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The use of amberlite XAD-2 columns for the determination of plasma aldosterone by radioimmunoassay
303
Clinica Chimico Acta, 63 (1975) 303-308 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7200
THE USE OF AMBERLITE XAD-2 COLUMNS FOR THE DETERMINATION OF PLASMA ALDOSTERONE BY RADIOIMMUNOASSAY
MARTIN
S. ROGINSKY,
ALLEN
I. PANETZ
and RICHARD
Department of Medicine, Nassau Counly Medical Center, and Brinkmann Instruments, Inc. Westbury, N. Y. 11590
(Received
D. GORDON
East Meadow, (C’.S.A.)
N. Y. 11554
March 19, 1975)
Summary
A rapid radioimmunoassay for the measurement of plasma aldosterone has been developed that uses an Amberlite XAD-2 column for both the extraction of aldosterone from plasma and for the on-column oxidation of aldosterone to form the gamma-lactone derivative. This unique time saving step allows a single technician to process a minimum of 25 specimens daily.
Introduction
Advances with the technique of radioimmunoassay have made possible the development of methods with the sensitivity required to quantitate the small amount of aldosterone present in blood [l-3]. Unfortunately, the need to separate aldosterone from the much greater concentrations of other steroids that cross-react with commercially available antibodies has created problems. Farmer et al. [4] succeeded in overcoming this problem by periodic acid oxidation of aldosterone to the gamma-lactone derivative, a step that facilitated the chemical elimination of other cross-reacting steroids. This procedure, however, as in most others that have been described, are time consuming, tedious and with poor recoveries. Amberlite XAD-2 resin columns have been used to eliminate time consuming extraction steps in the isolation of plasma and urinary steroids [5] . Chemical reactions to form derivatives directly on the resin column have not been previously described. In this report using a column of Amberlite XAD-2 resin, on-column oxidation of aldosterone with sodium metaperiodate has been successfully achieved. This oxidation step combined with the use of a commercially available antibody to the gamma-lactone derivative, has made possible the development of a rapid precise and reproducible method to measure the concentration of aldosterone in plasma.
304
Materials and methods Solvents. All organic solvents were spectroquality and were used as supplied. Glassware was either acid washed or disposable. Tris buffer. Tris buffer, 0.1 M was prepared by dissolving 12.114 g Tris, 3.0 glysozyme (3 X crystallized), 3.0 g bovine serum albumin, 2.0 g neomycin sulfate and 0.1 ml Hibitane (chlorhexedine, Imperial Chemical Company) in distilled water and diluted to 1 liter. Buffer pH was adjusted to 8.0 by the addition of 10 percent acetic acid and filtered. This buffer is stable for at least 3 months when stored at 4°C. Dextrun charcoal solution. Five grams Norit A charcoal and 0.5 g dextran T-70 were added to 100 ml Tris buffer. This solution is stable for two weeks when stored at 4°C. Sodium metaperiodate. An aqueous solution containing 1 g/100 ml was freshly prepared immediately prior to use. Antiserum. Lyophilized guinea pig serum from animals immunized to produce an antibody specific for aldosterone gamma-lactone was obtained from rF Laboratories (1930 Triway Lane, Houston, Texas 77043, U.S.A.). Ten milligrams of this antiserum was dissolved in 250 ml of 0.1 M Tris buffer. The mixture was stored at 4°C. Steroids. Aldosterone obtained from Steraloids, P.O. Box 0, Pawling, New York 12564, U.S.A. was used without further purification. A 4 mg/l stock solution in methanol and 4.0 ng/ml and 40 ng/ml working standards were prepared. [ 1,2-jH] Aldosterone 30 Ci/mM (New England Nuclear) was dissolved in benzene/ethanol (9 : 1, v/v) to a concentration of 2.0 X lo6 cpm/ml. Sample collection. Plasma or serum was collected and frozen until assayed. AmberZite XAD-2 columns. Columns were 5.5 cm in length and 1.0 cm in width prepacked with 2.1 grams of Amberlite XAD-2 resin of particle size 400-550 pm diameter obtained from Brinkmann Instruments, Inc., Westbury, N.Y., U.S.A. Columns were activated immediately prior to use with 3 ml of distilled water. Aldosterone isolation. One ml of undiluted serum, working standards (0.1 ml diluted to 1 ml with distilled water) or 0.25 ml of the [1,2-3H] aldosterone solution to be later used in the preparation of the trace [ 1,2-3 H] aldosterone-lactone were added to separate columns and allowed to drain completely. Columns were then washed consecutively with 6.0 ml 0.1 N sodium hydroxide, 3.0 ml water, 3.0 0.1 N acetic acid and 3.0 ml water followed by vacuum aspiration to remove residual water. A final 6.0 ml benzene wash to elute interfering compounds is followed by vacuum aspiration to remove residual benzene. Gamma-lactone preparation. 2.5 ml of sodium metaperiodate was added to each washed column. After the first drop passed through the column, the bottom of the column was capped, and the top of the reservoir was covered with Pamfilm@. Columns were incubated in the dark, at room temperature, for two hours. Following incubation, the columns were uncapped, allowed to drain, then washed sequentially with 10 ml of 9 percent sodium bicarbonate and 4.0 ml water. Residual water on the column was removed by aspiration. The gamma-lactone derivative was eluted with 15 ml ethyl acetate. These
305
eluates were taken to dryness with air at less than 60°C and then redissolved in 1.0 ml ethyl acetate except for the [ 1,2-3 H] aldosterone gamma-la&one dried eluate that was redissolved in 1.5 ml ethyl acetate. Antibody trace incubation mixture. One ml of the [1,2-3H] aldosterone gamma-lactone was taken to dryness under air at room temperature in a 100 ml beaker. Following addition of 50 ml of the antiserum the solution was allowed to equilibrate at 4°C. This mixture is then stable for two weeks at 4°C. For non-specific binding determinations, 0.25 ml of the [1,2-3H] aldosterone gamma-lactone was taken to dryness under air at room temperature, followed by the addition of 12.5 ml of 0.1 M Tris buffer without antibody. Radisimmunoassays. 0.5 ml of gamma-lactone derivative from unknowns were transferred to 12 mm X 75 mm disposable glass test tubes in duplicate. A standard curve was prepared from standards carried through the entire procedure using aliquots calculated to give 0, 4, 8, 12, 20, 40, 80, 120 and 240 pg. After taking to dryness under air at room temperature, 1.0 ml of the antibody trace incubation mixture was added and the tubes incubated 30 minutes at room temperature followed by 60 minutes in an ice bath. 0.2 ml dextrancharcoal solution was then added to each tube and briefly Vortex shaken. Tubes were allowed to settle. Centrifugation for 10 minutes at 3000 rpm allows for separation of the bound from free hormone. The supernat~t was then transferred to a counting vial and 10 ml Tnstagel scintillation mixture added for counting. Results
1. specificity Specificity of the antiserum and the effectiveness of oxidation using sodium metaperiodate to convert aldosterone to the gamma-la&one derivative while cross-reacting steroids are oxidized to etionic acid that subsequently are removed in the bicarbonate wash have been described by Farmer et al. (41. Oncolumn oxidation was evaluated by the addition of 100 pg/dl cortisol and 10 pg/dl corticosterone to 1.0 ml plasma from a bilaterally adrenalectomized subject which was then carried through the entire procedure. Such samples consistently gave results for aldosterone concentration of less than 8 pg/ml which is similar to what was observed with the assay blank. (The non-related binding component in serum.) 2.
Recovery
The recovery of aldosterone in 26 separate assays was determined by the addition of 1000 cpm [3H] aldosterone to plasma and carrying this sample through the entire procedure. Table I summarizes the procedural losses at various steps and compares them with the liquid/liquid extraction procedure of Farmer et al. [4].
3. Precision Standard curves as calculated by a program developed for a Wang 700A programmable calculator from the mathematical analysis of Rodbard and Lewald [6] showed extremely low variances. However, precision of this type
306 TABLE I COMPARISON
OF PROCEDURAL
LOSSES (PERCENT)
Procedural step
Liquid/liquid
XAD-2
Extraction Benzene wash Periodate oxidation Other losses
13 18 13 l(t-15
5 2 10 8-13
Total losses
54-59
25-30
on column
of assay depends relatively little on the precision of individual standard curves. When samples of plasma from an adrenalectomized subject to which either a low (50 pg) or high (250 pg) amount of aldosterone was added, the intra-assay standard deviation for the former was 3.0 pg (n = 10) and for the latter 2.6 pg (n = 10). 4. Assessment of the method Plasma aldosterone concentrations assayed with the present technique were compared to values obtained using liquid-liquid extraction procedure of Farmer (Table II). Individual samples were corrected for recovery as described above. The coefficient of variations was r = 0.947, p < 0.3. 5. Sensitivity The useful range of the standard curve was from 10 to 120 pg (Fig. 1). Below 8 pg could not be distinguished significantly from the water blank. Consequently, for lower levels, larger plasma samples than described are needed. 6. Normal range under various physiological conditions Plasma aldosterone was determined in 20 normal female controls on the fifth day of a balanced diet containing 120 mequivalents of Na and 90 mequivalents of K. Blood samples were obtained in the morning with the subjects recumbent since the night before and again after 4 hours in the upright position.
TABLE II PLASMA
ALDOSTERONE
IN pg/ml IN NORMAL
Upright position-diet 120 mequivaknts
SUBJECTS
Na. 90 mequivaknts
Subject
Liquid-liquid
XAD-2
A.L. N.F. C.E. M.R. A.P. A.M. S.S. F.M. R.R.
195 146 135 154 215 138 150 235 164
200 122 158 174 232 124 158 282 170
K.
on column
307
Picagrams Fig. 1. Standard curve for aldosterone gamma-la&me.
The mean ~tl S.D. recumbent plasma aldosterone was 79 + 36 pg/ml with a range of 40-125 pg/ml. In the upright position, the mean tl S.D. was 214 2 88 pg/ml with a range of 70-275 pg/ml. Discussion Since the measurement of aldosterone in plasma could provide more useful information for clinical diagnosis and investigation than does the estimation of the excretion of metabolites, the need has arisen for reliable, rapid methods for its determination. Difficulty in the precise quantitation of low levels of this hormone normally present in plasma, however, has presented problems. These problems have been time consuming isolation steps, low recovery of steroid or the necessity for an antibody of high specificity. We have demonstrated with our procedure that XAD-2 resin can be successful both in the extraction of aldosterone from plasma with good recovery, and in the subsequent oxidation of aldosterone on column with sodium metaperiodate, a step essential in the elimination of cross-reacting interfering steroids. The total recovery of 70-75 percent using Amberlite XAD-2 is considerably greater than other procedures using the gamma-lactone preparation. Comparative data obtained using the method of Farmer et al. [4] did not show any significant difference in the values observed. Liquid-liquid extraction such as used in Farmer’s method, would not remove steroid conjugates from the plasma whereas the XAD-2 column extracts both the free and conjugated steroids. Very little has been published on aldosterone conjugates in blood. Pasqualini [7] has commented that as much as 20 percent of aldosterone in plasma exists as the conjugates. Our data although not supporting this in no way is meant to refute this in that no extensive ~vestigation in a variety of clinical situations was explored. Hence,
308
we feel that the inclusion of the conjugates in the plasma aldosterone concentration does not affect the significance of this determination. In addition, the rapidity of the procedure through the elimination of liquid extractions, time consuming oxidation and overnight incubation with the antibody has resulted in a method whereby a single technician can process at least 25 separate plasma samples in a single day’s work. References 1
M.J.St.
2
B.T.
Cyr,
Martin
M.M. and
3
C. Gomez-Sanchez,
4
R.W.
Farmer,
5
H.L.
Bradlow,
6
D. Rodbard
7
J.R.
Sancho C.A.
D.C.
D.H. and
J.E.
11
and
P.Y.
Baulieu
Melby, N.M.
Howard
(1968)
Lewald,
in E.E.
J.C.
Steroids,
Kern
Brown.
Steroids,
Pasqualini,
and
Nugent.
Acta and
Clin.
21
Chem..
(1973)
Kaplan, and
18 (1972)
1395
169 J. Clin.
L.F.
Fabre,
Endocrinol., J. Clin.
36
(1973)
Endocrinol.,
795 36
(1973)
460
265 Endocrinol. P. Robe1
Suppl.. (eds),
147
(1970)
Aldosterone,
79 p. 131.
Blackwell,
Oxford.
1964
Clinica Chimico Acta, 63 (1975) 303-308 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7200
THE USE OF AMBERLITE XAD-2 COLUMNS FOR THE DETERMINATION OF PLASMA ALDOSTERONE BY RADIOIMMUNOASSAY
MARTIN
S. ROGINSKY,
ALLEN
I. PANETZ
and RICHARD
Department of Medicine, Nassau Counly Medical Center, and Brinkmann Instruments, Inc. Westbury, N. Y. 11590
(Received
D. GORDON
East Meadow, (C’.S.A.)
N. Y. 11554
March 19, 1975)
Summary
A rapid radioimmunoassay for the measurement of plasma aldosterone has been developed that uses an Amberlite XAD-2 column for both the extraction of aldosterone from plasma and for the on-column oxidation of aldosterone to form the gamma-lactone derivative. This unique time saving step allows a single technician to process a minimum of 25 specimens daily.
Introduction
Advances with the technique of radioimmunoassay have made possible the development of methods with the sensitivity required to quantitate the small amount of aldosterone present in blood [l-3]. Unfortunately, the need to separate aldosterone from the much greater concentrations of other steroids that cross-react with commercially available antibodies has created problems. Farmer et al. [4] succeeded in overcoming this problem by periodic acid oxidation of aldosterone to the gamma-lactone derivative, a step that facilitated the chemical elimination of other cross-reacting steroids. This procedure, however, as in most others that have been described, are time consuming, tedious and with poor recoveries. Amberlite XAD-2 resin columns have been used to eliminate time consuming extraction steps in the isolation of plasma and urinary steroids [5] . Chemical reactions to form derivatives directly on the resin column have not been previously described. In this report using a column of Amberlite XAD-2 resin, on-column oxidation of aldosterone with sodium metaperiodate has been successfully achieved. This oxidation step combined with the use of a commercially available antibody to the gamma-lactone derivative, has made possible the development of a rapid precise and reproducible method to measure the concentration of aldosterone in plasma.
304
Materials and methods Solvents. All organic solvents were spectroquality and were used as supplied. Glassware was either acid washed or disposable. Tris buffer. Tris buffer, 0.1 M was prepared by dissolving 12.114 g Tris, 3.0 glysozyme (3 X crystallized), 3.0 g bovine serum albumin, 2.0 g neomycin sulfate and 0.1 ml Hibitane (chlorhexedine, Imperial Chemical Company) in distilled water and diluted to 1 liter. Buffer pH was adjusted to 8.0 by the addition of 10 percent acetic acid and filtered. This buffer is stable for at least 3 months when stored at 4°C. Dextrun charcoal solution. Five grams Norit A charcoal and 0.5 g dextran T-70 were added to 100 ml Tris buffer. This solution is stable for two weeks when stored at 4°C. Sodium metaperiodate. An aqueous solution containing 1 g/100 ml was freshly prepared immediately prior to use. Antiserum. Lyophilized guinea pig serum from animals immunized to produce an antibody specific for aldosterone gamma-lactone was obtained from rF Laboratories (1930 Triway Lane, Houston, Texas 77043, U.S.A.). Ten milligrams of this antiserum was dissolved in 250 ml of 0.1 M Tris buffer. The mixture was stored at 4°C. Steroids. Aldosterone obtained from Steraloids, P.O. Box 0, Pawling, New York 12564, U.S.A. was used without further purification. A 4 mg/l stock solution in methanol and 4.0 ng/ml and 40 ng/ml working standards were prepared. [ 1,2-jH] Aldosterone 30 Ci/mM (New England Nuclear) was dissolved in benzene/ethanol (9 : 1, v/v) to a concentration of 2.0 X lo6 cpm/ml. Sample collection. Plasma or serum was collected and frozen until assayed. AmberZite XAD-2 columns. Columns were 5.5 cm in length and 1.0 cm in width prepacked with 2.1 grams of Amberlite XAD-2 resin of particle size 400-550 pm diameter obtained from Brinkmann Instruments, Inc., Westbury, N.Y., U.S.A. Columns were activated immediately prior to use with 3 ml of distilled water. Aldosterone isolation. One ml of undiluted serum, working standards (0.1 ml diluted to 1 ml with distilled water) or 0.25 ml of the [1,2-3H] aldosterone solution to be later used in the preparation of the trace [ 1,2-3 H] aldosterone-lactone were added to separate columns and allowed to drain completely. Columns were then washed consecutively with 6.0 ml 0.1 N sodium hydroxide, 3.0 ml water, 3.0 0.1 N acetic acid and 3.0 ml water followed by vacuum aspiration to remove residual water. A final 6.0 ml benzene wash to elute interfering compounds is followed by vacuum aspiration to remove residual benzene. Gamma-lactone preparation. 2.5 ml of sodium metaperiodate was added to each washed column. After the first drop passed through the column, the bottom of the column was capped, and the top of the reservoir was covered with Pamfilm@. Columns were incubated in the dark, at room temperature, for two hours. Following incubation, the columns were uncapped, allowed to drain, then washed sequentially with 10 ml of 9 percent sodium bicarbonate and 4.0 ml water. Residual water on the column was removed by aspiration. The gamma-lactone derivative was eluted with 15 ml ethyl acetate. These
305
eluates were taken to dryness with air at less than 60°C and then redissolved in 1.0 ml ethyl acetate except for the [ 1,2-3 H] aldosterone gamma-la&one dried eluate that was redissolved in 1.5 ml ethyl acetate. Antibody trace incubation mixture. One ml of the [1,2-3H] aldosterone gamma-lactone was taken to dryness under air at room temperature in a 100 ml beaker. Following addition of 50 ml of the antiserum the solution was allowed to equilibrate at 4°C. This mixture is then stable for two weeks at 4°C. For non-specific binding determinations, 0.25 ml of the [1,2-3H] aldosterone gamma-lactone was taken to dryness under air at room temperature, followed by the addition of 12.5 ml of 0.1 M Tris buffer without antibody. Radisimmunoassays. 0.5 ml of gamma-lactone derivative from unknowns were transferred to 12 mm X 75 mm disposable glass test tubes in duplicate. A standard curve was prepared from standards carried through the entire procedure using aliquots calculated to give 0, 4, 8, 12, 20, 40, 80, 120 and 240 pg. After taking to dryness under air at room temperature, 1.0 ml of the antibody trace incubation mixture was added and the tubes incubated 30 minutes at room temperature followed by 60 minutes in an ice bath. 0.2 ml dextrancharcoal solution was then added to each tube and briefly Vortex shaken. Tubes were allowed to settle. Centrifugation for 10 minutes at 3000 rpm allows for separation of the bound from free hormone. The supernat~t was then transferred to a counting vial and 10 ml Tnstagel scintillation mixture added for counting. Results
1. specificity Specificity of the antiserum and the effectiveness of oxidation using sodium metaperiodate to convert aldosterone to the gamma-la&one derivative while cross-reacting steroids are oxidized to etionic acid that subsequently are removed in the bicarbonate wash have been described by Farmer et al. (41. Oncolumn oxidation was evaluated by the addition of 100 pg/dl cortisol and 10 pg/dl corticosterone to 1.0 ml plasma from a bilaterally adrenalectomized subject which was then carried through the entire procedure. Such samples consistently gave results for aldosterone concentration of less than 8 pg/ml which is similar to what was observed with the assay blank. (The non-related binding component in serum.) 2.
Recovery
The recovery of aldosterone in 26 separate assays was determined by the addition of 1000 cpm [3H] aldosterone to plasma and carrying this sample through the entire procedure. Table I summarizes the procedural losses at various steps and compares them with the liquid/liquid extraction procedure of Farmer et al. [4].
3. Precision Standard curves as calculated by a program developed for a Wang 700A programmable calculator from the mathematical analysis of Rodbard and Lewald [6] showed extremely low variances. However, precision of this type
306 TABLE I COMPARISON
OF PROCEDURAL
LOSSES (PERCENT)
Procedural step
Liquid/liquid
XAD-2
Extraction Benzene wash Periodate oxidation Other losses
13 18 13 l(t-15
5 2 10 8-13
Total losses
54-59
25-30
on column
of assay depends relatively little on the precision of individual standard curves. When samples of plasma from an adrenalectomized subject to which either a low (50 pg) or high (250 pg) amount of aldosterone was added, the intra-assay standard deviation for the former was 3.0 pg (n = 10) and for the latter 2.6 pg (n = 10). 4. Assessment of the method Plasma aldosterone concentrations assayed with the present technique were compared to values obtained using liquid-liquid extraction procedure of Farmer (Table II). Individual samples were corrected for recovery as described above. The coefficient of variations was r = 0.947, p < 0.3. 5. Sensitivity The useful range of the standard curve was from 10 to 120 pg (Fig. 1). Below 8 pg could not be distinguished significantly from the water blank. Consequently, for lower levels, larger plasma samples than described are needed. 6. Normal range under various physiological conditions Plasma aldosterone was determined in 20 normal female controls on the fifth day of a balanced diet containing 120 mequivalents of Na and 90 mequivalents of K. Blood samples were obtained in the morning with the subjects recumbent since the night before and again after 4 hours in the upright position.
TABLE II PLASMA
ALDOSTERONE
IN pg/ml IN NORMAL
Upright position-diet 120 mequivaknts
SUBJECTS
Na. 90 mequivaknts
Subject
Liquid-liquid
XAD-2
A.L. N.F. C.E. M.R. A.P. A.M. S.S. F.M. R.R.
195 146 135 154 215 138 150 235 164
200 122 158 174 232 124 158 282 170
K.
on column
307
Picagrams Fig. 1. Standard curve for aldosterone gamma-la&me.
The mean ~tl S.D. recumbent plasma aldosterone was 79 + 36 pg/ml with a range of 40-125 pg/ml. In the upright position, the mean tl S.D. was 214 2 88 pg/ml with a range of 70-275 pg/ml. Discussion Since the measurement of aldosterone in plasma could provide more useful information for clinical diagnosis and investigation than does the estimation of the excretion of metabolites, the need has arisen for reliable, rapid methods for its determination. Difficulty in the precise quantitation of low levels of this hormone normally present in plasma, however, has presented problems. These problems have been time consuming isolation steps, low recovery of steroid or the necessity for an antibody of high specificity. We have demonstrated with our procedure that XAD-2 resin can be successful both in the extraction of aldosterone from plasma with good recovery, and in the subsequent oxidation of aldosterone on column with sodium metaperiodate, a step essential in the elimination of cross-reacting interfering steroids. The total recovery of 70-75 percent using Amberlite XAD-2 is considerably greater than other procedures using the gamma-lactone preparation. Comparative data obtained using the method of Farmer et al. [4] did not show any significant difference in the values observed. Liquid-liquid extraction such as used in Farmer’s method, would not remove steroid conjugates from the plasma whereas the XAD-2 column extracts both the free and conjugated steroids. Very little has been published on aldosterone conjugates in blood. Pasqualini [7] has commented that as much as 20 percent of aldosterone in plasma exists as the conjugates. Our data although not supporting this in no way is meant to refute this in that no extensive ~vestigation in a variety of clinical situations was explored. Hence,
308
we feel that the inclusion of the conjugates in the plasma aldosterone concentration does not affect the significance of this determination. In addition, the rapidity of the procedure through the elimination of liquid extractions, time consuming oxidation and overnight incubation with the antibody has resulted in a method whereby a single technician can process at least 25 separate plasma samples in a single day’s work. References 1
M.J.St.
2
B.T.
Cyr,
Martin
M.M. and
3
C. Gomez-Sanchez,
4
R.W.
Farmer,
5
H.L.
Bradlow,
6
D. Rodbard
7
J.R.
Sancho C.A.
D.C.
D.H. and
J.E.
11
and
P.Y.
Baulieu
Melby, N.M.
Howard
(1968)
Lewald,
in E.E.
J.C.
Steroids,
Kern
Brown.
Steroids,
Pasqualini,
and
Nugent.
Acta and
Clin.
21
Chem..
(1973)
Kaplan, and
18 (1972)
1395
169 J. Clin.
L.F.
Fabre,
Endocrinol., J. Clin.
36
(1973)
Endocrinol.,
795 36
(1973)
460
265 Endocrinol. P. Robe1
Suppl.. (eds),
147
(1970)
Aldosterone,
79 p. 131.
Blackwell,
Oxford.
1964