A Sensitive Radioenzymatic Assay for Dopamine, Norepinephrine, and Epinephrine in Plasma and Tissue NIRA BEN-JONATHAN AND JOHN C. PORTER Cecil H. and Ida Green Center for Reproductive Biology Sciences, the Department of Obstetrics and Gynecology, and the Department of Physiology, the University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235 ABSTRACT. A double-isotope, radioenzymatic assay for measuring dopamine, norepinephrine, and epinephrine in one sample is described. The assay procedure includes incubation, solvent extraction, and thin-layer chromatography. Dopamine, norepinephrine, and epinephrine were incubated with catechol-O-methyl transferase (COMT) and [3H]Sadenosyl methionine ([3H]SAM) and were converted to the O-methylated tritiated derivatives: [3H]methoxytyramine, [3H]normetanephrine, and [3H]metanephrine, respectively. After several extraction steps, the O-methylated products were purified by means of two-dimensional, thin-layer chromatography using silica gel. The thin-layer chromatographic system resulted in complete separation of the three O-methylated compounds with an overlap of only 1-2%. The assay was linear from 0 to 5 ng for each catecholamine and had a sensitivity of 10-30 pg. The
C
ATECHOLAMINES are best known as neurotransmitters which are produced and secreted by the central and autonomic nervous systems. In the last few decades, significant advances have been made in the knowledge of the biosynthesis of these compounds, their cellular and subcellular distribution, and the mechanisms which control their secretion and uptake by nerve endings (1-3). In addition to being neurotransmitters, catecholamines also function as hormones which are secreted mainly by the adrenal medulla. Moreover, these agents influence and modify, by yet unknown Received October 13, 1975. Please send correspondence to: John C. Porter, Ph.D., Department of Obstetrics and Gynecology, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, Texas 75235. Supported by grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AM01237) and the National Institute of Child Health and Human Development (HD08672).
addition of large amounts of plasma reduced the activity of COMT, but increasing the magnesium concentration in the incubation mixture and the addition of EGTA to plasma samples improved the recoveries. Each sample was corrected for losses incurred during extraction and chromatography by using [14C]methoxytyramine, [14C]normetanephrine, and [14C]metanephrine that were added at the end of incubation. Several catechol compounds known to be O-methylated by COMT were examined for crossreactivity. Of the substances tested, only dihydroxyphenylalanine (DOPA) exhibited cross-reactivity. However, the apparent 30% cross-reactivity of DOPA with dopamine was due to the presence of decarboxylase activity in the COMT preparation. As little as 50 fi\ of trunk plasma from decapitated rats was sufficient for the determination of the three catecholamines. (Endocrinology 98: 1497, 1976)
mechanisms, the secretion of a variety of hormones including hypothalamic releasing and inhibiting factors and pituitary tropic hormones. Still, the role of catecholamines in hormonal regulation is not fully understood even though neuronal involvement in certain endocrine functions is well documented (4-6). The lack of progress in understanding the hormonal aspects of catecholamines can be attributed to the absence of specific and sensitive methods for measuring these compounds in plasma. The fluorometric techniques (7-9), widely used for determining tissue levels of catecholamines, are inadequate for quantifying these substances in plasma. In 1968, Engelman et al. (10) introduced a radioenzymatic assay for measuring norepinephrine and epinephrine, utilizing catechol-O-methyl transferase (COMT). This assay is more sensitive than the fluorometric method; however, its sensitivity is still less than desirable. Several
1497
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 27 January 2016. at 07:49 For personal use only. No other uses without permission. . All rights reserved.
BEN-JONATHAN AND PORTER
1498
modifications of this radioenzymatic procedure have since been published (11-15), but none seems adequate for measuring dopamine, norepinephrine, and epinephrine simultaneously in small volumes of plasma. Materials and Methods A. Animals Adult male rats of the Long-Evans strain weighing 300-350 g were used. The animals were housed in an air-conditioned room and supplied with water and food ad lib. Ventromedial hypothalamic tissue (15-20 mg), the anterior pituitaries, posterior pituitaries, and a small portion of the spleen (20-25 mg) were immediately excised from rats killed by decapitation, and placed on dry ice. The tissues were homogenized in 30-50 vol (wt/vol) of icecold 0.1M perchloric acid. The homogenates were centrifuged at 10,000 x g for 20 min, and the supernatant fluid was stored at - 2 0 C. Protein determinations were done according to Lowry et al. (16), using crystalline bovine serum albumin as the standard. Trunk blood was collected from unanesthetized rats following decapitation. The blood was collected in cold heparinized tubes and centrifuged at 0 C, and the plasma was stored at - 2 0 C. Adrenal vein blood was collected according to the method of Porter (17). The rats were anesthetized with sodium pentobarbital (35-45 mg/kg BW) and supplied with oxygen during the experiment. The blood from the adrenal vein was collected in tubes at 0 C, and 7 samples of blood, each equivalent to a 10-min collection period, were obtained from eveiy animal. One set of rats was injected with 10 fjd of 0.15M NaCl containing 1 fxg of acetylcholine via a micro cannula inserted into the adrenal gland. Adrenal vein blood was collected from another set of rats which received no injection into the adrenal gland. The blood was centrifuged at 0 C, and the plasma was stored at - 2 0 C. B. Preparation of COMT All steps of purification of COMT were performed at 0-2 C. Rat livers (100 g) were homogenized in 4 vol (wt/vol) of 0.155M KC1, and the homogenate was centrifuged at 46,000 x g for 30
Endo • 1976 Vol 98 • No 6
min. The supernatant fluid was adjusted to pH 5.3 with 1M acetic acid. After centrifugation at 15,000 x g for 10 min, the supernatant fluid was adjusted to pH 6.8 with 0.5M phosphate buffer, pH 7.0, and then fractionated with ammonium sulfate (enzyme grade), as described by Nikodejevic et al. (18). The final precipitate from the ammonium sulfate fractionation was dissolved in 40 ml of 1 niM phosphate buffer, pH 7.0, and dialyzed overnight against three changes of 5 liters of 5 IHM phosphate buffer, pH 7.0, containing 0.1 mM dithiothreitol (Calbiochem). The dialysate was divided into 0.5 ml aliquots (each equivalent to 40 mg protein) and stored at —20 C. No apparent loss of enzyme activity was observed over a period of one year. C. Assay of dopamine, epinephrine
norepinephrine,
and
The assay procedure included incubation, solvent extraction, and thin-layer chromatography. Incubation and solvent extraction were performed in 12 x 75 mm disposable glass tubes. 1. Incubation. An assay using plasma included plasma blanks, unknown plasma samples, and plasma samples containing internal standards. For plasma blanks, 25-100 /xl of rat plasma which had been dialyzed for 24 h against 0.15M NaCl to remove endogenous catecholamines was used. The unknown samples consisted of 25-100 fx\ of heparinized rat plasma. Equivalent volumes of non-dialyzed, heparinized rat plasma containing 1 ng each of dopamine, L-norepinephrine, and L-epinephrine (Sigma; purity > 98%) served as internal standards. The volume in each tube was adjusted to 150 /oil with 0.1M perchloric acid. An assay using tissue extracts included blanks, tissue extracts, and tissue extracts containing internal standards. The blanks consisted of 150 lid of 0.1M perchloric acid. Aliquots of 25-100 (x\ of tissue extracts were used as samples. Equivalent volumes of tissue extracts containing 1 ng each of dopamine, norepinephrine, and epinephrine served as internal standards. The volume in each tube was adjusted to 150 /x\ with 0.1M perchloric acid. All tubes were kept at 0-2 C until the time of incubation. The O-methylation reaction was initiated by the addition of 50 /J of freshly prepared Reagent A. To prepare 2.7 ml of Reagent A
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 27 January 2016. at 07:49 For personal use only. No other uses without permission. . All rights reserved.
ASSAY FOR CATECHOLAMINES (sufficient for a 50-tube assay), the following substances were mixed in the order listed: 15 mg dithiothreitol; 125 /JL\ of 1M MgCl2; 1850 yul of 2M Tris-HCl buffer, pH 8.6 (Trizma base, Sigma Chemical Co.); 125 /AI of 0.05M EGTA [ethyleneglycol-bis-(/3-amino-ethylether) N,N'-tetra-acetic acid]; 500 fi\ of the COMT preparation; and 100 fx\ (50/xCi) of [3H-methyl]S-adenosyl methionine ([3H]SAM) having a specific activity of 11.2 Ci/ mmol (New England Nuclear). The tubes were incubated for 75 min at 37 C in a metabolic shaker at 40 cycles per min. The reaction was stopped by the addition of 0.3 ml of 0.5M borate buffer, pH 10. A mixture containing 10 /xg each of 3-methoxytyramine, normetanephrine, and metanephrine and 800 cpm each of [14C]methoxytyramine, [14C]normetanephrine, and [14C]metanephrine in 25 yu.1 of 0.1M acetic acid was added to each tube. 2. Solvent extraction. All extractions were done at room temperature by vigorous mixing for 1 min. After centrifugation (2000 x g) for 2 min, the aqueous phase was frozen in dry ice, and acetone and the organic phase were decanted. The O-methylated products were extracted from borate buffer into 3.5 ml of toluene:isoamyl alcohol (3:2, vol/vol). The organic phase was transferred to tubes containing 0.3 ml of 0.5M borate buffer, pH 10. After mixing and centrifugation, the organic phase was transferred to tubes containing 0.2 ml of 0.1M HCl as described by Cuello et al. (19). After extraction, the organic phase was then discarded; and the aqueous phase, containing the O-methylated products, was washed with 2.5 ml of ethyl acetate saturated with water. After separation of the phases, the organic phase was discarded. The O-methylated compounds in 0.1M HCl were stable at 4 C for one week at least. 3. Thin-layer chromatography. Plastic plates (10 x 10 cm) coated with silica gel without gypsum (Polygram Sil G, Brinkmann Instruments, Inc.) were used. The samples were dried at room temperature under nitrogen, which took 20-30 min. The residues were dissolved in 25 (A of acidified ethanol (95% ethanol and 5% of 0.1M HCl) and applied to the chromatographic plates Vi inch from one corner. Each tube was washed with 15 /xl of acidified ethanol, and the rinse was added to the plate. The plates were first developed in Solvent System I (chloroform:
1499
methanol:ammonium hydroxide; 120:70:10, vol/ vol). After drying, the plates were rotated 90 degrees and developed in Solvent System II (ethanol:diethyl ether:water:ammonium hydroxide; 150:90:48:12, vol/vol). When dry, the plates were exposed briefly to iodine vapor which revealed 3 spots corresponding to 3-methoxytyramine, normetanephrine, and metanephrine (Fig. 1). (Due to variation in the characteristics of different batches of chromatographic plates, it was sometimes necessary to alter slightly the methanol concentration in Solvent System I to achieve optimal separation of 3-methoxytyramine and normetanephrine.) Each spot, including the underlying plastic support, was cut out and placed in a scintillation vial containing 0.3 ml of 0.1M acetic acid. After vigorous shaking, 10 ml of Aquasol (New England Nuclear) was added, and the radioactivity was counted in a Packard Tri-Carb liquid scintillation spectrometer at 3 C. The counting conditions allowed for 30% counting efficiency for tritium, 79% efficiency for carbon-14, and 15-20% spill from the carbon-14 channel into the tritium channel. D. Preparation of l4C-labelled compounds The O-methylated, 14C-labelled compounds were prepared as described above, with the following changes. Instead of using [3H]SAM as the radioactive methyl donor, 0.2/u,Ci of [14C]SAM (specific activity of 57 mCi/mmol, New England Nuclear) was added to each tube. In addition, 200 ng of dopamine, norepinephrine, or epinephrine per tube was used. After incubation and extraction, the O-methylated, 14C-derivatives were purified by thin-layer chromatography. After visualization in UV light, the 14C-labelled compounds were extracted from the chromatographic plates with 0.1M acetic acid and stored at 2 C. The purity of the 14C-labelled tracers was checked periodically. E. Production of 14CO2from DL-3,4-dihydroxyphenylalanine-[carboxyl-uC}(V
C
ATECHOLAMINES are best known as neurotransmitters which are produced and secreted by the central and autonomic nervous systems. In the last few decades, significant advances have been made in the knowledge of the biosynthesis of these compounds, their cellular and subcellular distribution, and the mechanisms which control their secretion and uptake by nerve endings (1-3). In addition to being neurotransmitters, catecholamines also function as hormones which are secreted mainly by the adrenal medulla. Moreover, these agents influence and modify, by yet unknown Received October 13, 1975. Please send correspondence to: John C. Porter, Ph.D., Department of Obstetrics and Gynecology, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, Texas 75235. Supported by grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AM01237) and the National Institute of Child Health and Human Development (HD08672).
addition of large amounts of plasma reduced the activity of COMT, but increasing the magnesium concentration in the incubation mixture and the addition of EGTA to plasma samples improved the recoveries. Each sample was corrected for losses incurred during extraction and chromatography by using [14C]methoxytyramine, [14C]normetanephrine, and [14C]metanephrine that were added at the end of incubation. Several catechol compounds known to be O-methylated by COMT were examined for crossreactivity. Of the substances tested, only dihydroxyphenylalanine (DOPA) exhibited cross-reactivity. However, the apparent 30% cross-reactivity of DOPA with dopamine was due to the presence of decarboxylase activity in the COMT preparation. As little as 50 fi\ of trunk plasma from decapitated rats was sufficient for the determination of the three catecholamines. (Endocrinology 98: 1497, 1976)
mechanisms, the secretion of a variety of hormones including hypothalamic releasing and inhibiting factors and pituitary tropic hormones. Still, the role of catecholamines in hormonal regulation is not fully understood even though neuronal involvement in certain endocrine functions is well documented (4-6). The lack of progress in understanding the hormonal aspects of catecholamines can be attributed to the absence of specific and sensitive methods for measuring these compounds in plasma. The fluorometric techniques (7-9), widely used for determining tissue levels of catecholamines, are inadequate for quantifying these substances in plasma. In 1968, Engelman et al. (10) introduced a radioenzymatic assay for measuring norepinephrine and epinephrine, utilizing catechol-O-methyl transferase (COMT). This assay is more sensitive than the fluorometric method; however, its sensitivity is still less than desirable. Several
1497
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 27 January 2016. at 07:49 For personal use only. No other uses without permission. . All rights reserved.
BEN-JONATHAN AND PORTER
1498
modifications of this radioenzymatic procedure have since been published (11-15), but none seems adequate for measuring dopamine, norepinephrine, and epinephrine simultaneously in small volumes of plasma. Materials and Methods A. Animals Adult male rats of the Long-Evans strain weighing 300-350 g were used. The animals were housed in an air-conditioned room and supplied with water and food ad lib. Ventromedial hypothalamic tissue (15-20 mg), the anterior pituitaries, posterior pituitaries, and a small portion of the spleen (20-25 mg) were immediately excised from rats killed by decapitation, and placed on dry ice. The tissues were homogenized in 30-50 vol (wt/vol) of icecold 0.1M perchloric acid. The homogenates were centrifuged at 10,000 x g for 20 min, and the supernatant fluid was stored at - 2 0 C. Protein determinations were done according to Lowry et al. (16), using crystalline bovine serum albumin as the standard. Trunk blood was collected from unanesthetized rats following decapitation. The blood was collected in cold heparinized tubes and centrifuged at 0 C, and the plasma was stored at - 2 0 C. Adrenal vein blood was collected according to the method of Porter (17). The rats were anesthetized with sodium pentobarbital (35-45 mg/kg BW) and supplied with oxygen during the experiment. The blood from the adrenal vein was collected in tubes at 0 C, and 7 samples of blood, each equivalent to a 10-min collection period, were obtained from eveiy animal. One set of rats was injected with 10 fjd of 0.15M NaCl containing 1 fxg of acetylcholine via a micro cannula inserted into the adrenal gland. Adrenal vein blood was collected from another set of rats which received no injection into the adrenal gland. The blood was centrifuged at 0 C, and the plasma was stored at - 2 0 C. B. Preparation of COMT All steps of purification of COMT were performed at 0-2 C. Rat livers (100 g) were homogenized in 4 vol (wt/vol) of 0.155M KC1, and the homogenate was centrifuged at 46,000 x g for 30
Endo • 1976 Vol 98 • No 6
min. The supernatant fluid was adjusted to pH 5.3 with 1M acetic acid. After centrifugation at 15,000 x g for 10 min, the supernatant fluid was adjusted to pH 6.8 with 0.5M phosphate buffer, pH 7.0, and then fractionated with ammonium sulfate (enzyme grade), as described by Nikodejevic et al. (18). The final precipitate from the ammonium sulfate fractionation was dissolved in 40 ml of 1 niM phosphate buffer, pH 7.0, and dialyzed overnight against three changes of 5 liters of 5 IHM phosphate buffer, pH 7.0, containing 0.1 mM dithiothreitol (Calbiochem). The dialysate was divided into 0.5 ml aliquots (each equivalent to 40 mg protein) and stored at —20 C. No apparent loss of enzyme activity was observed over a period of one year. C. Assay of dopamine, epinephrine
norepinephrine,
and
The assay procedure included incubation, solvent extraction, and thin-layer chromatography. Incubation and solvent extraction were performed in 12 x 75 mm disposable glass tubes. 1. Incubation. An assay using plasma included plasma blanks, unknown plasma samples, and plasma samples containing internal standards. For plasma blanks, 25-100 /xl of rat plasma which had been dialyzed for 24 h against 0.15M NaCl to remove endogenous catecholamines was used. The unknown samples consisted of 25-100 fx\ of heparinized rat plasma. Equivalent volumes of non-dialyzed, heparinized rat plasma containing 1 ng each of dopamine, L-norepinephrine, and L-epinephrine (Sigma; purity > 98%) served as internal standards. The volume in each tube was adjusted to 150 /oil with 0.1M perchloric acid. An assay using tissue extracts included blanks, tissue extracts, and tissue extracts containing internal standards. The blanks consisted of 150 lid of 0.1M perchloric acid. Aliquots of 25-100 (x\ of tissue extracts were used as samples. Equivalent volumes of tissue extracts containing 1 ng each of dopamine, norepinephrine, and epinephrine served as internal standards. The volume in each tube was adjusted to 150 /x\ with 0.1M perchloric acid. All tubes were kept at 0-2 C until the time of incubation. The O-methylation reaction was initiated by the addition of 50 /J of freshly prepared Reagent A. To prepare 2.7 ml of Reagent A
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 27 January 2016. at 07:49 For personal use only. No other uses without permission. . All rights reserved.
ASSAY FOR CATECHOLAMINES (sufficient for a 50-tube assay), the following substances were mixed in the order listed: 15 mg dithiothreitol; 125 /JL\ of 1M MgCl2; 1850 yul of 2M Tris-HCl buffer, pH 8.6 (Trizma base, Sigma Chemical Co.); 125 /AI of 0.05M EGTA [ethyleneglycol-bis-(/3-amino-ethylether) N,N'-tetra-acetic acid]; 500 fi\ of the COMT preparation; and 100 fx\ (50/xCi) of [3H-methyl]S-adenosyl methionine ([3H]SAM) having a specific activity of 11.2 Ci/ mmol (New England Nuclear). The tubes were incubated for 75 min at 37 C in a metabolic shaker at 40 cycles per min. The reaction was stopped by the addition of 0.3 ml of 0.5M borate buffer, pH 10. A mixture containing 10 /xg each of 3-methoxytyramine, normetanephrine, and metanephrine and 800 cpm each of [14C]methoxytyramine, [14C]normetanephrine, and [14C]metanephrine in 25 yu.1 of 0.1M acetic acid was added to each tube. 2. Solvent extraction. All extractions were done at room temperature by vigorous mixing for 1 min. After centrifugation (2000 x g) for 2 min, the aqueous phase was frozen in dry ice, and acetone and the organic phase were decanted. The O-methylated products were extracted from borate buffer into 3.5 ml of toluene:isoamyl alcohol (3:2, vol/vol). The organic phase was transferred to tubes containing 0.3 ml of 0.5M borate buffer, pH 10. After mixing and centrifugation, the organic phase was transferred to tubes containing 0.2 ml of 0.1M HCl as described by Cuello et al. (19). After extraction, the organic phase was then discarded; and the aqueous phase, containing the O-methylated products, was washed with 2.5 ml of ethyl acetate saturated with water. After separation of the phases, the organic phase was discarded. The O-methylated compounds in 0.1M HCl were stable at 4 C for one week at least. 3. Thin-layer chromatography. Plastic plates (10 x 10 cm) coated with silica gel without gypsum (Polygram Sil G, Brinkmann Instruments, Inc.) were used. The samples were dried at room temperature under nitrogen, which took 20-30 min. The residues were dissolved in 25 (A of acidified ethanol (95% ethanol and 5% of 0.1M HCl) and applied to the chromatographic plates Vi inch from one corner. Each tube was washed with 15 /xl of acidified ethanol, and the rinse was added to the plate. The plates were first developed in Solvent System I (chloroform:
1499
methanol:ammonium hydroxide; 120:70:10, vol/ vol). After drying, the plates were rotated 90 degrees and developed in Solvent System II (ethanol:diethyl ether:water:ammonium hydroxide; 150:90:48:12, vol/vol). When dry, the plates were exposed briefly to iodine vapor which revealed 3 spots corresponding to 3-methoxytyramine, normetanephrine, and metanephrine (Fig. 1). (Due to variation in the characteristics of different batches of chromatographic plates, it was sometimes necessary to alter slightly the methanol concentration in Solvent System I to achieve optimal separation of 3-methoxytyramine and normetanephrine.) Each spot, including the underlying plastic support, was cut out and placed in a scintillation vial containing 0.3 ml of 0.1M acetic acid. After vigorous shaking, 10 ml of Aquasol (New England Nuclear) was added, and the radioactivity was counted in a Packard Tri-Carb liquid scintillation spectrometer at 3 C. The counting conditions allowed for 30% counting efficiency for tritium, 79% efficiency for carbon-14, and 15-20% spill from the carbon-14 channel into the tritium channel. D. Preparation of l4C-labelled compounds The O-methylated, 14C-labelled compounds were prepared as described above, with the following changes. Instead of using [3H]SAM as the radioactive methyl donor, 0.2/u,Ci of [14C]SAM (specific activity of 57 mCi/mmol, New England Nuclear) was added to each tube. In addition, 200 ng of dopamine, norepinephrine, or epinephrine per tube was used. After incubation and extraction, the O-methylated, 14C-derivatives were purified by thin-layer chromatography. After visualization in UV light, the 14C-labelled compounds were extracted from the chromatographic plates with 0.1M acetic acid and stored at 2 C. The purity of the 14C-labelled tracers was checked periodically. E. Production of 14CO2from DL-3,4-dihydroxyphenylalanine-[carboxyl-uC}(V
