SOLVOLYSABLE DEOXYCORTICOSTERONE CONJUGATES IN HUMAN URINE THE LATE
Endocrine Unit,
C. L. COPE
AND
S. LOIZOU
Department of Medicine, Royal Postgraduate
Medical School, Du Cane Road, London, W12 OHS
(Received 29 April 1975) SUMMARY
The nature of the urinary conjugate converted by solvolysis, to free unconjugated deoxycorticosterone (DOC) was studied. A comparison of 11 solvolysis techniques has shown that the method employed in this study yielded 86 % of the highest yield by any of the techniques tried. Three successive chromatographic systems on paper showed that no appreciable amounts of contaminants were present in the free DOC eluates, following solvolysis. By preparing authentic [3H]DOC sulphate and subjecting it to solvolysis it was shown that more than 90 % of the tritiated DOC was recovered, after chromatography of the free DOC extract. This suggests that much of the solvolysable DOC in human urine is present in the form of the sulphate conjugate. The levels of DOC, excreted as the solvolysable conjugate in a variety of urine specimens, were shown to be much higher than those of free DOC, the former being 4\m=.\8 to 127 times higher than the amount of the latter. This highly variable ratio suggests that the site of production of solvolysable DOC is different from that for free DOC. The only correlation between free and solvolysable DOC was shown in dexamethasone-suppressed patients, in whom the mean percentage remaining after suppression was 30\m=.\6 % for free DOC, 24\m=.\1 % for solvolysable DOC and 22\m=.\2 % for cortisol. As solvolysable DOC is present in much larger amounts in urine, care is necessary in the storage of urine samples in which free DOC estimates are to be made, as we found that urine specimens left at room temperature for 1 week could show rises of as much as 400 % of their starting free DOC levels.
INTRODUCTION
Although the mineralocorticoid 11-deoxycorticosterone (DOC) was one of the first steroids of adrenal origin to be isolated (Reichstein & Euw, 1938), interest in its possible role in the human body rapidly waned after the discovery of the presence of cortisol and aldosterone in the plasma, because for many years DOC could not be detected in either plasma or urine. More recent techniques, however, have made possible the estimation of DOC in plasma both by double isotope derivative (Oddie, Coughlan & Scoggins, 1972) and by radioimmuno¬ assay methods (Arnold & James, 1971). Additional interest in this mineralocorticoid has been aroused by the discovery of a link between overproduction of DOC and many hypokaliaemic disorders, both in normotensive and in hypertensive subjects (Biglieri, 1965; Brown, Ferriss, Fraser, Lever, Love, Robertson & Wilson, 1972). Recently Cope & Loizou (1975) have used a radioimmunoassay technique to detect the presence of free unconjugated DOC in normal urine, and to estimate its amount which ranged from 13-7 to 76-7 ng daily with a mean of 41 ng. The quantity
excreted was not increased in uncomplicated hypertension (mean 28-8 ng), and was in the normal range in ten out of twelve cases of active Cushing's syndrome due to adrenal hyper¬ plasia (mean 38-1 ng). The excretion of free DOC was however found to be above the normal range in eight out of nine subjects suffering from hypokaliaemia, their outputs ranged from 145 to 1820 ng daily. These findings thus support the belief that there is a link between disordered potassium metabolism and the adrenal production of DOC. During the course of this present work it was found that a much greater quantity of DOC is present in normal urine in a form which can be converted into free DOC by the process of solvolysis. This communication reports on the excretion of solvolysable DOC in normal, hypertensive, hyperadrenal and some hypokaliaemic subjects and considers the relationship of this solvolysable fraction to the free DOC excreted by the same subjects. MATERIALS
Deoxycorticosterone (21-hydroxy-4-pregnene-3,20-dione) was obtained from Koch-Light Laboratories, Colnbrook, Bucks, and [l,2-3H]deoxycorticosterone from New England Nuclear Corp., Dreieichenhain, West Germany. The stated activity of the latter was 45 Ci/mmol and its radiopurity, tested at intervals by chromatography with inert DOC as carrier, exceeded 97 %. Anti-deoxycorticosterone serum was received as a generous gift from Professor V. H. T. James. It had been prepared in rabbits by the method of Arnold & James (1971) using a 3-oxime conjugate. Measurements of its cross-reactivity with other steroids have been reported by Cope & Loizou (1975); specificity for DOC was found to be high. Organic solvents were of Analar grade. Benzene and ethyl acetate used as solvents were redistilled before use. All dichloromethane was redistilled and was in addition freshly washed twice with an equal volume of water immediately before use. Ethanol (RR) was purified by refluxing with sodium hydroxide and silver nitrate before final distillation through ' a Vigreux column. Dextran 70 was obtained from Pharmacia Ltd. Charcoal was Norit trioxide from N.V. Norit Amsterdam. special Pyridine-sulphur Algemeine Maatschappij, complex was obtained from R. N. Emmanuel Ltd of Wembley, London, as a dry powder. METHODS
Solvolysis of urine The method of Burstein & Lieberman (1958) was used. To aliquots of urine representing 1/48 of a 24 h urine collection was added sodium chloride to a concentration of 20 % and the whole was then adjusted to pH 1 -0 with 20 % sulphuric acid. Two and a half volumes of ethyl acetate were then added together with an internal standard of 20000 c.p.m. [l,2-3H]DOC. After thorough mixing the whole was incubated for 24 h at 40 °C in the dark with occasional further mixing. After the incubation, the ethyl acetate layer was separated, washed first with an equal volume of 1 -0 M-sodium hydroxide to neutralize the acid, then washed once each with 1/7 volume of 0-1 M-sodium hydroxide, 1/7 volume of 0-1 M-acetic acid and finally 1/7 volume of distilled water. The combined soda washings were backwashed with 1/3 volume of ethyl acetate. The combined ethyl acetate extract was freed of water by passage through anhydrous sodium sulphate and was evaporated to dryness under reduced pressure on a water-bath at 78 °C. The residue was dissolved in 4 ml benzeneethanol (9:1, v/v).
Extraction
DOC from urine The extraction procedure was similar to that described above but the solvolysis stage was omitted and the first wash was with 1/7 volume of 0-1 M-sodium hydroxide in place of the 1-0 M. Details were as described by Cope & Loizou (1975).
offree
Purification of extracted DOC Extracts of both free and solvolysed DOC were routinely purified before assay by paper chromatography first on Bush B3 and then on Bush A systems (Bush, 1952). Elution of the appropriate DOC zone was into benzene-ethanol (9:1, v/v). Radioimmunoassay of DOC Details of the method of assay of free unconjugated DOC were as described by Cope & Loizou (1973, 1975). The anti-DOC serum was used in a final dilution of 1 in 50000 in a 0-1 M-borate buffer at pH 80 and containing 5 g crystalline bovine plasma albumin/1. Dextran-coated charcoal was used to separate free from bound fractions. The standard curve was drawn from tubes containing zero, 005, 0-1, 0-2, 0-5 and 10 ng DOC. The bound fraction in the zero tube averaged 46 %. This assay has been shown by Cope & Loizou (1975) to have a coefficient of variation for replicate estimations at the zero point of 2-28 %, a sensitivity sufficient to detect 7 pg DOC, a coefficient of variation between assays at two different concentrations of 4-2 % and the ability to recover added DOC with an accuracy of 103 ± 5 (s.d.) %. Preparation of[l,2-3H]DOC sulphate
[l,2-3H]Deoxycorticosterone sulphate of low specific activity was prepared by dissolving pure DOC in dry benzene, adding [l,2-3H]DOC and evaporating the solvent. The residue was dissolved in dry pyridine and reacted with pyridine-sulphur trioxide complex for 5 days at room temperature in the dark. The resultant [3H]DOC sulphate was freed from reagents and from unchanged DOC by paper chromatography using the Lewbart & Schneider (1955) systems II (acid) and III (alkaline). In the former the RF was 0-18 and in the latter about 0-70. The overall yield was 35 % and the specific activity of the final product was 4170 d.p.m. DOC sulphate//¿g. The product was shown to contain ether sulphate by the méthylène blue reaction of Roy (1956) and Crepy & Rulleau-Meslin (1960). It was demonstrated to be free of unconjugated DOC by (1) absence of reaction to the anti-DOC serum, (2) low solubility in dichloro¬ methane and ethyl acetate, the partition coefficients against water being 3 and 1-5% respectively, (3) negligible tritium in the DOC zone of the B3 chromatogram, (4) absence of detectable DOC in the DOC zone of the B3 chromatogram, and finally (5) when run on an activated Florisil column using the solvents of Baulieu & Emilozzi (1962), 97 % of the recovered tritium was found in the 'sulphate' fraction. RESULTS
Extent
of solvolysis Since conjugates undergoing solvolysis was not fully determined, an appro¬ could not be employed. Deoxycorticosterone sulphate was probably internal standard priate a major component of this fraction and its behaviour is considered below, but other con¬ jugates with different properties may well have been present in addition. A comparison has therefore been made of the yields of solvolysable DOC obtained by a variety of solvolysis methods on a single normal urine sample. The results are shown in the nature of the
Table 1. Seven replicate assays of this urine sample by the 24 h solvolysis of Burstein & Lieberman (1958), which has been used routinely in this study, gave a mean of 1700 + 121 (s.d.) ng daily. The mean for the best nine solvolysis techniques on the same urine was 1793 ng daily, i.e. 105 % of the routine method. Intra-assay variation (n 8) for urinary A eluate. 6-5 in eluate and 7-6 in the second Bush or DOC the B3 was solvolysable % % =
Table 1.
Solvolysable deoxycorticosterone excretion determined in the same urine sample by 11 methods B3 eluate
Bush A eluate
Method*
(ng/day)
(ng/day)
BL24h BL48h BL72h BL96h BL168h BUT BL 24 Kornel 24 BL Am. 24 THF Mean EE24h EE BL 24
1886
1694 1824 1881 1963 1982 1517 1968 1790 1540 1793
2030 2072 2163 2592 1934 2496 1713 1987 2098 296 979
% of mean (%ofB3) BL 24 (1700) Bush A 90 90 90 91
76-5 78-5 79 104 77-5 86-3
100 107 110 115 116 89 115 105 91 105 14 46-5
% of mean of 9 methods
(1793) 94 102 105 109 110 85 110 100 86 100
* Description of methods : BL, Burstein & Lieberman (1958) solvolysis, pH 10, 20 % NaCl, 2-5 vols ethyl
acetate for 24, 48, 72, 96 168 h. BUT BL 24, butanol extract solvolysed as above (BL) for 24 h. Kornel 24, butanol extract of urine saturated with ammonium sulphate, dried and solvolysed by the Kornel (1965) method. BL Am. 24, Burstein & Lieberman solvolysis pH 10, saturated ammonium sulphate, 2-5 vols ethyl acetate for 24 h. THF, the tetrahydrofuran technique of Jacobson & Lieberman (1962). EE 24 h, urine pH 10, saturated ammonium sulphate. Solvolysis with 4 vols ether: ethanol (3:1, v/v) at or
room
temperature.
EE BL 24, urine pH 10, saturated ammonium sulphate extracted with 4 vols ether:ethanol Dried and solvolysed in 4 vols ethyl acetate for 24 h.
(3:1, v/v).
Purity of extracted solvolysed DOC Evidence for the adequate purity of free DOC extracted from unsolvolysed urine has been published by Cope & Loizou (1975). The eluate from the first or B3 chromatograms often contains impurity reacting with the antiserum but this is removed by the second chromato¬ gram in the Bush A system, constancy of specific activity on further chromatography being thereby achieved. That no significant amounts of additional contaminants were created by the solvolysis to reach the Bush A eluate is shown in Table 2. The DOC content of five different urine samples was assayed in eluate from both the second (Bush A) chromatogram and after a third chromatogram on the E4 system of Eberlein & Bongiovanni (1955). The mean alteration in specific activity in the third eluate was only 1 %. Reagent blanks For urinary-free DOC, Cope & Loizou (1975) found the reagent blank to average 18 pg/ assay tube, this being equivalent under the conditions ofthat assay to 3-6 ng/1. Urine extracts from a patient with panhypopituitarism gave values indistinguishable from the reagent blank. The process of solvolysis tends to increase the blank value. When 25 ml portions of water were submitted to the solvolysis procedure, and the product was run on the B3 and
Table 2. Purity
of extracted solvolysed deoxycorticosterone Estimated excretion (ng/day)
Sample
Bush A
E4 eluate
1 2 3 4 5
355 1700 2037 2075 4375
431 1400 1946 2042 4587
Relative sp.act.
(Bush A
Mean
=
=
100)
82-5 1210 104-5 1010 95-5 100-9
Bush A systems, the final reagent blank averaged 21 pg/tube, this being equivalent to 10-20 ng/1 depending on the dilution of the eluate. This blank thus represents less than 2 % of the mean solvolysable DOC found in normal urine samples. The lowest value for solvolysable DOC excretion which we have recorded in a human urine sample was in the above mentioned panhypopituitary subject in whom the apparent daily excretion was 55 ng after subtraction of the reagent blank.
Recovery of tritium of the internal standard
[3H]DOC used as internal standard monitors the recovery of all DOC liberated in the solvolysis vessel. Recovery of this tritium in the final Bush A eluate averaged 29-7 ± 6-4 % (n 55). This may be compared with the mean of 40-3 ± 6-8 % in the Bush A eluate of free DOC extracts. Much of the loss in the solvolysis procedure is incurred in the washing of the ethyl acetate extract. Behaviour of [3H]DOC sulphate Because of the high probability that DOC sulphate is a major component of the solvolysable DOC fraction it was relevant to compare its behaviour with that of the actual solvolysable DOC fraction. When authentic [3H]DOC sulphate was added to urine in amounts of 0-55 /¿g/100 ml and subjected to solvolysis for 24 h at 40 °C, more than 90 % of the tritium entered the ethyl acetate phase, and all of this was found on chromatography to be free DOC. The recovery of [3H]DOC sulphate added to urine was 93-3 ± 11% (n 10). When butanol a was mixture added of normal urine and the was to extract [3H]DOC sulphate the Emilozzi of Baulieu & activated Florisil column solvents an passed through using (1962), 95% of the tritium recovered was found in the 'sulphate' fraction, but when an equal amount of the same butanol extract without added [3H]DOC sulphate was passed through an identical column and the effluent fractions were assayed for DOC, the total recovery was 99-3 % of the DOC content of the original extract, but only 76 % of this was present in the sulphate fraction. When the same butanol extract was hydrolysed with /ff-glucuronidase from Patella vulgata, which also contains much sulphatase, 92 % was recovered as free DOC suggesting that little if any additional DOC is present as glucuronide. Normal excretion rates of solvolysable DOC Excretion of solvolysable DOC was measured in 14 normal human subjects of both sexes (7 men and 7 women). Results varied widely from 0-20 to 2-75 /¿g daily, the mean being 1-16/¿g daily. The scatter of values was much greater in women than men. In women the values were lower and ranged from 0-20 to 1-28 µg daily, whereas in men the range was 1-5 to 2-75 µg daily; of the seven female subjects, three were taking the contraceptive pill and the scatter of their solvolysable DOC excretions was not different from that of the other
The
=
=
four.
Essential hypertension DOC excretions found in 14 subjects suffering from essential solvolysable hypertension without evidence of general metabolic disturbance was also wide, ranging from 0-18 to 3-2 /¿g daily, the mean for the group being 1-22 µg daily. The scatter was again greater in women than in men, the two highest and the two lowest values all being found in women. The mean free DOC excretion for this group was 28-8 ng daily. Owing to the wide scatter the difference of the hypertensive group from the normal was not statistically significant, nor was the difference between the sexes. The range of
Table 3. Levels
of urinary free cortisol and free and solvolysable deoxycorticosterone (DOC) Cushing's syndrome before and after dexamethasone suppression
in patients with
Solvolysable DOC (ng/day)
Free DOC
(ng/day) Patient M.O. D.A. B.U. R.O. F.L. Mean
Before
After
115-7 58-3 58-9 1790 890
34 9-8 7-9 99 34
% remaining
Before
After
29-6 16-8 13-4 550 38-2
2260 2750 1180 3050 1840
1150 140 150 1150 260
30-6% Urinary free cortisol (/¿g/day)
Patient M.O. D.A. B.U. R.O. F.L. Mean
Before 688 160 200 299 2586
Endocrine Unit,
C. L. COPE
AND
S. LOIZOU
Department of Medicine, Royal Postgraduate
Medical School, Du Cane Road, London, W12 OHS
(Received 29 April 1975) SUMMARY
The nature of the urinary conjugate converted by solvolysis, to free unconjugated deoxycorticosterone (DOC) was studied. A comparison of 11 solvolysis techniques has shown that the method employed in this study yielded 86 % of the highest yield by any of the techniques tried. Three successive chromatographic systems on paper showed that no appreciable amounts of contaminants were present in the free DOC eluates, following solvolysis. By preparing authentic [3H]DOC sulphate and subjecting it to solvolysis it was shown that more than 90 % of the tritiated DOC was recovered, after chromatography of the free DOC extract. This suggests that much of the solvolysable DOC in human urine is present in the form of the sulphate conjugate. The levels of DOC, excreted as the solvolysable conjugate in a variety of urine specimens, were shown to be much higher than those of free DOC, the former being 4\m=.\8 to 127 times higher than the amount of the latter. This highly variable ratio suggests that the site of production of solvolysable DOC is different from that for free DOC. The only correlation between free and solvolysable DOC was shown in dexamethasone-suppressed patients, in whom the mean percentage remaining after suppression was 30\m=.\6 % for free DOC, 24\m=.\1 % for solvolysable DOC and 22\m=.\2 % for cortisol. As solvolysable DOC is present in much larger amounts in urine, care is necessary in the storage of urine samples in which free DOC estimates are to be made, as we found that urine specimens left at room temperature for 1 week could show rises of as much as 400 % of their starting free DOC levels.
INTRODUCTION
Although the mineralocorticoid 11-deoxycorticosterone (DOC) was one of the first steroids of adrenal origin to be isolated (Reichstein & Euw, 1938), interest in its possible role in the human body rapidly waned after the discovery of the presence of cortisol and aldosterone in the plasma, because for many years DOC could not be detected in either plasma or urine. More recent techniques, however, have made possible the estimation of DOC in plasma both by double isotope derivative (Oddie, Coughlan & Scoggins, 1972) and by radioimmuno¬ assay methods (Arnold & James, 1971). Additional interest in this mineralocorticoid has been aroused by the discovery of a link between overproduction of DOC and many hypokaliaemic disorders, both in normotensive and in hypertensive subjects (Biglieri, 1965; Brown, Ferriss, Fraser, Lever, Love, Robertson & Wilson, 1972). Recently Cope & Loizou (1975) have used a radioimmunoassay technique to detect the presence of free unconjugated DOC in normal urine, and to estimate its amount which ranged from 13-7 to 76-7 ng daily with a mean of 41 ng. The quantity
excreted was not increased in uncomplicated hypertension (mean 28-8 ng), and was in the normal range in ten out of twelve cases of active Cushing's syndrome due to adrenal hyper¬ plasia (mean 38-1 ng). The excretion of free DOC was however found to be above the normal range in eight out of nine subjects suffering from hypokaliaemia, their outputs ranged from 145 to 1820 ng daily. These findings thus support the belief that there is a link between disordered potassium metabolism and the adrenal production of DOC. During the course of this present work it was found that a much greater quantity of DOC is present in normal urine in a form which can be converted into free DOC by the process of solvolysis. This communication reports on the excretion of solvolysable DOC in normal, hypertensive, hyperadrenal and some hypokaliaemic subjects and considers the relationship of this solvolysable fraction to the free DOC excreted by the same subjects. MATERIALS
Deoxycorticosterone (21-hydroxy-4-pregnene-3,20-dione) was obtained from Koch-Light Laboratories, Colnbrook, Bucks, and [l,2-3H]deoxycorticosterone from New England Nuclear Corp., Dreieichenhain, West Germany. The stated activity of the latter was 45 Ci/mmol and its radiopurity, tested at intervals by chromatography with inert DOC as carrier, exceeded 97 %. Anti-deoxycorticosterone serum was received as a generous gift from Professor V. H. T. James. It had been prepared in rabbits by the method of Arnold & James (1971) using a 3-oxime conjugate. Measurements of its cross-reactivity with other steroids have been reported by Cope & Loizou (1975); specificity for DOC was found to be high. Organic solvents were of Analar grade. Benzene and ethyl acetate used as solvents were redistilled before use. All dichloromethane was redistilled and was in addition freshly washed twice with an equal volume of water immediately before use. Ethanol (RR) was purified by refluxing with sodium hydroxide and silver nitrate before final distillation through ' a Vigreux column. Dextran 70 was obtained from Pharmacia Ltd. Charcoal was Norit trioxide from N.V. Norit Amsterdam. special Pyridine-sulphur Algemeine Maatschappij, complex was obtained from R. N. Emmanuel Ltd of Wembley, London, as a dry powder. METHODS
Solvolysis of urine The method of Burstein & Lieberman (1958) was used. To aliquots of urine representing 1/48 of a 24 h urine collection was added sodium chloride to a concentration of 20 % and the whole was then adjusted to pH 1 -0 with 20 % sulphuric acid. Two and a half volumes of ethyl acetate were then added together with an internal standard of 20000 c.p.m. [l,2-3H]DOC. After thorough mixing the whole was incubated for 24 h at 40 °C in the dark with occasional further mixing. After the incubation, the ethyl acetate layer was separated, washed first with an equal volume of 1 -0 M-sodium hydroxide to neutralize the acid, then washed once each with 1/7 volume of 0-1 M-sodium hydroxide, 1/7 volume of 0-1 M-acetic acid and finally 1/7 volume of distilled water. The combined soda washings were backwashed with 1/3 volume of ethyl acetate. The combined ethyl acetate extract was freed of water by passage through anhydrous sodium sulphate and was evaporated to dryness under reduced pressure on a water-bath at 78 °C. The residue was dissolved in 4 ml benzeneethanol (9:1, v/v).
Extraction
DOC from urine The extraction procedure was similar to that described above but the solvolysis stage was omitted and the first wash was with 1/7 volume of 0-1 M-sodium hydroxide in place of the 1-0 M. Details were as described by Cope & Loizou (1975).
offree
Purification of extracted DOC Extracts of both free and solvolysed DOC were routinely purified before assay by paper chromatography first on Bush B3 and then on Bush A systems (Bush, 1952). Elution of the appropriate DOC zone was into benzene-ethanol (9:1, v/v). Radioimmunoassay of DOC Details of the method of assay of free unconjugated DOC were as described by Cope & Loizou (1973, 1975). The anti-DOC serum was used in a final dilution of 1 in 50000 in a 0-1 M-borate buffer at pH 80 and containing 5 g crystalline bovine plasma albumin/1. Dextran-coated charcoal was used to separate free from bound fractions. The standard curve was drawn from tubes containing zero, 005, 0-1, 0-2, 0-5 and 10 ng DOC. The bound fraction in the zero tube averaged 46 %. This assay has been shown by Cope & Loizou (1975) to have a coefficient of variation for replicate estimations at the zero point of 2-28 %, a sensitivity sufficient to detect 7 pg DOC, a coefficient of variation between assays at two different concentrations of 4-2 % and the ability to recover added DOC with an accuracy of 103 ± 5 (s.d.) %. Preparation of[l,2-3H]DOC sulphate
[l,2-3H]Deoxycorticosterone sulphate of low specific activity was prepared by dissolving pure DOC in dry benzene, adding [l,2-3H]DOC and evaporating the solvent. The residue was dissolved in dry pyridine and reacted with pyridine-sulphur trioxide complex for 5 days at room temperature in the dark. The resultant [3H]DOC sulphate was freed from reagents and from unchanged DOC by paper chromatography using the Lewbart & Schneider (1955) systems II (acid) and III (alkaline). In the former the RF was 0-18 and in the latter about 0-70. The overall yield was 35 % and the specific activity of the final product was 4170 d.p.m. DOC sulphate//¿g. The product was shown to contain ether sulphate by the méthylène blue reaction of Roy (1956) and Crepy & Rulleau-Meslin (1960). It was demonstrated to be free of unconjugated DOC by (1) absence of reaction to the anti-DOC serum, (2) low solubility in dichloro¬ methane and ethyl acetate, the partition coefficients against water being 3 and 1-5% respectively, (3) negligible tritium in the DOC zone of the B3 chromatogram, (4) absence of detectable DOC in the DOC zone of the B3 chromatogram, and finally (5) when run on an activated Florisil column using the solvents of Baulieu & Emilozzi (1962), 97 % of the recovered tritium was found in the 'sulphate' fraction. RESULTS
Extent
of solvolysis Since conjugates undergoing solvolysis was not fully determined, an appro¬ could not be employed. Deoxycorticosterone sulphate was probably internal standard priate a major component of this fraction and its behaviour is considered below, but other con¬ jugates with different properties may well have been present in addition. A comparison has therefore been made of the yields of solvolysable DOC obtained by a variety of solvolysis methods on a single normal urine sample. The results are shown in the nature of the
Table 1. Seven replicate assays of this urine sample by the 24 h solvolysis of Burstein & Lieberman (1958), which has been used routinely in this study, gave a mean of 1700 + 121 (s.d.) ng daily. The mean for the best nine solvolysis techniques on the same urine was 1793 ng daily, i.e. 105 % of the routine method. Intra-assay variation (n 8) for urinary A eluate. 6-5 in eluate and 7-6 in the second Bush or DOC the B3 was solvolysable % % =
Table 1.
Solvolysable deoxycorticosterone excretion determined in the same urine sample by 11 methods B3 eluate
Bush A eluate
Method*
(ng/day)
(ng/day)
BL24h BL48h BL72h BL96h BL168h BUT BL 24 Kornel 24 BL Am. 24 THF Mean EE24h EE BL 24
1886
1694 1824 1881 1963 1982 1517 1968 1790 1540 1793
2030 2072 2163 2592 1934 2496 1713 1987 2098 296 979
% of mean (%ofB3) BL 24 (1700) Bush A 90 90 90 91
76-5 78-5 79 104 77-5 86-3
100 107 110 115 116 89 115 105 91 105 14 46-5
% of mean of 9 methods
(1793) 94 102 105 109 110 85 110 100 86 100
* Description of methods : BL, Burstein & Lieberman (1958) solvolysis, pH 10, 20 % NaCl, 2-5 vols ethyl
acetate for 24, 48, 72, 96 168 h. BUT BL 24, butanol extract solvolysed as above (BL) for 24 h. Kornel 24, butanol extract of urine saturated with ammonium sulphate, dried and solvolysed by the Kornel (1965) method. BL Am. 24, Burstein & Lieberman solvolysis pH 10, saturated ammonium sulphate, 2-5 vols ethyl acetate for 24 h. THF, the tetrahydrofuran technique of Jacobson & Lieberman (1962). EE 24 h, urine pH 10, saturated ammonium sulphate. Solvolysis with 4 vols ether: ethanol (3:1, v/v) at or
room
temperature.
EE BL 24, urine pH 10, saturated ammonium sulphate extracted with 4 vols ether:ethanol Dried and solvolysed in 4 vols ethyl acetate for 24 h.
(3:1, v/v).
Purity of extracted solvolysed DOC Evidence for the adequate purity of free DOC extracted from unsolvolysed urine has been published by Cope & Loizou (1975). The eluate from the first or B3 chromatograms often contains impurity reacting with the antiserum but this is removed by the second chromato¬ gram in the Bush A system, constancy of specific activity on further chromatography being thereby achieved. That no significant amounts of additional contaminants were created by the solvolysis to reach the Bush A eluate is shown in Table 2. The DOC content of five different urine samples was assayed in eluate from both the second (Bush A) chromatogram and after a third chromatogram on the E4 system of Eberlein & Bongiovanni (1955). The mean alteration in specific activity in the third eluate was only 1 %. Reagent blanks For urinary-free DOC, Cope & Loizou (1975) found the reagent blank to average 18 pg/ assay tube, this being equivalent under the conditions ofthat assay to 3-6 ng/1. Urine extracts from a patient with panhypopituitarism gave values indistinguishable from the reagent blank. The process of solvolysis tends to increase the blank value. When 25 ml portions of water were submitted to the solvolysis procedure, and the product was run on the B3 and
Table 2. Purity
of extracted solvolysed deoxycorticosterone Estimated excretion (ng/day)
Sample
Bush A
E4 eluate
1 2 3 4 5
355 1700 2037 2075 4375
431 1400 1946 2042 4587
Relative sp.act.
(Bush A
Mean
=
=
100)
82-5 1210 104-5 1010 95-5 100-9
Bush A systems, the final reagent blank averaged 21 pg/tube, this being equivalent to 10-20 ng/1 depending on the dilution of the eluate. This blank thus represents less than 2 % of the mean solvolysable DOC found in normal urine samples. The lowest value for solvolysable DOC excretion which we have recorded in a human urine sample was in the above mentioned panhypopituitary subject in whom the apparent daily excretion was 55 ng after subtraction of the reagent blank.
Recovery of tritium of the internal standard
[3H]DOC used as internal standard monitors the recovery of all DOC liberated in the solvolysis vessel. Recovery of this tritium in the final Bush A eluate averaged 29-7 ± 6-4 % (n 55). This may be compared with the mean of 40-3 ± 6-8 % in the Bush A eluate of free DOC extracts. Much of the loss in the solvolysis procedure is incurred in the washing of the ethyl acetate extract. Behaviour of [3H]DOC sulphate Because of the high probability that DOC sulphate is a major component of the solvolysable DOC fraction it was relevant to compare its behaviour with that of the actual solvolysable DOC fraction. When authentic [3H]DOC sulphate was added to urine in amounts of 0-55 /¿g/100 ml and subjected to solvolysis for 24 h at 40 °C, more than 90 % of the tritium entered the ethyl acetate phase, and all of this was found on chromatography to be free DOC. The recovery of [3H]DOC sulphate added to urine was 93-3 ± 11% (n 10). When butanol a was mixture added of normal urine and the was to extract [3H]DOC sulphate the Emilozzi of Baulieu & activated Florisil column solvents an passed through using (1962), 95% of the tritium recovered was found in the 'sulphate' fraction, but when an equal amount of the same butanol extract without added [3H]DOC sulphate was passed through an identical column and the effluent fractions were assayed for DOC, the total recovery was 99-3 % of the DOC content of the original extract, but only 76 % of this was present in the sulphate fraction. When the same butanol extract was hydrolysed with /ff-glucuronidase from Patella vulgata, which also contains much sulphatase, 92 % was recovered as free DOC suggesting that little if any additional DOC is present as glucuronide. Normal excretion rates of solvolysable DOC Excretion of solvolysable DOC was measured in 14 normal human subjects of both sexes (7 men and 7 women). Results varied widely from 0-20 to 2-75 /¿g daily, the mean being 1-16/¿g daily. The scatter of values was much greater in women than men. In women the values were lower and ranged from 0-20 to 1-28 µg daily, whereas in men the range was 1-5 to 2-75 µg daily; of the seven female subjects, three were taking the contraceptive pill and the scatter of their solvolysable DOC excretions was not different from that of the other
The
=
=
four.
Essential hypertension DOC excretions found in 14 subjects suffering from essential solvolysable hypertension without evidence of general metabolic disturbance was also wide, ranging from 0-18 to 3-2 /¿g daily, the mean for the group being 1-22 µg daily. The scatter was again greater in women than in men, the two highest and the two lowest values all being found in women. The mean free DOC excretion for this group was 28-8 ng daily. Owing to the wide scatter the difference of the hypertensive group from the normal was not statistically significant, nor was the difference between the sexes. The range of
Table 3. Levels
of urinary free cortisol and free and solvolysable deoxycorticosterone (DOC) Cushing's syndrome before and after dexamethasone suppression
in patients with
Solvolysable DOC (ng/day)
Free DOC
(ng/day) Patient M.O. D.A. B.U. R.O. F.L. Mean
Before
After
115-7 58-3 58-9 1790 890
34 9-8 7-9 99 34
% remaining
Before
After
29-6 16-8 13-4 550 38-2
2260 2750 1180 3050 1840
1150 140 150 1150 260
30-6% Urinary free cortisol (/¿g/day)
Patient M.O. D.A. B.U. R.O. F.L. Mean
Before 688 160 200 299 2586
