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Further modification of a fluorometric method for analyzing brain amines
Zlf~:ROZHE~Il~:.~L,jOURUhL
Further
23, 9- 11, (1978)
modification of a Fluorometric for Analyzing Brain Amines
Method
ALFRED E. CIARLOSE
INTRODUCTION This laboratory has attempted to duplicate a fluorometric analysis that was described by Maickel ~1crl. (4). The attempt resulted in failure, due to the difficulty in handling very small samples and due to the presence of an interfering substance(s) when measuring the norepinephrine (NE) fluorophor. In addition, their method did not describe measurement of dopamine (DA) in the same sample and this was considered a necessity in our analysis. Serotonin (5-HT) analysis was described and no difficulty was encountered in duplicating this portion. Another method described by Chang (II did relate the ability to measure NE and DA, but not S-HT. Therefore, by modifying and combining both methods f 1,4), an analysis was reported that enables one to measure (using slightly larger sample volumes) 5-HT, NE, and DA in one sample (3). In the latter report, and probably in Chang’s f/i method, an alumina step eliminated the interfering substance(s). The source of the interference had been investigated for some years. resulting in failure, as cited above. However, some new ideas suggested a reevaluation of the problem, since solving the problem would result in a considerable saving of time (by elimination of the alumina step). The following pieces of data were accumulated over the course of many experiments: (a) although all three amines are stored in 0.1 N hydrochloric acid. increased sensitivity of NE and DA measurement results if ail subsequent dilutions are done with 0.2 N acetic acid, especially since 0.2 N acetic acid dilutions have no effect on 5-HT sensitivity; (b) rr-butanoi was found to be the major cause of interference, with I?-heptane also contributing interference. This was noted by a consistent. approximate 30% increase of fluorescence units for internal standards (exposed to ,I-butanol and r?-heptane) compared to external standards, and this fact was further corroborated by noting increased excitation and emission spectra of NE and DA exposed to /r-butanol and /z-heptane compared to the spectra produced by NE and DA not exposed to the organic compounds; (c) a second extraction step with n-heptane had no effect on tz-butanol‘s interference. 9
10
ALFRED
MATERIALS
E. CIARLONE
AND METHODS
All reagents were either reagent or fluorescence grade. Double deionized and distilled water was used where indicated. n-Butanol (Regis Chemical Company, Morton Grove, Illinois) was acidified as described by Chang (I), n-Heptane (Regis Chemical Company), 0.1 N and 10 N Hydrochloric acid, 5 N Sodium hydroxide, 0.2 N and 5 N Acetic acid, 0.1 N Iodine, as described by Chang (I) or Ciarlone (3), 0.1 M EDTA, as described by Chang (1) or Ciarlone (31, 25% Alkaline sulfite, as described by Chang (I) or Ciarlone (3), o-Phthalaldehyde (OPT) 4 mg% (Regis Chemical Company), 5-HT hydrogen oxalate (Regis Chemical Company) diluted to 1000 pug/ml (as free base) with 0.1 N HCl, NE hydrochloride (Regis Chemical Company) diluted to 1000 pglml (as free base) with 0.1 N HCl, DA hydrochloride (Calbiochem) diluted to 1000 pg/ml (as free base) with 0.1 N HCl. Homogenization of tissue and recovery of the three amines from tissue homogenates has been described previously (I, 2, 4). Duplicate internal standard tubes were carried in parallel with the tissue homogenates. Internal standards containing three concentrations of the three amines were diluted in 0.2 N acetic acid and prepared so that the total volume in each tube was 0.3 ml. These tubes would receive 3 ml of butanol. The homogenates and internal standard tubes were centrifuged at 1000 R for 5 min. Two and one-half milliliters of the supernatant fluid were transferred to tubes containing 1.6 ml of 0.2 N acetic acid and 5 ml of heptane. All tubes were placed on a vortex mixer for 30 set and centrifuged at 1000g for 5 min. The organic supernatant phase was discarded. Two tenths of milliliter of the aqueous phase was transferred to tubes (for continuation of 5-HT analysis) and 1 ml was transferred to tubes for the analysis of NE and DA. The latter tubes may be stored in ice. Three external standards for 5-HT were prepared in duplicate, in 0.2 N acetic acid and to a total volume of 0.2 ml. To all 5-HT tubes, including a reagent blank (consisting of 0.2 ml of 0.2 N acetic acid), 1.2 ml of OPT were added and mixed well. All tubes were placed in a boiling water bath for 10 min, cooled in tap water and read in a spectrophotofluorometer. Excitation and emission were 355 and 470 nm, respectively (uncorrected). Continnution of the NE and DA ancrlysis. At this time, external stan-
FLL’ORORIETRIC
ANALYSIS
11
OF AhlINES
TABLE I RTCOVERI. OF 5-HT. NE, ~SD DA Original
Predicted concentration”
Measured concentration’
Recovery*’
S-HT 3.0 1.5 0.75
0.25 0.125 0.0625
0.25 0.125 0.0625
100
0.6 0.3 0. I5
0.25 0.125 0.0625
0.23 0.1 14 0.057
91
1.2 0.6 0.3
0.5 0.25 0.125
0.43 0.215 0.108
86
NE
DA
” Original microgram concentration added to internal standards. e Predicted microgram concentration (due to known volume losses) in internal standards. I’ Actual microgram concentration measured and interpolated from external standards. ” Measured concentration in internal standards / external standards x 100.
dards were prepared for NE and DA using three concentrations and in duplicate, in 0.2 N acetic acid and to a volume of 1.6 ml per tube. The butanol and heptane interference was accounted for by adding 2.5 ml of butanol and 5 ml of heptane to the tubes. All tubes were placed on a vortex mixer for 30 set and centrifuged at 1000g for 5 min. The organic supernatant phase was aspirated and discarded and I ml of the aqueous phase was transferred to tubes. The remainder of the assay procedure (oxidation) should be followed according to the method described previously (3). This oxidation procedure was originally described by Chang (I). The remainder of reagents listed above, but not used, are to be used for the oxidation of NE and DA. RESULTS AND DISCUSSION
Table 1 shows the results of a typical analysis. For that particular day, 5-HT recovery was loo%, NE was 91%, and DA was 86%. The analysis was repeated nine more times with the result that 5-HT recovery was consistently 100%. NE was in a range of 91- 100%. and DA in a range of 86-93s. This method is relatively easy to accomplish and it is relatively fast. The elimination of the alumina step results in a l/3 reduction in time to perform the analysis. The method also retains the slightly larger sample
12
ALFKED
E. CIARLONE
volumes, allowing the use of standard cuvets rather than microcuvets. Greater ease of handling the slightly larger volumes was also advantageous in this laboratory. Last, it should also be noted, and it has been reported previously (3), that this method can be further modified to measure epinephrine (I) and 5-hydroxyindole-3-acetic acid (51, the major metabolite of 5-HT. SUMMARY A method is presented for the fluorometric analysis of serotonin, norepinephrine, and dopamine. The method is relatively easy. relatively fast. and uses manageable sample volumes. The method has the additional advantage of saving 113the time. compared to the time involved to accomplish a similar analysis that was reported previously.
REFERENCES I. 2. 3.
4.
5.
Chang. C. C.. A sensitive method for spectrophotofluorometric assay of catecholamines. I/jr. J. Ner,~ophcr,r,lrr~,[)/, 3, 643-649 (1964). Ciarlone. A. E., Influence of Convulsive Doses of Lidocaine on Central Biogenic Amines. Ph.D. Thesis, University of Pittsburgh. 1974. Ciarlone. A. E.. Modification of a spectrophotofluorometric method of analyzing serotonin, norepinephrine. and dopamine in one brain sample. Mic~roc~hc~m.J. 21, 349-3.54 (1976). Maickel, R. P.. Cox. Jr.. R. H.. Saillant. .I.. and Miller. F. P., A method for the determination of serotonin and norepinephrine in discrete areas of rat brain. /III. .I. Ricrc,ophrr,mcr~o/. 7, 275-281 (1968). Miller. F. P.. Cox, Jr.. R. H.. Snodgrass. N. R.. and Maickel. R. P.. Comparative effects of p-chlorophenylalanine. [I-chloro-amphetamine and p-chloro-N-methylamphetamine on rat brain norepinephrine. 5-hydroxytryptamine, and 5-hydroxyindole-3-acetic acid. Biochrm. Plrtrrn~t~ol. 19, 435-442 ( 1970).
Further
23, 9- 11, (1978)
modification of a Fluorometric for Analyzing Brain Amines
Method
ALFRED E. CIARLOSE
INTRODUCTION This laboratory has attempted to duplicate a fluorometric analysis that was described by Maickel ~1crl. (4). The attempt resulted in failure, due to the difficulty in handling very small samples and due to the presence of an interfering substance(s) when measuring the norepinephrine (NE) fluorophor. In addition, their method did not describe measurement of dopamine (DA) in the same sample and this was considered a necessity in our analysis. Serotonin (5-HT) analysis was described and no difficulty was encountered in duplicating this portion. Another method described by Chang (II did relate the ability to measure NE and DA, but not S-HT. Therefore, by modifying and combining both methods f 1,4), an analysis was reported that enables one to measure (using slightly larger sample volumes) 5-HT, NE, and DA in one sample (3). In the latter report, and probably in Chang’s f/i method, an alumina step eliminated the interfering substance(s). The source of the interference had been investigated for some years. resulting in failure, as cited above. However, some new ideas suggested a reevaluation of the problem, since solving the problem would result in a considerable saving of time (by elimination of the alumina step). The following pieces of data were accumulated over the course of many experiments: (a) although all three amines are stored in 0.1 N hydrochloric acid. increased sensitivity of NE and DA measurement results if ail subsequent dilutions are done with 0.2 N acetic acid, especially since 0.2 N acetic acid dilutions have no effect on 5-HT sensitivity; (b) rr-butanoi was found to be the major cause of interference, with I?-heptane also contributing interference. This was noted by a consistent. approximate 30% increase of fluorescence units for internal standards (exposed to ,I-butanol and r?-heptane) compared to external standards, and this fact was further corroborated by noting increased excitation and emission spectra of NE and DA exposed to /r-butanol and /z-heptane compared to the spectra produced by NE and DA not exposed to the organic compounds; (c) a second extraction step with n-heptane had no effect on tz-butanol‘s interference. 9
10
ALFRED
MATERIALS
E. CIARLONE
AND METHODS
All reagents were either reagent or fluorescence grade. Double deionized and distilled water was used where indicated. n-Butanol (Regis Chemical Company, Morton Grove, Illinois) was acidified as described by Chang (I), n-Heptane (Regis Chemical Company), 0.1 N and 10 N Hydrochloric acid, 5 N Sodium hydroxide, 0.2 N and 5 N Acetic acid, 0.1 N Iodine, as described by Chang (I) or Ciarlone (3), 0.1 M EDTA, as described by Chang (1) or Ciarlone (31, 25% Alkaline sulfite, as described by Chang (I) or Ciarlone (3), o-Phthalaldehyde (OPT) 4 mg% (Regis Chemical Company), 5-HT hydrogen oxalate (Regis Chemical Company) diluted to 1000 pug/ml (as free base) with 0.1 N HCl, NE hydrochloride (Regis Chemical Company) diluted to 1000 pglml (as free base) with 0.1 N HCl, DA hydrochloride (Calbiochem) diluted to 1000 pg/ml (as free base) with 0.1 N HCl. Homogenization of tissue and recovery of the three amines from tissue homogenates has been described previously (I, 2, 4). Duplicate internal standard tubes were carried in parallel with the tissue homogenates. Internal standards containing three concentrations of the three amines were diluted in 0.2 N acetic acid and prepared so that the total volume in each tube was 0.3 ml. These tubes would receive 3 ml of butanol. The homogenates and internal standard tubes were centrifuged at 1000 R for 5 min. Two and one-half milliliters of the supernatant fluid were transferred to tubes containing 1.6 ml of 0.2 N acetic acid and 5 ml of heptane. All tubes were placed on a vortex mixer for 30 set and centrifuged at 1000g for 5 min. The organic supernatant phase was discarded. Two tenths of milliliter of the aqueous phase was transferred to tubes (for continuation of 5-HT analysis) and 1 ml was transferred to tubes for the analysis of NE and DA. The latter tubes may be stored in ice. Three external standards for 5-HT were prepared in duplicate, in 0.2 N acetic acid and to a total volume of 0.2 ml. To all 5-HT tubes, including a reagent blank (consisting of 0.2 ml of 0.2 N acetic acid), 1.2 ml of OPT were added and mixed well. All tubes were placed in a boiling water bath for 10 min, cooled in tap water and read in a spectrophotofluorometer. Excitation and emission were 355 and 470 nm, respectively (uncorrected). Continnution of the NE and DA ancrlysis. At this time, external stan-
FLL’ORORIETRIC
ANALYSIS
11
OF AhlINES
TABLE I RTCOVERI. OF 5-HT. NE, ~SD DA Original
Predicted concentration”
Measured concentration’
Recovery*’
S-HT 3.0 1.5 0.75
0.25 0.125 0.0625
0.25 0.125 0.0625
100
0.6 0.3 0. I5
0.25 0.125 0.0625
0.23 0.1 14 0.057
91
1.2 0.6 0.3
0.5 0.25 0.125
0.43 0.215 0.108
86
NE
DA
” Original microgram concentration added to internal standards. e Predicted microgram concentration (due to known volume losses) in internal standards. I’ Actual microgram concentration measured and interpolated from external standards. ” Measured concentration in internal standards / external standards x 100.
dards were prepared for NE and DA using three concentrations and in duplicate, in 0.2 N acetic acid and to a volume of 1.6 ml per tube. The butanol and heptane interference was accounted for by adding 2.5 ml of butanol and 5 ml of heptane to the tubes. All tubes were placed on a vortex mixer for 30 set and centrifuged at 1000g for 5 min. The organic supernatant phase was aspirated and discarded and I ml of the aqueous phase was transferred to tubes. The remainder of the assay procedure (oxidation) should be followed according to the method described previously (3). This oxidation procedure was originally described by Chang (I). The remainder of reagents listed above, but not used, are to be used for the oxidation of NE and DA. RESULTS AND DISCUSSION
Table 1 shows the results of a typical analysis. For that particular day, 5-HT recovery was loo%, NE was 91%, and DA was 86%. The analysis was repeated nine more times with the result that 5-HT recovery was consistently 100%. NE was in a range of 91- 100%. and DA in a range of 86-93s. This method is relatively easy to accomplish and it is relatively fast. The elimination of the alumina step results in a l/3 reduction in time to perform the analysis. The method also retains the slightly larger sample
12
ALFKED
E. CIARLONE
volumes, allowing the use of standard cuvets rather than microcuvets. Greater ease of handling the slightly larger volumes was also advantageous in this laboratory. Last, it should also be noted, and it has been reported previously (3), that this method can be further modified to measure epinephrine (I) and 5-hydroxyindole-3-acetic acid (51, the major metabolite of 5-HT. SUMMARY A method is presented for the fluorometric analysis of serotonin, norepinephrine, and dopamine. The method is relatively easy. relatively fast. and uses manageable sample volumes. The method has the additional advantage of saving 113the time. compared to the time involved to accomplish a similar analysis that was reported previously.
REFERENCES I. 2. 3.
4.
5.
Chang. C. C.. A sensitive method for spectrophotofluorometric assay of catecholamines. I/jr. J. Ner,~ophcr,r,lrr~,[)/, 3, 643-649 (1964). Ciarlone. A. E., Influence of Convulsive Doses of Lidocaine on Central Biogenic Amines. Ph.D. Thesis, University of Pittsburgh. 1974. Ciarlone. A. E.. Modification of a spectrophotofluorometric method of analyzing serotonin, norepinephrine. and dopamine in one brain sample. Mic~roc~hc~m.J. 21, 349-3.54 (1976). Maickel, R. P.. Cox. Jr.. R. H.. Saillant. .I.. and Miller. F. P., A method for the determination of serotonin and norepinephrine in discrete areas of rat brain. /III. .I. Ricrc,ophrr,mcr~o/. 7, 275-281 (1968). Miller. F. P.. Cox, Jr.. R. H.. Snodgrass. N. R.. and Maickel. R. P.. Comparative effects of p-chlorophenylalanine. [I-chloro-amphetamine and p-chloro-N-methylamphetamine on rat brain norepinephrine. 5-hydroxytryptamine, and 5-hydroxyindole-3-acetic acid. Biochrm. Plrtrrn~t~ol. 19, 435-442 ( 1970).