate buffer 0.05M, pH 7.8, 1 ml of heparinized plasma from a healthy woman taking oral contraceptives, drawn at 8 AM after a 2 mg dexamethasone dose at midnight (total plasma F: 0.2 /xg/100 ml), 70 ng (6 Mill, cpm) 3H-cortisol, 140 ng cortisol. The solution was stable for at least 2 weeks at 4C. Tracer solution for charcoal cortisol uptake: 10 ml methanol, 140 ng (12 Mill, cpm) 3Hcortisol, 360 ng cortisol. The solution was stored at - 2 0 C. Dialysis tracer solution: 700 ng (60 Mill, cpm) 3 H-cortisol in 1 ml methanol. This solution was stored at - 2 0 C. 2) Total plasma F Total plasma F was determined by a modification of the competitive protein binding assay described by Nugent and Mayes (8). Heparinized plasma, 0.025-0.1 ml, was extracted with 2 ml of dichloromethane by shaking on a vortex mixer for 60 s. A series of duplicate F standards were prepared using 0, 2, 5, 7.5, 10, 15 and 20 ng F in methanol. 1 ml aliquots of the extracts and the standards were dried in test tubes at 45 C in an air stream. After evaporation was completed (5-10 min), 1 ml of the CBG-tracer solution was added to each tube. The samples were shaken for 5 min at 45 C and then transferred to an ice-water bath. After 30 min 0.5 ml of the charcoal-dextran suspension was added at 0 C, the tubes shaken and kept at 0 C for 5 min, then centrifuged at 4 C for 10 min at 3500 x g. Duplicate 0.5 ml aliquots were then transferred to counting vials at 0 C, solubilized in Insta-Gel (Packard) and counted in a liquid scintillation spectrometer for a period of time sufficient to result in a counting error of less than 2%. A standard curve was prepared using the percent radioactivity present in the supernatant. No correction was made for extraction efficiency. 3) Charcoal cortisol uptake (CFU) Heparinized plasma, 0.1 ml, and 0.1 ml tracer solution were added to 4 ml of borate buffer 0.05M, pH 7.8. The tubes were inverted 10 times to assure thorough mixing. In preliminary experiments the composition of the tracer solution was optimized for maximal distinction be-
463
tween the charcoal uptakes of plasmas with different CBG content or F levels. A total (labeled and unlabeled) F concentration of 50 ng/ml was found to be optimal. The maximal methanol concentration in the sample was 2.5%. Duplicate 1 ml aliquots were incubated for 10 min at 37 C, then transferred to an ice-water bath. The rest of the procedure (charcoal addition, centrifugation, counting and reaction times) was carried out as in the method for total plasma F. In preliminary experiments different incubation times after charcoal addition were tested. Isotope uptake by charcoal increased from the zero time value by 3.4% during the first 10 min and by 12.7% at 60 min. A 5-min incubation at 0 C was found to give maximum reproducibility. Four tubes from different samples with water added instead of charcoal suspension were included in every assay for "total counts." The result was expressed as percent of "total counts" present in the supernatant. CFU in percent was defined as 100-(percentage in supernatant). 4) Charcoal cortisol uptake ratio (CFUR) CFUR was calculated as the ratio of the uptakes of the unknown sample and of that of a reference serum pool determined in the same assay. Quadruplicate samples of the reference serum were included in each assay run, one pair of reference samples was placed at the beginning and one at the end of the assay rack. The two values obtained for the reference serum were averaged. 5) Equilibrium dialysis Dialysis was performed according to a modification of the method of Doe et al. (9). 16 x 100 Visking tubing VA" and 1" diameter (Union Carbide) was soaked for 24 h in phosphate buffer, 0.1M, pH 7.4, rinsed and tested for leaks. Plasma samples were used undiluted and diluted 1:2, 1:5, and 1:10 with the same buffer. 10 fi\ of the dialysis tracer solution per ml of undiluted plasma was added to the samples. The maximum methanol concentration in the sample was 1%. The samples were incubated at 37 C in a water bath for 1 h. Duplicate 1, 2 and 5 ml quantities were then placed into the dialysis bags, the bags tied twice at both ends, and dialysis carried out for 24 h at 37 C against 3, 5, 10, and 20 ml of the same buffer. No preservative was added to the buffer as
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464
BAUMANN, RAPPAPORT, LEMARCHAND-BERAUD AND FELBER
bacterial growth was found to be minimal during the times employed. After completion of dialysis, the bags were weighed, and 0.1 ml aliquots of dialysand and dialysate were placed in scintillation vials, solubilized and counted as described. The nature of the labeled compounds was determined by radiochromatography in the dialysate and dialysand. The empty bags were washed with buffer and dichloromethane, the combined washings and the bag were counted for adsorbed radioactivity. Percent free F was expressed as the ratio of cpm concentration in the dialysate over that of the dialysand. Free plasma F concentration was calculated as (total plasma F) x (% free F). 6)
Radiochromatography
Ascending chromatography of original 3H-cortisol, dialysate and dialysand was performed on Whatman no. 3 paper in benzene:methanol: water = 4:2:1, (vol:vol:vol). Developed strips were first scanned in a strip scanner and then cut into 10 mm wide transverse sections. Each section was eluted with toluene and the eluate was counted in a liquid scintillation spectrometer. 7) Free cortisol index (FFI) Free Cortisol Index was calculated as (total plasma F) x (charcoal. F uptake ratio). The same reference serum pool was used as a control in all assays, i.e., total plasma F, CFU and dialysis. 8) Subjects Heparinized blood samples from 18 young normal volunteers (15 male, 3 female) were drawn at different times throughout the day. None was taking any medication. Similar blood samples were obtained from 17 healthy young females taking oral contraceptives for at least 6 months prior to the time of study. In addition, plasma samples from 12 women at different stages of their pregnancy (19 to 38 weeks) were studied. 10 subjects of group I were studied in the same way during ACTH tests after informed consent had been given. 1 mg of jQ'-^-ACTH (Synacthen CIBA) was injected intra-
JCE & M . 1975 Vol 40 • No 3
muscularly at 9 AM and blood samples were taken 2, 4 and 6 h after injection. Results 1) Cortisol, charcoal cortisol uptake, free cortisol index and Free Cortisol The mean values, ranges, and standard deviations of total plasma cortisol (F), charcoal cortisol uptake in percent (CFU), charcoal cortisol uptake ratio (CFUR), free cortisol index (FFI)3, percent free cortisol (% FF) and free cortisol concentration (FF) for the different groups at standard clock times are summarized in Tables 1 and 2. The individual measurements, obtained throughout the day, are given in Fig. 1. The average values from multiple assays for the reference serum were: F 8.76 fig/100 ml, CFU 66.2%, CFUR 1.00 by definition, FFI 8.76%, FF 7.1%, FF 0.62 /xg/100 ml. 2) Equilibrium dialysis During the dialysis experiments it was found that equilibrium was reached after 12 to 16 h. After this time identical results were obtained when tracer was added to the inside or to the outside of the dialysis bag and with dialysis carried out over a total period of 72 h. No significant fluid shifts were detectable by comparison of the weights of the bags before and after dialysis. Adsorption of isotope to the dialysis membrane was found to be negligible (0.01% of total radioactivity). Radiochromatography of the dialysate and the dialyzed plasma after completion of dialysis showed a single peak with the Rf of the cortisol originally used. No difference in percent dialyzable isotope was found when different volumes of plasma and outer buffer were used (1 ml to 5 ml and 3 ml to 20 ml, respec-
3 To avoid confusion with true concentrations of FF, we elected to express FFI as a dimensionless parameter, although formally it has the dimension of >g/100 ml."
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7.4-22.0 (12.9 ± 4.1)
1.0-12.0
0.8-5.8
21.0-43.8(29.0 + 6.4)
8:00 AM
5 : 0 0 PM
Midnight
ACTH
Clock time
0.98-1.20 (1.13 ± 0.08)
0.80-0.98 (0.91 ± 0.07)
0.72-1.13 (0.97 ± 0.11)
8-10 AM
b) Pregnant
19.8-54.8 (33.5 ± 10.1)
6.2-22.9 (12.9 ± 7.1)
5 : 0 0 PM
28.1-41.8 (35.8 ± 3.9)'
20.4-47.9 (29.0 ± 12.7)
33.3-53.1 (38.1 ± 9.7)
%
/xg/100 ml
19.8-61.2 (34.6 ± 13.1)
CFU
Total F
8 : 0 0 AM
contraceptives
a) Oral
65.4-81.6 (74.5 ± 4.8)
52.6-62.3 (60.1 ± 4.5)
47.2-76.9 (65.7 ± 8.5)
0.92-1.16 (1.05 ± 0.08)
CFUR
(2.4 ± 2.0)
(5.3 ± 2.7)
24.4-45.8 (32.4 ± 6.0)
0.7-5.3
1.1-11.7
9.0-29.3 (13.9 ± 5.2)
FFP
(7.9 ± 2.6)
10.9-15.6 (13.1 ± 1.5)
4.8-12.3
4.1-12.6 (10.0 ± 3.2)
5.3-15.1 (10.6 ± 2.8)
%FF
2.58-6.47(3.9
0.42-0.63 (0.54 ± 0.06)
0.32-0.72 (0.44 ± 0.18)
0.49-0.80 (0.61 ± 0.09)
CFUR
(5.0 ± 1.6)
10.7-26.6(17.8 + 4.5)
4.0-7.3
9.8-35.6 (20.6 ± 6.8)
FFI
3.0-12.4 (6.8 ± 2.6)
4 . 1 - 9.2 (5.4 ± 2.6)
2.7- 9.6 (5.7 ± 1.9)
% FF
1.21-3.57 (2.17 ± 0.78)
0.46-1.81 (0.63 ± 0.32)
0.8 -3.25 (2.04 ± 0.78)
Hg/100 ml
FF
±1.13)
0.08-0.47 (0.20 ± 0.17)
0.21-1.43 (0.46 ± 0.27)
0.54-2.80 (1.38 ± 0.63)
fig/100 ml
FF
T A B L E 2. Plasma cortisol parameters at standard clock times in normal females on oral contraceptives and in pregnant subjects (range, mean ± SD)
(2.6 ± 2.2)
(5.5 ±2.4)
%
/xg/100 ml
Time
60.6-77.8 (70.1 ± 5.0)
CFU
Total F
Clock
TABLE 1. Plasma cortisol parameters at standard clock times and after standard ACTH stimulation in normal subjects (range, mean ± SD;
or
W X
D
I—I
o r z
H i—i
50
n o
5
BAUMANN, RAPPAPORT, LEMARCHAND-BERAUD AND FELBER
466
JCE & M • 1975 Vol 40 • No 3
•
60
• •
E
• * • »
»
§ 50
Nor mals ACTH Estrogens Pregnant
-4
Ho
*
40
A
o •
a. UI
A
§30
•
w V
! *
I
S
gjl.2
• •
>
3 20 a.
S io 1 A
I1-6 - 1
•
§0.8
•
8 0.4 -
FIG. 1. Total plasma cortisol, "Charcoal cortisol uptake ratio," "Free Cortisol Index," and Free Cortisol Concentration at various clock times in normal, ACTH stimulated, estrogentreated and pregnant subjects.
a:
5 o
n
A
6 -
60
_
50
5_
A A
E
A
o
2
40
• V
20
• A
V V
co
A*4 ***
ro
• •
E CORTISOL
oo
•
•
A A A A
4 -
A
I
UI 1
10
n
m
• m
V V
A A A
V
•
• • • •
*
ae
1
«
••
•r
0
with a mean x-intercept of 18.1 ± 3.24 100 ml in the normal and ACTH-stimulated subjects. It was 51.2 jug/100 ml in one "pill plasma" where enough points were accumulated to permit Scatchard analysis. Since maximal binding capacity is independent of temperature and can be deter3) Relationship between free cortisol index mined at non-equilibrium conditions (10), and free cortisol concentration these values represent CBG maximal bindA close positive correlation between FFI ing capacity. Scatchard analysis of the refand FF was found (P < 0.001), as shown erence serum under equilibrium conditions at 37 C gave an apparent maximal binding in Fig. 3. capacity of 21.0 /ag/100 ml and an apparent association constant of 2.24 x 107 liters/mol 4) CBG binding characteristics for CBG-cortisol interaction. Scatchard analysis of the values obtained yielded a low capacity binding component SD.
tively). Dilution of plasma, however, had a marked effect on percent dialyzable F, even when the amount of tracer added was adjusted according to the protein dilution in the sample (Fig. 2). The experiments were therefore carried out with undiluted plasma.
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467
FREE CORTISOL INDEX IN HUMAN PLASMA
5) Accuracy, precision and reproducibility The recovery of F in routine plasma F determinations in our laboratory averages 92%, the coefficient of variation averages 7%. Intra-assay variability for CFU was determined from 6 replicate determinations of the reference serum in the same assay. The coefficient of variation found was 2.1%. Interassay variability for CFU was determined using 9 different plasmas with total F concentrations ranging from 1.0 to 26.8 /xg/100 ml in 4 different assays. A mean coefficient of variation of 3.5% was found (range 1.2-6.8%). Since the addition of charcoal to the reaction mixture resulted in mild non-equilibrium conditions, strict adherence to the time schedules after charcoal addition was necessary to achieve this degree of quality. An important factor affecting interassay reproducibility of CFU was found to be the composition of the tracer solution added, although this is partially corrected by the use of CFUR. The tracer solution was therefore stored tightly sealed at —20 C to minimize evaporation of solvent and care was taken to prepare different batches in an identical fashion.
30
25
*
20
15
10
undiluted
1:2
1:5
1:10
plasma dilution
FIG. 2. Two representative examples of the effect of plasma dilution on percent dialyzable cortisol. Diluent was 0.1M phosphate buffer, pH 7.4. Dialysis was carried out at 37 C for 24 h against the same buffer. The amount of cortisol tracer added was adjusted in proportion to plasma dilution (7, 3.5, 1.4 and 0.7 ng/ml).
FIG. 3. Correlation between "Free Cortisol Index" and free cortisol concentration as determined by equilibrium dialysis in normal, ACTH-stimulated, estrogen-treated and pregnant subjects.
estrogen! pregnant
p
ABSTRACT. A rapid and simple method for the estimation of free (nonprotein-bound) cortisol in human plasma is described. Total plasma cortisol (F) was determined by a competitive protein binding assay. An estimate of the saturation of Corticosteroid Binding Globulin was obtained by the use of "charcoal cortisol uptake" (CFU). "Charcoal cortisol uptake ratio" (CFUR) was defined as the ratio of the CFU of the unknown plasma to that of a reference serum pool. Free cortisol index (FFI) was calculated as (total plasma F) x (CFUR). Percent free cortisol
M
ETHODS currently employed in the clinical laboratory for the determination of cortisol (F) in plasma measure total (free and protein bound) unconjugated F. Approximately 90% of circulating F is bound to plasma proteins in man, mainly to Corticosteroid Binding Globulin (CBG) or Transcortin and to albumin (1-4). It is, however, widely accepted that the unbound (free) rather than the protein-bound moiety is biologically active (5-7). Total plasma F is influenced by the concentration of binding proteins, particularly CBG, while free F is less dependent on such factors, being controlled within narrow limits by pituitary ACTH secretion. A variety of drugs and diseases are known to alter CBG and albumin content of plasma, notably estrogens, which have been shown to increase CBG and F concentration threefold. With a large portion of the population taking oral contraReceived September 12, 1974. Address reprint requests to Dr. J. P. Felber. 1 A preliminary report of this work has appeared in the program of the 56th Annual Meeting of the Endocrine Society, No. 316, 1974. 2 Present address: Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014.
ceptives, this often renders interpretation of plasma F values difficult. For these reasons, the measurement of free F would be preferable, but methods heretofore used, such as dialysis, ultrafiltration or gel chromatography are time consuming, cumbersome and generally not suitable for routine laboratory use. This report describes a simple and rapid method for the estimation of plasma free F by means of analysis of CBG saturation in conjunction with total F determination. A similar method for the estimation of free thyroxine ("free thyroxine index") has found wide application as a routine clinical test. Materials and Methods 1) Reagents 1,2-3H-Cortisol (51.7 Ci/mmol) was purchased from New England Nuclear Company and examined for purity by ascending paper chromatography. It was found to be > 95% pure. Unlabeled cortisol was obtained from Merck, Darmstadt, Germany. The reference serum pool was obtained in lyophilized form from Hyland Laboratories, Costa Mesa, California. All other chemicals were reagent grade. Dextran-coated charcoal suspension was prepared as follows: 150 ml borate buffer 0.05M, pH 7.8, 500 mg Norit A (Fisher), 50 mg dextran
462
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FREE CORTISOL INDEX IN HUMAN PLASMA T 70 (Pharmacia). The suspension was found to be stable for at least 4 weeks at 4 C. CBG-tracer solution: 150 ml borate buffer 0.05M, pH 7.8, 1 ml of heparinized plasma from a healthy woman taking oral contraceptives, drawn at 8 AM after a 2 mg dexamethasone dose at midnight (total plasma F: 0.2 /xg/100 ml), 70 ng (6 Mill, cpm) 3H-cortisol, 140 ng cortisol. The solution was stable for at least 2 weeks at 4C. Tracer solution for charcoal cortisol uptake: 10 ml methanol, 140 ng (12 Mill, cpm) 3Hcortisol, 360 ng cortisol. The solution was stored at - 2 0 C. Dialysis tracer solution: 700 ng (60 Mill, cpm) 3 H-cortisol in 1 ml methanol. This solution was stored at - 2 0 C. 2) Total plasma F Total plasma F was determined by a modification of the competitive protein binding assay described by Nugent and Mayes (8). Heparinized plasma, 0.025-0.1 ml, was extracted with 2 ml of dichloromethane by shaking on a vortex mixer for 60 s. A series of duplicate F standards were prepared using 0, 2, 5, 7.5, 10, 15 and 20 ng F in methanol. 1 ml aliquots of the extracts and the standards were dried in test tubes at 45 C in an air stream. After evaporation was completed (5-10 min), 1 ml of the CBG-tracer solution was added to each tube. The samples were shaken for 5 min at 45 C and then transferred to an ice-water bath. After 30 min 0.5 ml of the charcoal-dextran suspension was added at 0 C, the tubes shaken and kept at 0 C for 5 min, then centrifuged at 4 C for 10 min at 3500 x g. Duplicate 0.5 ml aliquots were then transferred to counting vials at 0 C, solubilized in Insta-Gel (Packard) and counted in a liquid scintillation spectrometer for a period of time sufficient to result in a counting error of less than 2%. A standard curve was prepared using the percent radioactivity present in the supernatant. No correction was made for extraction efficiency. 3) Charcoal cortisol uptake (CFU) Heparinized plasma, 0.1 ml, and 0.1 ml tracer solution were added to 4 ml of borate buffer 0.05M, pH 7.8. The tubes were inverted 10 times to assure thorough mixing. In preliminary experiments the composition of the tracer solution was optimized for maximal distinction be-
463
tween the charcoal uptakes of plasmas with different CBG content or F levels. A total (labeled and unlabeled) F concentration of 50 ng/ml was found to be optimal. The maximal methanol concentration in the sample was 2.5%. Duplicate 1 ml aliquots were incubated for 10 min at 37 C, then transferred to an ice-water bath. The rest of the procedure (charcoal addition, centrifugation, counting and reaction times) was carried out as in the method for total plasma F. In preliminary experiments different incubation times after charcoal addition were tested. Isotope uptake by charcoal increased from the zero time value by 3.4% during the first 10 min and by 12.7% at 60 min. A 5-min incubation at 0 C was found to give maximum reproducibility. Four tubes from different samples with water added instead of charcoal suspension were included in every assay for "total counts." The result was expressed as percent of "total counts" present in the supernatant. CFU in percent was defined as 100-(percentage in supernatant). 4) Charcoal cortisol uptake ratio (CFUR) CFUR was calculated as the ratio of the uptakes of the unknown sample and of that of a reference serum pool determined in the same assay. Quadruplicate samples of the reference serum were included in each assay run, one pair of reference samples was placed at the beginning and one at the end of the assay rack. The two values obtained for the reference serum were averaged. 5) Equilibrium dialysis Dialysis was performed according to a modification of the method of Doe et al. (9). 16 x 100 Visking tubing VA" and 1" diameter (Union Carbide) was soaked for 24 h in phosphate buffer, 0.1M, pH 7.4, rinsed and tested for leaks. Plasma samples were used undiluted and diluted 1:2, 1:5, and 1:10 with the same buffer. 10 fi\ of the dialysis tracer solution per ml of undiluted plasma was added to the samples. The maximum methanol concentration in the sample was 1%. The samples were incubated at 37 C in a water bath for 1 h. Duplicate 1, 2 and 5 ml quantities were then placed into the dialysis bags, the bags tied twice at both ends, and dialysis carried out for 24 h at 37 C against 3, 5, 10, and 20 ml of the same buffer. No preservative was added to the buffer as
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464
BAUMANN, RAPPAPORT, LEMARCHAND-BERAUD AND FELBER
bacterial growth was found to be minimal during the times employed. After completion of dialysis, the bags were weighed, and 0.1 ml aliquots of dialysand and dialysate were placed in scintillation vials, solubilized and counted as described. The nature of the labeled compounds was determined by radiochromatography in the dialysate and dialysand. The empty bags were washed with buffer and dichloromethane, the combined washings and the bag were counted for adsorbed radioactivity. Percent free F was expressed as the ratio of cpm concentration in the dialysate over that of the dialysand. Free plasma F concentration was calculated as (total plasma F) x (% free F). 6)
Radiochromatography
Ascending chromatography of original 3H-cortisol, dialysate and dialysand was performed on Whatman no. 3 paper in benzene:methanol: water = 4:2:1, (vol:vol:vol). Developed strips were first scanned in a strip scanner and then cut into 10 mm wide transverse sections. Each section was eluted with toluene and the eluate was counted in a liquid scintillation spectrometer. 7) Free cortisol index (FFI) Free Cortisol Index was calculated as (total plasma F) x (charcoal. F uptake ratio). The same reference serum pool was used as a control in all assays, i.e., total plasma F, CFU and dialysis. 8) Subjects Heparinized blood samples from 18 young normal volunteers (15 male, 3 female) were drawn at different times throughout the day. None was taking any medication. Similar blood samples were obtained from 17 healthy young females taking oral contraceptives for at least 6 months prior to the time of study. In addition, plasma samples from 12 women at different stages of their pregnancy (19 to 38 weeks) were studied. 10 subjects of group I were studied in the same way during ACTH tests after informed consent had been given. 1 mg of jQ'-^-ACTH (Synacthen CIBA) was injected intra-
JCE & M . 1975 Vol 40 • No 3
muscularly at 9 AM and blood samples were taken 2, 4 and 6 h after injection. Results 1) Cortisol, charcoal cortisol uptake, free cortisol index and Free Cortisol The mean values, ranges, and standard deviations of total plasma cortisol (F), charcoal cortisol uptake in percent (CFU), charcoal cortisol uptake ratio (CFUR), free cortisol index (FFI)3, percent free cortisol (% FF) and free cortisol concentration (FF) for the different groups at standard clock times are summarized in Tables 1 and 2. The individual measurements, obtained throughout the day, are given in Fig. 1. The average values from multiple assays for the reference serum were: F 8.76 fig/100 ml, CFU 66.2%, CFUR 1.00 by definition, FFI 8.76%, FF 7.1%, FF 0.62 /xg/100 ml. 2) Equilibrium dialysis During the dialysis experiments it was found that equilibrium was reached after 12 to 16 h. After this time identical results were obtained when tracer was added to the inside or to the outside of the dialysis bag and with dialysis carried out over a total period of 72 h. No significant fluid shifts were detectable by comparison of the weights of the bags before and after dialysis. Adsorption of isotope to the dialysis membrane was found to be negligible (0.01% of total radioactivity). Radiochromatography of the dialysate and the dialyzed plasma after completion of dialysis showed a single peak with the Rf of the cortisol originally used. No difference in percent dialyzable isotope was found when different volumes of plasma and outer buffer were used (1 ml to 5 ml and 3 ml to 20 ml, respec-
3 To avoid confusion with true concentrations of FF, we elected to express FFI as a dimensionless parameter, although formally it has the dimension of >g/100 ml."
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7.4-22.0 (12.9 ± 4.1)
1.0-12.0
0.8-5.8
21.0-43.8(29.0 + 6.4)
8:00 AM
5 : 0 0 PM
Midnight
ACTH
Clock time
0.98-1.20 (1.13 ± 0.08)
0.80-0.98 (0.91 ± 0.07)
0.72-1.13 (0.97 ± 0.11)
8-10 AM
b) Pregnant
19.8-54.8 (33.5 ± 10.1)
6.2-22.9 (12.9 ± 7.1)
5 : 0 0 PM
28.1-41.8 (35.8 ± 3.9)'
20.4-47.9 (29.0 ± 12.7)
33.3-53.1 (38.1 ± 9.7)
%
/xg/100 ml
19.8-61.2 (34.6 ± 13.1)
CFU
Total F
8 : 0 0 AM
contraceptives
a) Oral
65.4-81.6 (74.5 ± 4.8)
52.6-62.3 (60.1 ± 4.5)
47.2-76.9 (65.7 ± 8.5)
0.92-1.16 (1.05 ± 0.08)
CFUR
(2.4 ± 2.0)
(5.3 ± 2.7)
24.4-45.8 (32.4 ± 6.0)
0.7-5.3
1.1-11.7
9.0-29.3 (13.9 ± 5.2)
FFP
(7.9 ± 2.6)
10.9-15.6 (13.1 ± 1.5)
4.8-12.3
4.1-12.6 (10.0 ± 3.2)
5.3-15.1 (10.6 ± 2.8)
%FF
2.58-6.47(3.9
0.42-0.63 (0.54 ± 0.06)
0.32-0.72 (0.44 ± 0.18)
0.49-0.80 (0.61 ± 0.09)
CFUR
(5.0 ± 1.6)
10.7-26.6(17.8 + 4.5)
4.0-7.3
9.8-35.6 (20.6 ± 6.8)
FFI
3.0-12.4 (6.8 ± 2.6)
4 . 1 - 9.2 (5.4 ± 2.6)
2.7- 9.6 (5.7 ± 1.9)
% FF
1.21-3.57 (2.17 ± 0.78)
0.46-1.81 (0.63 ± 0.32)
0.8 -3.25 (2.04 ± 0.78)
Hg/100 ml
FF
±1.13)
0.08-0.47 (0.20 ± 0.17)
0.21-1.43 (0.46 ± 0.27)
0.54-2.80 (1.38 ± 0.63)
fig/100 ml
FF
T A B L E 2. Plasma cortisol parameters at standard clock times in normal females on oral contraceptives and in pregnant subjects (range, mean ± SD)
(2.6 ± 2.2)
(5.5 ±2.4)
%
/xg/100 ml
Time
60.6-77.8 (70.1 ± 5.0)
CFU
Total F
Clock
TABLE 1. Plasma cortisol parameters at standard clock times and after standard ACTH stimulation in normal subjects (range, mean ± SD;
or
W X
D
I—I
o r z
H i—i
50
n o
5
BAUMANN, RAPPAPORT, LEMARCHAND-BERAUD AND FELBER
466
JCE & M • 1975 Vol 40 • No 3
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§ 50
Nor mals ACTH Estrogens Pregnant
-4
Ho
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40
A
o •
a. UI
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§30
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3 20 a.
S io 1 A
I1-6 - 1
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§0.8
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8 0.4 -
FIG. 1. Total plasma cortisol, "Charcoal cortisol uptake ratio," "Free Cortisol Index," and Free Cortisol Concentration at various clock times in normal, ACTH stimulated, estrogentreated and pregnant subjects.
a:
5 o
n
A
6 -
60
_
50
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E
A
o
2
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• V
20
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E CORTISOL
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with a mean x-intercept of 18.1 ± 3.24 100 ml in the normal and ACTH-stimulated subjects. It was 51.2 jug/100 ml in one "pill plasma" where enough points were accumulated to permit Scatchard analysis. Since maximal binding capacity is independent of temperature and can be deter3) Relationship between free cortisol index mined at non-equilibrium conditions (10), and free cortisol concentration these values represent CBG maximal bindA close positive correlation between FFI ing capacity. Scatchard analysis of the refand FF was found (P < 0.001), as shown erence serum under equilibrium conditions at 37 C gave an apparent maximal binding in Fig. 3. capacity of 21.0 /ag/100 ml and an apparent association constant of 2.24 x 107 liters/mol 4) CBG binding characteristics for CBG-cortisol interaction. Scatchard analysis of the values obtained yielded a low capacity binding component SD.
tively). Dilution of plasma, however, had a marked effect on percent dialyzable F, even when the amount of tracer added was adjusted according to the protein dilution in the sample (Fig. 2). The experiments were therefore carried out with undiluted plasma.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 16:36 For personal use only. No other uses without permission. . All rights reserved.
467
FREE CORTISOL INDEX IN HUMAN PLASMA
5) Accuracy, precision and reproducibility The recovery of F in routine plasma F determinations in our laboratory averages 92%, the coefficient of variation averages 7%. Intra-assay variability for CFU was determined from 6 replicate determinations of the reference serum in the same assay. The coefficient of variation found was 2.1%. Interassay variability for CFU was determined using 9 different plasmas with total F concentrations ranging from 1.0 to 26.8 /xg/100 ml in 4 different assays. A mean coefficient of variation of 3.5% was found (range 1.2-6.8%). Since the addition of charcoal to the reaction mixture resulted in mild non-equilibrium conditions, strict adherence to the time schedules after charcoal addition was necessary to achieve this degree of quality. An important factor affecting interassay reproducibility of CFU was found to be the composition of the tracer solution added, although this is partially corrected by the use of CFUR. The tracer solution was therefore stored tightly sealed at —20 C to minimize evaporation of solvent and care was taken to prepare different batches in an identical fashion.
30
25
*
20
15
10
undiluted
1:2
1:5
1:10
plasma dilution
FIG. 2. Two representative examples of the effect of plasma dilution on percent dialyzable cortisol. Diluent was 0.1M phosphate buffer, pH 7.4. Dialysis was carried out at 37 C for 24 h against the same buffer. The amount of cortisol tracer added was adjusted in proportion to plasma dilution (7, 3.5, 1.4 and 0.7 ng/ml).
FIG. 3. Correlation between "Free Cortisol Index" and free cortisol concentration as determined by equilibrium dialysis in normal, ACTH-stimulated, estrogen-treated and pregnant subjects.
estrogen! pregnant
p
