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Microchemical urinalysis
MICROCHEMICALJOURNAL 16, 443-449 (1971)
Microchemical VIII. Determination BENJAMIN Department
Urinalysis
of Urinary
IT-Hydroxycorticosteroids
W. GRUNBAUM
of Physiology-Anatomy, Berkeley, California Received
March
AND NELLO University 94720
1
PACE
of California,
18, 1971
The urinary excretion of 17-hydroxycorticosteroids (17-HCS) is a reflection of the physiological activity of the adrenal cortex. Chemically, the 17-HCS are derivatives of cyclopentanoperhydrophenanthrene. They all have in common ketonic groups at carbon atoms 3 and 20, a double bond at the 4 (5) position in ring A and a hydroxyl group at carbon atom 21. They also have a characterizing 17-cy-hydroxyl group. The proximity of the oxygens on carbons 17, 20, and 21 constitutes a dihydroxyacetone configuration, which reacts with phenylhydrazine to produce a colored complex. The major 17-HCS produced by the adrenal cortex and circulating in the blood are hydrocortisone and cortisone in the unconjugated active form. Eventually the 17-HCS are reduced in the liver to the tetrahydroderivatives at carbon atom 3 and at which point they are conjugated with glucuronic acid or sulfate into the inactive water-soluble form. As such they appear in the urine. The glucuronide esters of 17-HCS eliminated in the urine account for 90-95% of the total 17-HCS (2), the sulfate esters accounting for the remainder. In the microprocedure described in this paper, based mainly on the method of Silber (7), the urine is hydrolyzed enzymatically and is then extracted and purified in a manner that eliminates both nonsteroidal metabolites as well as steroids not classed as 17-HCS. The fraction containing the purified 17-HCS is subjected to a reaction in which 17, 21-dihydroxy-20-ketosteroid rearranges in an ethanolic acid medium to form a 21-aldehyde (6), which then in the presence of phenylhydrazine forms the yellow phenylhydrazone. MATERIALS
AND METHODS
Equipment
1. Beckman DU spectrophotometer, equipped with a microap1 This study was supported by NASA Grant NGl.-05-003-024. 443
444
GRUNBAUM
2.
3. 4.
5.
AND
PACE
erture plate, which allows selection of an aperture that coincides with small volumes, and a microcell holder. Cuvettes, type 17 (about 2 mm wide on the inside and a 10 mm light path; outside dimensions: 25 mm high, 12.5 mm wide). (Available from Precision Cells, Inc., 401 Broadway, New York, New York.) Kimax culture tubes, screw top, 9 X 100 and 10 X 125 mm, and screw caps with Teflon liner. Repipets, (a) five 1 ml, for dispensing: water, P-glucuronidase, 0.1 N sodium hydroxide, sulfuric acid blanks and phenylhydrazine reagent; (b) one 5 ml for methylene dichloride; and (c) one 10 ml for carbon tetrachloride. Grunbaum pipets, 100 :J and 1 ml.
Items 4 and 5, are available Berkeley, California 947 10.
from Labindustries,
1802 2nd St.,
6. A device for automatic transfer of liquids (Fig. 1). This is an
7. 8. 9. 10. 11.
12.
improved model of the one published earier (3). The present form of the device is much easier to assemble, and has a control port which permits regulation of flow by use of the index finger. When transferring a clear solution from one container to another, the disposable filter can be omitted. By use of this device, a liquid fraction can be removed completely without loss due to spillage or use of transfer pipets. Water bath with a capacity for boiling water. Shaking machine, heavy duty. Vortex mixer. Centrifuge, International type SB, size 1. Assortment of volumetric flasks and graduated Pyrex measuring pipets, one each of 1, 2, and 10 ml. pH Meter with a combination microelectrode.
Reagents 1. Phosphate buffer, pH 6.5: KH,PO1, 3.353 g, and Na,HPO,, 1.647 g dissolved in 250 ml of distilled water. 2. /&Glucuronidase, type 1, Sigma Chemical Co., St. Louis, MO. To prepare 250 ml of buffered enzyme, weigh 1.35 g of /3glucuronidase and dissolve it with 250 ml of phosphate buffer. Dissolve the enzyme gently to prevent foaming. The buffered enzyme is divided into aliquots of 25 or 50 ml in polyethylene bottles and stored in the freezer until use. 3. Ethanol, “Gold Shield” (Commercial Solvents Corp. ) . 4. Hydrocortisone, the standard for 17-HCS. (Sigma Chemical
DETERMINATION
Filtrate or upper
OF URINARY
17-HYDROXYCORTICOSTEROIDS
445
phose
Precipitate or
lower
phose
NOT
FIG.
1. Device
5. 6. 7.
8.
for
Elastomer-coated
TO SCALE
automatic
transfer
of liquids,
filtration, and extraction of solids.
Co., St. Louis, MO.) Place 50.0 mg hydrocortisone into a loo-ml volumetric Aask. Dissolve with about 10 ml of absolute ethanol and make up to volume with distilled water. This is the stock standard, 500 ;Lg/ml. If stored in the refrigerator, well-stoppered, the stock standard remains stable for 3 months. Just prior to use, three working standards are prepared as follows: Transfer 100, 200, and 300 ~1 stock standard with a lOO-1~1Grunbaum pipet into respective lo-ml volumetric flasks and dilute with distilled water to mark. This results in three working standards of hydrocortisone concentration of 5, 10, and 15 /kg/ml. Sulfuric acid, reagent grade (Baker and Adamson). Prepare a 24 N solution by carefully adding, while mixing in ice bath, 320 ml coned H.&SO,to 180 ml distilled water. Sulfuric acid “blank” reagent. Add 100 ml of 24 N H,SO, to 50 ml of absolute ethanol slowly while continuously mixing and cooling. Prepare fresh before each use. Phenylhydrazine hydrochloride was recrystallized from hot ethanol, dried over calcium chloride in vacuum, and stored in a brown bottle in a desiccator. Dissolve 21.65 mg with 50 ml of “blank” reagent. Prepare fresh before use. Carbon tetrachloride, reagent grade (Eastman Organic Chemicals).
446
GRUNBAUM
AND PACE
9. Dichloromethane (Matheson, Coleman, Bell). The purity of the dichloromethane is critical. The reagent from this company gave us low and constant blank readings. 10. Sodium hydroxide, 0.1 N. Dissolve 2 g of reagent grade NaOH with 500 ml of distilled water. Procedure A. Enzymatic hydrolysis. With a Grunbaum pipet, transfer l-ml aliquots of urine into a 10 X 125mm screw-cap culture tube. (Up to 40 samples can be run simultaneously by one analyst.) Using the pH meter and microelectrode, adjust hydrogen-ion concentration to pH 6.5 by adding minimal amounts of either 1 N NaOH or HCl, depending on the original pH. This is best done by clamping the culture tube to a ring stand over a magnetic stirrer, placing a micro Teflon-coated stirrer in the tube and titrating with acid or base through a fine polyethylene tubing whose end is submerged in the urine. The polyethylene tubing, in turn, is attached to a length of capillary silicon rubber tubing through which delivers the acid or base from a reservoir. The flow is easily controlled by a spring clamp. The pH adjustment is thus very fast and accurate, and the urine sample is diluted minimally. To each tube was added, with a Repipet, 1 ml (250 units) of ,@-glucuronidasesolution. The tubes were capped tightly, mixed on a Vortex mixer and incubated overnight (16 hours) in a 37°C water bath. Hydrocortisone working standards in three different concentrations and a water blank were similarly prepared.
B. Wushing. With a Repipet, 6 ml of carbon tetrachloride were added to each tube. The screw caps were tightly attached and tubes subjected to mechanical shaking for 3 minutes. They were then centrifuged at 2000 rpm for 5 minutes. The upper aqueous phase (about 2 ml) was transferred to another clean set of culture tubes of the same size, using the device shown in Fig. 1. By narrowing the inlet orifice of the transfer device, a very clean separation of the two phases is achieved. A second washing with 6 ml carbon tetrachloride was made as above. After the second centrifugation, 1 ml of the upper aqueous layer was transferred into clean 10 X loo-mm screw-cap tubes, using a l-ml Grunbaum pipet. C. Extraction. To the l-ml samples were added accurately 5 ml of dichloromethane with a Repipet. The contents were mixed by use of a mechanical shaker for about 30 seconds. After centrifugation at 2000 rpm for 5 minutes, the upper aqueous phase was discarded by careful aspiration.
DETERMINATION
OF URINARY
17-HYDROXYCORTICOSTEROIDS
447
To the remaining dichloromethane phase were added 0.4 ml of freshly prepared 0.1 N NaOH. The contents were mixed on a Vortex mixer and centrifuged for 3 minutes at 2000 rpm. The NaOH layer was discarded by aspiration. The dichloromethane is very volatile, and the tubes must be tightly capped except when actually working with one. Using a calibrated 2-ml volumetric pipet, an aliquot of the dichloromethane extract was transferred into each of two 90 X loo-mm glass culture tubes. For each original sample, standard and blank, there were now two tubes. To one set of tubes were added 0.5 ml of sulfuric acid “blank reagent”. These tubes were used later to adjust the spectrophotometer to zero for the respective tubes to which were added 0.5 ml of phenylhydrazine hydrochloride reagent. The tubes were capped and contents mixed vigorously on a Vortex mixer and then centrifuged for 3 minutes at 2000 rpm. Following centrifugation, the entire upper dichloromethane phase was discarded by aspiration, and the tubes were recapped. The tubes were left for color development for about 16 hours (overnight) at room temperature, and preferably in the dark. Using the Beckman DU spectrophotometer and microcuvettes, each sample was measured against its own blank at three different wavelenths, 380, 410, and 440 nm. Maximum absorption is at 410 nm. The 380- and 440-nm wavelengths are used to correct the 410-nm reading for interfering substances (I). D. Color
development.
1. Determine net absorbance of each sample at three different wavelengths, 380, 410, and 440 nm, by subtracting the absorbance of the reagent blank at the respective wavelengths. 2. Corrected O.D. 410“,,, = 2(net O.D. 410nm) - (net O.D. 380n,,,+ net O.D. *a0.,,). Corrected O.D. 41,,nmof sample 3. Corrected O.D. 110,Imof standard X concn of standard = concn of 17-HCS in sample. 4. An alternative to above calculation is to read the concentration of unknown samples from a standard curve, such as that shown in Fig. 2. E. Calculation.
RESULTS AND DISCUSSION
With the procedure described, a technician could conveniently determine 17-HCS in 40 urine samples at a time. This was possible because of the very simplified procedures used in sampling, extraction, and
448
GRUNBAUM
0
FIG.
10
20 Hydrocortisone,
AND
PACE
30 pg/ml
40
50
2. Calibration line for standard solutions of hydrocortisone.
transfers. The standard use of 1 ml of urine from man or monkey was selected as a matter of practical convenience, Smaller amounts of urine could be used, but then the reproducibility of replicate samples would not be as good. The standard curve shown in fig. 2 shows the degree of linearity in the concentration range 5 to 50 pg/ml for hydrocortisone. The absorbance is a linear function of this concentration. The maximum variation of replicate samples in routine analyses from day to day is indicated. If the absorbance of standards fell outside these limits, fresh standards were prepared. In a recovery study, three different monkey urine samples having concentrations of 17-HCS from 8 to 16 pg/ml were each fortified with 5, 10, and 15 ALg/rnlof hydrocortisone. The maxium error in the recovery of the added hydrocortisone was t8 % , which is about the same as the variation in replicates shown in Fig. 1. For the size of urine sample used and the relative complexity of the procedure, and including the variation in absorbance of the blank, a maximum analytical error of ~8% appears acceptable. In the procedure described, the mean value for adult human males was about 11 pg/ml of urine. This corresponds to the average values given in the Handbook of Clinical Laboratory Data (4). For male monkeys, the mean value was about half that of the adult human males. In this procedure only the 17-HCS glucuronate complex is measured. However, in determining urinary 17-HCS, the individual variability between subjects must be considered. For instance, the ratio of 17-HCS complexed with glucuronic acid compared to that with sulfate may vary
DETERMINATION
OF URINARY
17-HYDROXYCORTICOSTEROIDS
between individuals. This appears to be especially sential hypertension
449
true in cases of es-
(5).
If it becomes necessary to determine the urinary sulfate-37-HCS complex separately, the procedure becomes very lengthy and involved. Solvolysis of the steroid-sulfate complex can be used on the urinary fraction remaining after the action of ,&glucuronidase (5). Another ap-
proach would be through the enzyme sulfatase. Fractionation of the hydrolysate after the action of the enzyme p-glucuronidase by thin-layer chromatography, in a manner similar to our
method described for the 17-ketosteroids (3), would permit both the individualization and quantification of the major components of the urinary 17-HCS and their relative amounts. Furthermore, minor 17-HCS fractions would be detected and the degree of “impurities”, if any,
would be evident. SUMMARY A procedure is described which uilizes 1 ml of human or monkey urine for the determination of 17-hydroxycorticosteroids. In this procedure, only the steroids conjugated with glucuronic acid are determined. The steps involve an enzymatic hydrolysis, followed by the extraction of the free steroids, with subsequent washing of the extract to rid it of interfering substances, and finally development of a colored compound by the use of phenylhydrazine. REFERENCES 1. Allen, W. M., A simple method for analyzing complicated adsorption curves, of use in the calorimetric determination of urinary steroids. J. C/in. Endocrinol. Metab. 10, 71-83 (1950). 2. Dutton, G. .I., ed., “Glucuronic Acid Free and Combined,” p. 533. Academic Press, New York, 1966. 3. Grunbaum, B. W., and Pace, N., Microchemical urinalysis. V. Quantitative analysis of individual urinary 17.ketosteroids by thin-layer chromatography. Microckem. J. 15, 103-121 (1970). 4. “Handbook of Clinical Laboratory Data,” (H. C. Damm, ed.), p. 158. The Chemical Rubber Co., Cleveland, Ohio, 1965. 5. Kornel, L., and Takeda, R., Studies on steroid conjugates: V. Urinary 17hydroxycorticosteroids in essential hypertension. J. C/in. Endocrinol. 27, 233-241 (1967). 6. Mattox, V. R., Steroids derived from bile acids XV-the formation of glyoxal sidechain Cl’ from steroids with dihydroxyacetone and &I(’ keto sidechain. .I. Amer. Ckem. Sot. 74, 43404347 (1952). 7. Silber, R. H., Free and conjugated 17-hydroxycorticosteroids in urine. in “Standard Methods of Clinical Chemistry” (D. Seligson, ed.), Vol. 4, p. 113. Academic Press, New York, 1963.
Microchemical VIII. Determination BENJAMIN Department
Urinalysis
of Urinary
IT-Hydroxycorticosteroids
W. GRUNBAUM
of Physiology-Anatomy, Berkeley, California Received
March
AND NELLO University 94720
1
PACE
of California,
18, 1971
The urinary excretion of 17-hydroxycorticosteroids (17-HCS) is a reflection of the physiological activity of the adrenal cortex. Chemically, the 17-HCS are derivatives of cyclopentanoperhydrophenanthrene. They all have in common ketonic groups at carbon atoms 3 and 20, a double bond at the 4 (5) position in ring A and a hydroxyl group at carbon atom 21. They also have a characterizing 17-cy-hydroxyl group. The proximity of the oxygens on carbons 17, 20, and 21 constitutes a dihydroxyacetone configuration, which reacts with phenylhydrazine to produce a colored complex. The major 17-HCS produced by the adrenal cortex and circulating in the blood are hydrocortisone and cortisone in the unconjugated active form. Eventually the 17-HCS are reduced in the liver to the tetrahydroderivatives at carbon atom 3 and at which point they are conjugated with glucuronic acid or sulfate into the inactive water-soluble form. As such they appear in the urine. The glucuronide esters of 17-HCS eliminated in the urine account for 90-95% of the total 17-HCS (2), the sulfate esters accounting for the remainder. In the microprocedure described in this paper, based mainly on the method of Silber (7), the urine is hydrolyzed enzymatically and is then extracted and purified in a manner that eliminates both nonsteroidal metabolites as well as steroids not classed as 17-HCS. The fraction containing the purified 17-HCS is subjected to a reaction in which 17, 21-dihydroxy-20-ketosteroid rearranges in an ethanolic acid medium to form a 21-aldehyde (6), which then in the presence of phenylhydrazine forms the yellow phenylhydrazone. MATERIALS
AND METHODS
Equipment
1. Beckman DU spectrophotometer, equipped with a microap1 This study was supported by NASA Grant NGl.-05-003-024. 443
444
GRUNBAUM
2.
3. 4.
5.
AND
PACE
erture plate, which allows selection of an aperture that coincides with small volumes, and a microcell holder. Cuvettes, type 17 (about 2 mm wide on the inside and a 10 mm light path; outside dimensions: 25 mm high, 12.5 mm wide). (Available from Precision Cells, Inc., 401 Broadway, New York, New York.) Kimax culture tubes, screw top, 9 X 100 and 10 X 125 mm, and screw caps with Teflon liner. Repipets, (a) five 1 ml, for dispensing: water, P-glucuronidase, 0.1 N sodium hydroxide, sulfuric acid blanks and phenylhydrazine reagent; (b) one 5 ml for methylene dichloride; and (c) one 10 ml for carbon tetrachloride. Grunbaum pipets, 100 :J and 1 ml.
Items 4 and 5, are available Berkeley, California 947 10.
from Labindustries,
1802 2nd St.,
6. A device for automatic transfer of liquids (Fig. 1). This is an
7. 8. 9. 10. 11.
12.
improved model of the one published earier (3). The present form of the device is much easier to assemble, and has a control port which permits regulation of flow by use of the index finger. When transferring a clear solution from one container to another, the disposable filter can be omitted. By use of this device, a liquid fraction can be removed completely without loss due to spillage or use of transfer pipets. Water bath with a capacity for boiling water. Shaking machine, heavy duty. Vortex mixer. Centrifuge, International type SB, size 1. Assortment of volumetric flasks and graduated Pyrex measuring pipets, one each of 1, 2, and 10 ml. pH Meter with a combination microelectrode.
Reagents 1. Phosphate buffer, pH 6.5: KH,PO1, 3.353 g, and Na,HPO,, 1.647 g dissolved in 250 ml of distilled water. 2. /&Glucuronidase, type 1, Sigma Chemical Co., St. Louis, MO. To prepare 250 ml of buffered enzyme, weigh 1.35 g of /3glucuronidase and dissolve it with 250 ml of phosphate buffer. Dissolve the enzyme gently to prevent foaming. The buffered enzyme is divided into aliquots of 25 or 50 ml in polyethylene bottles and stored in the freezer until use. 3. Ethanol, “Gold Shield” (Commercial Solvents Corp. ) . 4. Hydrocortisone, the standard for 17-HCS. (Sigma Chemical
DETERMINATION
Filtrate or upper
OF URINARY
17-HYDROXYCORTICOSTEROIDS
445
phose
Precipitate or
lower
phose
NOT
FIG.
1. Device
5. 6. 7.
8.
for
Elastomer-coated
TO SCALE
automatic
transfer
of liquids,
filtration, and extraction of solids.
Co., St. Louis, MO.) Place 50.0 mg hydrocortisone into a loo-ml volumetric Aask. Dissolve with about 10 ml of absolute ethanol and make up to volume with distilled water. This is the stock standard, 500 ;Lg/ml. If stored in the refrigerator, well-stoppered, the stock standard remains stable for 3 months. Just prior to use, three working standards are prepared as follows: Transfer 100, 200, and 300 ~1 stock standard with a lOO-1~1Grunbaum pipet into respective lo-ml volumetric flasks and dilute with distilled water to mark. This results in three working standards of hydrocortisone concentration of 5, 10, and 15 /kg/ml. Sulfuric acid, reagent grade (Baker and Adamson). Prepare a 24 N solution by carefully adding, while mixing in ice bath, 320 ml coned H.&SO,to 180 ml distilled water. Sulfuric acid “blank” reagent. Add 100 ml of 24 N H,SO, to 50 ml of absolute ethanol slowly while continuously mixing and cooling. Prepare fresh before each use. Phenylhydrazine hydrochloride was recrystallized from hot ethanol, dried over calcium chloride in vacuum, and stored in a brown bottle in a desiccator. Dissolve 21.65 mg with 50 ml of “blank” reagent. Prepare fresh before use. Carbon tetrachloride, reagent grade (Eastman Organic Chemicals).
446
GRUNBAUM
AND PACE
9. Dichloromethane (Matheson, Coleman, Bell). The purity of the dichloromethane is critical. The reagent from this company gave us low and constant blank readings. 10. Sodium hydroxide, 0.1 N. Dissolve 2 g of reagent grade NaOH with 500 ml of distilled water. Procedure A. Enzymatic hydrolysis. With a Grunbaum pipet, transfer l-ml aliquots of urine into a 10 X 125mm screw-cap culture tube. (Up to 40 samples can be run simultaneously by one analyst.) Using the pH meter and microelectrode, adjust hydrogen-ion concentration to pH 6.5 by adding minimal amounts of either 1 N NaOH or HCl, depending on the original pH. This is best done by clamping the culture tube to a ring stand over a magnetic stirrer, placing a micro Teflon-coated stirrer in the tube and titrating with acid or base through a fine polyethylene tubing whose end is submerged in the urine. The polyethylene tubing, in turn, is attached to a length of capillary silicon rubber tubing through which delivers the acid or base from a reservoir. The flow is easily controlled by a spring clamp. The pH adjustment is thus very fast and accurate, and the urine sample is diluted minimally. To each tube was added, with a Repipet, 1 ml (250 units) of ,@-glucuronidasesolution. The tubes were capped tightly, mixed on a Vortex mixer and incubated overnight (16 hours) in a 37°C water bath. Hydrocortisone working standards in three different concentrations and a water blank were similarly prepared.
B. Wushing. With a Repipet, 6 ml of carbon tetrachloride were added to each tube. The screw caps were tightly attached and tubes subjected to mechanical shaking for 3 minutes. They were then centrifuged at 2000 rpm for 5 minutes. The upper aqueous phase (about 2 ml) was transferred to another clean set of culture tubes of the same size, using the device shown in Fig. 1. By narrowing the inlet orifice of the transfer device, a very clean separation of the two phases is achieved. A second washing with 6 ml carbon tetrachloride was made as above. After the second centrifugation, 1 ml of the upper aqueous layer was transferred into clean 10 X loo-mm screw-cap tubes, using a l-ml Grunbaum pipet. C. Extraction. To the l-ml samples were added accurately 5 ml of dichloromethane with a Repipet. The contents were mixed by use of a mechanical shaker for about 30 seconds. After centrifugation at 2000 rpm for 5 minutes, the upper aqueous phase was discarded by careful aspiration.
DETERMINATION
OF URINARY
17-HYDROXYCORTICOSTEROIDS
447
To the remaining dichloromethane phase were added 0.4 ml of freshly prepared 0.1 N NaOH. The contents were mixed on a Vortex mixer and centrifuged for 3 minutes at 2000 rpm. The NaOH layer was discarded by aspiration. The dichloromethane is very volatile, and the tubes must be tightly capped except when actually working with one. Using a calibrated 2-ml volumetric pipet, an aliquot of the dichloromethane extract was transferred into each of two 90 X loo-mm glass culture tubes. For each original sample, standard and blank, there were now two tubes. To one set of tubes were added 0.5 ml of sulfuric acid “blank reagent”. These tubes were used later to adjust the spectrophotometer to zero for the respective tubes to which were added 0.5 ml of phenylhydrazine hydrochloride reagent. The tubes were capped and contents mixed vigorously on a Vortex mixer and then centrifuged for 3 minutes at 2000 rpm. Following centrifugation, the entire upper dichloromethane phase was discarded by aspiration, and the tubes were recapped. The tubes were left for color development for about 16 hours (overnight) at room temperature, and preferably in the dark. Using the Beckman DU spectrophotometer and microcuvettes, each sample was measured against its own blank at three different wavelenths, 380, 410, and 440 nm. Maximum absorption is at 410 nm. The 380- and 440-nm wavelengths are used to correct the 410-nm reading for interfering substances (I). D. Color
development.
1. Determine net absorbance of each sample at three different wavelengths, 380, 410, and 440 nm, by subtracting the absorbance of the reagent blank at the respective wavelengths. 2. Corrected O.D. 410“,,, = 2(net O.D. 410nm) - (net O.D. 380n,,,+ net O.D. *a0.,,). Corrected O.D. 41,,nmof sample 3. Corrected O.D. 110,Imof standard X concn of standard = concn of 17-HCS in sample. 4. An alternative to above calculation is to read the concentration of unknown samples from a standard curve, such as that shown in Fig. 2. E. Calculation.
RESULTS AND DISCUSSION
With the procedure described, a technician could conveniently determine 17-HCS in 40 urine samples at a time. This was possible because of the very simplified procedures used in sampling, extraction, and
448
GRUNBAUM
0
FIG.
10
20 Hydrocortisone,
AND
PACE
30 pg/ml
40
50
2. Calibration line for standard solutions of hydrocortisone.
transfers. The standard use of 1 ml of urine from man or monkey was selected as a matter of practical convenience, Smaller amounts of urine could be used, but then the reproducibility of replicate samples would not be as good. The standard curve shown in fig. 2 shows the degree of linearity in the concentration range 5 to 50 pg/ml for hydrocortisone. The absorbance is a linear function of this concentration. The maximum variation of replicate samples in routine analyses from day to day is indicated. If the absorbance of standards fell outside these limits, fresh standards were prepared. In a recovery study, three different monkey urine samples having concentrations of 17-HCS from 8 to 16 pg/ml were each fortified with 5, 10, and 15 ALg/rnlof hydrocortisone. The maxium error in the recovery of the added hydrocortisone was t8 % , which is about the same as the variation in replicates shown in Fig. 1. For the size of urine sample used and the relative complexity of the procedure, and including the variation in absorbance of the blank, a maximum analytical error of ~8% appears acceptable. In the procedure described, the mean value for adult human males was about 11 pg/ml of urine. This corresponds to the average values given in the Handbook of Clinical Laboratory Data (4). For male monkeys, the mean value was about half that of the adult human males. In this procedure only the 17-HCS glucuronate complex is measured. However, in determining urinary 17-HCS, the individual variability between subjects must be considered. For instance, the ratio of 17-HCS complexed with glucuronic acid compared to that with sulfate may vary
DETERMINATION
OF URINARY
17-HYDROXYCORTICOSTEROIDS
between individuals. This appears to be especially sential hypertension
449
true in cases of es-
(5).
If it becomes necessary to determine the urinary sulfate-37-HCS complex separately, the procedure becomes very lengthy and involved. Solvolysis of the steroid-sulfate complex can be used on the urinary fraction remaining after the action of ,&glucuronidase (5). Another ap-
proach would be through the enzyme sulfatase. Fractionation of the hydrolysate after the action of the enzyme p-glucuronidase by thin-layer chromatography, in a manner similar to our
method described for the 17-ketosteroids (3), would permit both the individualization and quantification of the major components of the urinary 17-HCS and their relative amounts. Furthermore, minor 17-HCS fractions would be detected and the degree of “impurities”, if any,
would be evident. SUMMARY A procedure is described which uilizes 1 ml of human or monkey urine for the determination of 17-hydroxycorticosteroids. In this procedure, only the steroids conjugated with glucuronic acid are determined. The steps involve an enzymatic hydrolysis, followed by the extraction of the free steroids, with subsequent washing of the extract to rid it of interfering substances, and finally development of a colored compound by the use of phenylhydrazine. REFERENCES 1. Allen, W. M., A simple method for analyzing complicated adsorption curves, of use in the calorimetric determination of urinary steroids. J. C/in. Endocrinol. Metab. 10, 71-83 (1950). 2. Dutton, G. .I., ed., “Glucuronic Acid Free and Combined,” p. 533. Academic Press, New York, 1966. 3. Grunbaum, B. W., and Pace, N., Microchemical urinalysis. V. Quantitative analysis of individual urinary 17.ketosteroids by thin-layer chromatography. Microckem. J. 15, 103-121 (1970). 4. “Handbook of Clinical Laboratory Data,” (H. C. Damm, ed.), p. 158. The Chemical Rubber Co., Cleveland, Ohio, 1965. 5. Kornel, L., and Takeda, R., Studies on steroid conjugates: V. Urinary 17hydroxycorticosteroids in essential hypertension. J. C/in. Endocrinol. 27, 233-241 (1967). 6. Mattox, V. R., Steroids derived from bile acids XV-the formation of glyoxal sidechain Cl’ from steroids with dihydroxyacetone and &I(’ keto sidechain. .I. Amer. Ckem. Sot. 74, 43404347 (1952). 7. Silber, R. H., Free and conjugated 17-hydroxycorticosteroids in urine. in “Standard Methods of Clinical Chemistry” (D. Seligson, ed.), Vol. 4, p. 113. Academic Press, New York, 1963.