Life Sciences, Vol . 25, Printed in the U .S .A .
pp . 775-782
Pergamon Press
A SIMPLE, SENSITIVE METHOD FOR MEASURING 3,4-DIHYDROXYPHENYLACETIC ACID AND HOMOVANILLIC ACID IN RAT BRAIN TISSUE USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTIONI Franz Hefti Laboratory of Neuroendocrine Regulation Department of Nutrition and Food Science Massachusetts Institute of Technology Cambridge, Massachusetts 02139 (Received in final form July
23, 1979)
Summa~ A sensitive, specific, and very simple method, using high-performance liquid chromatography combined with electrochemical detection, measured 3,4-dihydroxy phenylacetic acid and homovanillic acid in small areas of rat brain. The dopamine metabolites were extracted with diethyl ether and a known amount of vanillic acid as internal standard, and were separated on a microparticulate reverse-phase column using a sodium acetate buffer (pH 5 .0) as mobile phase. 3,4-Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) are the major metabolites of the neurotransmitter dopamine . Measurement of their concentrations in the brain provides a good estimate of in vivo dopamine release without the necessity of using drugs to inhibit specific enzymes (1,2) . The widely used fluorimetric methods (3,4) can measure these compounds only in large rat brain areas ; automated procedures are more sensitive and can measure amounts as small as about 2 ng (5) . Enzymatic-isotopic methods (6-8) are even more sensitive (10-100 pg) ; however, they are relatively complex and are available for DOPAC only . Sensitivities in the ng range have been obtained with gas chromatographic techniques (9-11), which require derivatiaation of the compounds before analysis . This report describes a simple, rapid method for the measurement of DOPAC and HVA in small areas of rat brain . It is based on the combination of high-performance liquid chromatography (HPLC) and electrochemical detection, a technique successfully used by Adams and associates (12,13) for catecholamine measurement, and is sensitive to less than 100 pg . Materials and Methods The stainless steel HPLC equipment and the electrochemical 1 These studies were supported in part by grants from the National Institutes of Health and the National Aeronautics and Space Administration (to Dr . R. J. Wurtman) . Dr . F. Hefti is a Fellow of the Swiss National Science Foundation . 0024-3205/79/090775-0702 .00/0 Copyright (c) 1979 Pergamon Press Ltd
77 6
HPLC Method for DOPAC and HVA
Vol . 25, No . 9, 1979
detector (Bioanalytical Systems Inc ., West Lafayette, IN) consisted of a single piston pump with electronically controlled flow rate, pulse dampener, loop injection valve, and electrochemical detector (LC-4) . A 125 um gasket was used in the flow cell to give a volume of approximately 10 u1 . The carbon paste electrode was packed according to Keller et al . (13), using CP-O type carbon paste . A Clg -reverse-phase column (3 .9 x 300 mm, uBondapak ; Waters Associates, Milford, MA) was protected by a short precolumn (2 x 69 mm) containing C lg -reverse-phase particles (diamoter 30 um : Whatman, Clifton, NJ) . For analysis of a larger number of samples, the injection valve was replaced by an automatic sample injector (WISP 710A ; Waters) . After the analyses, 50~ isopropanol was run through the system to prevent bacterial growth . The following chemicals and drugs were used : DOPAC and HVA (Regis Chemical Co ., Morton Grove, IL) ; vanillic acid (VA), provenecid, and apomorphine (Sigma Chemical Co ., St . Louis, t40) ; per chloric acid and diethyl ether (Baker Analyzed Reagents, Phillipsburg, t7 J) ; isopropanol (HPLC grade ; Fisher Scientific Co ., Fair Lawn, NJ) . Haloperidol was a gift from McNeil Labs (Fort Washington, PA) . Distilled and deionized water (Millipore Q system ; Millipore Corp ., Bedford, MA) was used for the HPLC buffer . Male Sprague-Dawley rats, 150-200 g (Charles River Breeding Laboratories, Wilmington, MA), were killed by decapitation . After removal of the brain, different areas were dissected by hand and quickly frozen on dry ice . Brain tissue was homogenized in approximately 20 volumes of 0 .1 M perchloric acid . Homogenization and all further extraction steps were done at 0-4°C . Homogenates were centrifuged at 5000 x ~ for 10 minutes and aliquots (100-500 ul) of the supernatant were transferred to conical, plastic, 1,5-m1 tubes (Eppendorf ; Brinkmann Instruments, Inc ., Westbury, NY) for extraction . Perchloric acid (0 .1 M) was added to the final volume of 500 ul . VA (5 ng) in 10 ul of 0 .1 td perchloric acid was added as internal standard . Diethyl ether (750 ul) was then pipetted to the tubes, which were closed immediately, shaken for 15 seconds on a vortex shaker, and briefly centrifuged to separate the phases, Five hundred ul of the ether phase were then carefully transferred to another set of Eppendorf tubes and dried in a rotary evaporator (Savant Instruments, Hicksville, 11Y) for 15 minutes . The residue was reconstituted in 100 ul of 0 .05 to sodium acetate buffer (pH 5 .0) and stored frozen up to one week until further analysis . The metabolites and the internal standard were separated on the C lg -reverse-phase column with 0 .05 M sodium acetate buffer (pit 5 .0) as mobile phase . The buffer was filtered (0 .45 um Millipore filters) and degassed before use . The system was run at a flow rate of 1 .6 ml/minute, yielding a pressure of 1400-2000 psi . The electrochemical detector was set at +0 .7 V, usually at sensitivities between 1 and 5 nA/V . The system was kept at ambient temperature ; 10-80 ul of the extracted samples were injected . Several standards containing equal amounts (0 .1-50 ng) of DOPAC, HVA, and VA also were extracted and analyzed . These standards were used to calculate the ratios of the peak heights of DOPAC/VA and HVA/VA . These ratios were shown to be constant up to 100 ng ; therefore, the means of the ratios obtained from all standards were calculated . DOPAC concentrations of tissue samples F~ere then calculated according to the equation :
HPLC Method for DOPAC and HVA
Vol . 25, No . 9, 1979
DOPAC (ng/ml)=
777
5 ng x peak height DOPAC peak height VA x standard ratio DOPAC/VA x mg tissue
HVA concentrations were calculated similarly . To assess the interference of impurities from the extraction procedure, 500 ul 0 .1 M perchloric acid were extracted and analyzed as a blank . Results and Discussion This reverse-phase system allows baseline separation of DOPAC, HVA, and the internal standard, VA . Fig . 1 shows a typical chromatogram from a rat striatal sample ; typical retention times were 5 .8 minutes for DOPAC, 17 .6 minutes for HVA, and 13 .8 minutes for VA . c~
â S
0.5 nA
y min
FIG . 1
Typical Chromatogram of Tissue Sample Containing Nucleus Accumbens and Olfactory Tubercle of an Untreated Rat The tissue was homogenized in 700 ul of 0 .1 M perchloric acid ; 500 ul were carried through the extraction and 60 ul of the final solution (100 ul) were injected . Chromatographic conditions : C lg -reverse-phase column ; O .Q5 M sodium acetate buffer (pH 5 .0) ; 1 .6 ml/minute flow rate ; 1500 psi ; electrochemical detector +0 .7 V; ambient temperature .
778
HPLC Method for DOPAC and HVA
Vol . 25, No . 9, 1979
ra ~Ô Z Y
FIG . 2 Standard Curve Various amount of DOPAC and HVA were extracted and and analyzed, together with 5 ng of the internal standard, VA . The abscissa shows the ratio of the peak heights of DOPAC and HVA to the peak height of the internal standard in each sample . One ng of both DOPAC and HVA gave a signal of 0 .20 nA when 75 ul of the final extract were injected . The variation of the baseline was lower than 0 .005 nA . The other acidic catecholamine metabolites, 3,4-dihydroxymandelic acid and vanillylmandelic acid, which might interfere with DOPAC and HVA detection, were eluted at 2 .3 and 3 .2 minutes, respectively, and were clearly separated from DOPAC. Occasionally an additional peak was eluted 2 minutes after DOPAC . Its height was always about one-tenth of the DOPAC peak, and it could represent a degradation product of DOPAC. Since the height of this peak was in a constant ratio to the DOPAC peak in all samples and standards, its appearance did not affect the assay's reliability . A small, unidentified peak sometimes appeared 0 .5 minutes before DOPAC . This peak was the only one observed in blanks and therefore was caused by an impurity originating in the extraction pro cedure . 5-Hydroxyindole acetic acid, which was extracted with
Vol . 25, No . 9, 1979
HPLC Method for DOPAC and HVA
779
specially prepared ether (14) eluted between VA and HVA (15 .9 minutes) and did not interfere with the separation . In the brain areas studied, no peak corresponding to this compound was detected . DOPAC and HVA extraction with diethyl ether is a very simple and efficient purification step that separates these compounds from any material interfering with the electrochemical detection process . Recoveries are smaller than those achieved with ethyl acetate (4,5) ; however, the method's sensitivity is high enough to compensate for this difference and the use of an internal standard corrects for possible variations in recoveries . The mean corrected recoveries were 318 for DOPAC, 60$ for HVA, and 89$ for VA, and were constant up to at least 100 ng . Given the relatively short retention times of DOPAC and HVA obtained with this separation, about 70 samples could be analyzed per day when the automatic sample injector was used . A higher temperature (up to 60 °C) during the column separation further reduced the the retention times without affecting the quality of the separation . Fig. 2 shows a standard curve obtained with standards carried through the extraction . The detector response was linear up to 100 ng of DOPAC and HVA . Using peak areas instead of peak heights for the calculation did not improve the reliability of the assay . The method's sensitivity is largely dependent upon the performance of the electrochemical detector . The carbon paste electrode was replaced weekly to ensure a good baseline . With directly injected standards, 5 pg of DOPAC and HVA could be detected ; with standards carried through the extraction procedure, a limit sensitivity (defined as the amount giving a peak twice as high as the fluctuation of the baseline) of 50-100 pg was achieved . No special effort was made to increase the sensitivity to lower levels ; sensitivity could possibly be increased by using slower flow rates, greater buffer strength, and smaller column diameters . Table 1 lists DOPAC and HVA concentrationa .found in different rat brain regions rich in dopaminergic innervation . The concentrations are in the range of those previously reported (5-7) . TABLE 1 DOPAC and HVA Concentrations in Rat Brain Areas Rich in Dopaminergic Innervation DOPAC (ng/~J)
(ng/mg)
Substantia nigra
0 .15 + 0 .03
0 .18 + 0 .03
Corpus striatum
1 .95 + 0 .13
0 .69 + 0 .06
Nucleus accumbens + olfactory tubercle
0 .80 + 0 .12
0 .30 + 0 .03
Prefrontal cortex
0 .05 + 0 .01
0 .04 + 0 .01
HVA
Data are given as means + S .E .M . ; n = 5 or 6 .
780
HPLC Method for DOPAC and HVA
Voi . 25, No . 9, 1979
In all tested areas, only peaks corresponding to DOPAC and HVA were observed . No endogenous VA could be detected, which permitted its use as an internal standard . Endogenous VA should be tested in each new area to be analyzed . Table 2 shows a pharmacological experiment confirming the reliability of the method : as previously reported (1,2), haloperidol and probenecid increased the levels of the dopamine metabolites, whereas apomorphine reduced them . TABLE 2 Effects of Probenecid, Haloperidol, and Apomorphine on Striatal DOPAC and HVA Concentrations DOPAC (nq/mg)
HVA (ng/mg)
Saline (n = 6)
15 .8 + 1 .0
5 .1 + 0 .3
Acetic acid (n = 4)
16 .2 + 2 .6
4 .2 + 0 .8
Probenecid, 200 mg/kg, 1 hour (n = 7)
19 .6 + 1 .O a
11 .0 + 1 .5 b
Haloperidol, 5 mg/kg, 30 minutes (n = 6)
33 .8 + 2 .6 b
14 .4 + 1 .6 b
Apomorphine, 1 mg/kg, 30 minutes (n = 6)
13 .8 + 1 .2
3 .1 + 0 .3b
Haloperidol was dissolved in a few drops of concenThe trated acetic acid and then diluted with saline . vehicle was injected as control . Proteins were determined according to the method of Lowry _et _al . (15) . Data are presented as means + S .E .M . aP < 0 .05, differs from control . bP < 0 .01, differs from control . Acknowledgements The author thanks Miss C . Watkins and Dr . B . Glaeser for technical assistance and Dr . R.J . Wurtman for his support and interest . References 1 . J . KORF, L . GRASDIJK and B .H .C . WESTERINK, J. Pieurochem . _26 : 579-584 (1976) . 2 . R .H . ROTH, L.C . MURRIN and J .R . WALTERS, Eur . J . Pharmacol . _36 : 163-171 (1976) . 3 . G .F . MURPHY, D . ROBINSON and D .F . SHARMAN, Br . J . Pharmacol . 36 : 107-115 (1969) . 4. J .R . WALTERS and R.H . ROTH, Biochem . Pharmacol . 21 : 2111-2121 (1972) . 5 . B .H .C . WESTERINK and J . KORF, Eur . J . Pharmacol . _38 : 281-291 (1976) . 6 . A. ARGIOLAS, F . FADDA, E . STEFANINI and G.L . GESSA, J . Neurochem . 29 : 599-601 (1977) .
Vol . 25, No, 9, 1979
HPLC Method for DOPAC and HVA
781
7, J.W . KEBABIAN, J.M . SAAVEDRA and J . AXELROD, J. Neurochem, _28 : 795-801 (1977) . 8 . C .F, SALLER and M,J . ZIGMOND, Life Sci . 23 : 117-1130 (1978) . 9 . J .D .M . PEARSON and D .F, SHARMAN, Br . J . PTiarmacol . _53 : 143148 (1975) . 10 . E . WATSON, B . TRAVIS and S, WILK, Life Sci, _15 : 2167-2178 (1974) . 11, D .S . WALKER, P .W . DETTMAR, K . TAYLOR, G .M, SHILOCK and A . COWAN, J . Neurochem 30 : 929-931 (1978) . 12, L .J . FELICE, J.D . FELICSand P .T . KISSINGER, J . Neurochem . _31 : 1461-1465 (1978) . 13 . R . KELLER, A, OKE, I . MEFFORD and R .N . ADAMS, Life Sci . _19 : 959~1004 (1976) . 14, S . UDENFRIEND, E . TITUS and H . WEISSBACH, J, Biol . Chem . _216 : 499-512 (1955) . 15 . O .H, LOWRY, N.J . ROSEBROUGH, A. FARR and R. J . RANDALL, J . Biol, Chem . 193 : 265-275 (1951) .
pp . 775-782
Pergamon Press
A SIMPLE, SENSITIVE METHOD FOR MEASURING 3,4-DIHYDROXYPHENYLACETIC ACID AND HOMOVANILLIC ACID IN RAT BRAIN TISSUE USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTIONI Franz Hefti Laboratory of Neuroendocrine Regulation Department of Nutrition and Food Science Massachusetts Institute of Technology Cambridge, Massachusetts 02139 (Received in final form July
23, 1979)
Summa~ A sensitive, specific, and very simple method, using high-performance liquid chromatography combined with electrochemical detection, measured 3,4-dihydroxy phenylacetic acid and homovanillic acid in small areas of rat brain. The dopamine metabolites were extracted with diethyl ether and a known amount of vanillic acid as internal standard, and were separated on a microparticulate reverse-phase column using a sodium acetate buffer (pH 5 .0) as mobile phase. 3,4-Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) are the major metabolites of the neurotransmitter dopamine . Measurement of their concentrations in the brain provides a good estimate of in vivo dopamine release without the necessity of using drugs to inhibit specific enzymes (1,2) . The widely used fluorimetric methods (3,4) can measure these compounds only in large rat brain areas ; automated procedures are more sensitive and can measure amounts as small as about 2 ng (5) . Enzymatic-isotopic methods (6-8) are even more sensitive (10-100 pg) ; however, they are relatively complex and are available for DOPAC only . Sensitivities in the ng range have been obtained with gas chromatographic techniques (9-11), which require derivatiaation of the compounds before analysis . This report describes a simple, rapid method for the measurement of DOPAC and HVA in small areas of rat brain . It is based on the combination of high-performance liquid chromatography (HPLC) and electrochemical detection, a technique successfully used by Adams and associates (12,13) for catecholamine measurement, and is sensitive to less than 100 pg . Materials and Methods The stainless steel HPLC equipment and the electrochemical 1 These studies were supported in part by grants from the National Institutes of Health and the National Aeronautics and Space Administration (to Dr . R. J. Wurtman) . Dr . F. Hefti is a Fellow of the Swiss National Science Foundation . 0024-3205/79/090775-0702 .00/0 Copyright (c) 1979 Pergamon Press Ltd
77 6
HPLC Method for DOPAC and HVA
Vol . 25, No . 9, 1979
detector (Bioanalytical Systems Inc ., West Lafayette, IN) consisted of a single piston pump with electronically controlled flow rate, pulse dampener, loop injection valve, and electrochemical detector (LC-4) . A 125 um gasket was used in the flow cell to give a volume of approximately 10 u1 . The carbon paste electrode was packed according to Keller et al . (13), using CP-O type carbon paste . A Clg -reverse-phase column (3 .9 x 300 mm, uBondapak ; Waters Associates, Milford, MA) was protected by a short precolumn (2 x 69 mm) containing C lg -reverse-phase particles (diamoter 30 um : Whatman, Clifton, NJ) . For analysis of a larger number of samples, the injection valve was replaced by an automatic sample injector (WISP 710A ; Waters) . After the analyses, 50~ isopropanol was run through the system to prevent bacterial growth . The following chemicals and drugs were used : DOPAC and HVA (Regis Chemical Co ., Morton Grove, IL) ; vanillic acid (VA), provenecid, and apomorphine (Sigma Chemical Co ., St . Louis, t40) ; per chloric acid and diethyl ether (Baker Analyzed Reagents, Phillipsburg, t7 J) ; isopropanol (HPLC grade ; Fisher Scientific Co ., Fair Lawn, NJ) . Haloperidol was a gift from McNeil Labs (Fort Washington, PA) . Distilled and deionized water (Millipore Q system ; Millipore Corp ., Bedford, MA) was used for the HPLC buffer . Male Sprague-Dawley rats, 150-200 g (Charles River Breeding Laboratories, Wilmington, MA), were killed by decapitation . After removal of the brain, different areas were dissected by hand and quickly frozen on dry ice . Brain tissue was homogenized in approximately 20 volumes of 0 .1 M perchloric acid . Homogenization and all further extraction steps were done at 0-4°C . Homogenates were centrifuged at 5000 x ~ for 10 minutes and aliquots (100-500 ul) of the supernatant were transferred to conical, plastic, 1,5-m1 tubes (Eppendorf ; Brinkmann Instruments, Inc ., Westbury, NY) for extraction . Perchloric acid (0 .1 M) was added to the final volume of 500 ul . VA (5 ng) in 10 ul of 0 .1 td perchloric acid was added as internal standard . Diethyl ether (750 ul) was then pipetted to the tubes, which were closed immediately, shaken for 15 seconds on a vortex shaker, and briefly centrifuged to separate the phases, Five hundred ul of the ether phase were then carefully transferred to another set of Eppendorf tubes and dried in a rotary evaporator (Savant Instruments, Hicksville, 11Y) for 15 minutes . The residue was reconstituted in 100 ul of 0 .05 to sodium acetate buffer (pH 5 .0) and stored frozen up to one week until further analysis . The metabolites and the internal standard were separated on the C lg -reverse-phase column with 0 .05 M sodium acetate buffer (pit 5 .0) as mobile phase . The buffer was filtered (0 .45 um Millipore filters) and degassed before use . The system was run at a flow rate of 1 .6 ml/minute, yielding a pressure of 1400-2000 psi . The electrochemical detector was set at +0 .7 V, usually at sensitivities between 1 and 5 nA/V . The system was kept at ambient temperature ; 10-80 ul of the extracted samples were injected . Several standards containing equal amounts (0 .1-50 ng) of DOPAC, HVA, and VA also were extracted and analyzed . These standards were used to calculate the ratios of the peak heights of DOPAC/VA and HVA/VA . These ratios were shown to be constant up to 100 ng ; therefore, the means of the ratios obtained from all standards were calculated . DOPAC concentrations of tissue samples F~ere then calculated according to the equation :
HPLC Method for DOPAC and HVA
Vol . 25, No . 9, 1979
DOPAC (ng/ml)=
777
5 ng x peak height DOPAC peak height VA x standard ratio DOPAC/VA x mg tissue
HVA concentrations were calculated similarly . To assess the interference of impurities from the extraction procedure, 500 ul 0 .1 M perchloric acid were extracted and analyzed as a blank . Results and Discussion This reverse-phase system allows baseline separation of DOPAC, HVA, and the internal standard, VA . Fig . 1 shows a typical chromatogram from a rat striatal sample ; typical retention times were 5 .8 minutes for DOPAC, 17 .6 minutes for HVA, and 13 .8 minutes for VA . c~
â S
0.5 nA
y min
FIG . 1
Typical Chromatogram of Tissue Sample Containing Nucleus Accumbens and Olfactory Tubercle of an Untreated Rat The tissue was homogenized in 700 ul of 0 .1 M perchloric acid ; 500 ul were carried through the extraction and 60 ul of the final solution (100 ul) were injected . Chromatographic conditions : C lg -reverse-phase column ; O .Q5 M sodium acetate buffer (pH 5 .0) ; 1 .6 ml/minute flow rate ; 1500 psi ; electrochemical detector +0 .7 V; ambient temperature .
778
HPLC Method for DOPAC and HVA
Vol . 25, No . 9, 1979
ra ~Ô Z Y
FIG . 2 Standard Curve Various amount of DOPAC and HVA were extracted and and analyzed, together with 5 ng of the internal standard, VA . The abscissa shows the ratio of the peak heights of DOPAC and HVA to the peak height of the internal standard in each sample . One ng of both DOPAC and HVA gave a signal of 0 .20 nA when 75 ul of the final extract were injected . The variation of the baseline was lower than 0 .005 nA . The other acidic catecholamine metabolites, 3,4-dihydroxymandelic acid and vanillylmandelic acid, which might interfere with DOPAC and HVA detection, were eluted at 2 .3 and 3 .2 minutes, respectively, and were clearly separated from DOPAC. Occasionally an additional peak was eluted 2 minutes after DOPAC . Its height was always about one-tenth of the DOPAC peak, and it could represent a degradation product of DOPAC. Since the height of this peak was in a constant ratio to the DOPAC peak in all samples and standards, its appearance did not affect the assay's reliability . A small, unidentified peak sometimes appeared 0 .5 minutes before DOPAC . This peak was the only one observed in blanks and therefore was caused by an impurity originating in the extraction pro cedure . 5-Hydroxyindole acetic acid, which was extracted with
Vol . 25, No . 9, 1979
HPLC Method for DOPAC and HVA
779
specially prepared ether (14) eluted between VA and HVA (15 .9 minutes) and did not interfere with the separation . In the brain areas studied, no peak corresponding to this compound was detected . DOPAC and HVA extraction with diethyl ether is a very simple and efficient purification step that separates these compounds from any material interfering with the electrochemical detection process . Recoveries are smaller than those achieved with ethyl acetate (4,5) ; however, the method's sensitivity is high enough to compensate for this difference and the use of an internal standard corrects for possible variations in recoveries . The mean corrected recoveries were 318 for DOPAC, 60$ for HVA, and 89$ for VA, and were constant up to at least 100 ng . Given the relatively short retention times of DOPAC and HVA obtained with this separation, about 70 samples could be analyzed per day when the automatic sample injector was used . A higher temperature (up to 60 °C) during the column separation further reduced the the retention times without affecting the quality of the separation . Fig. 2 shows a standard curve obtained with standards carried through the extraction . The detector response was linear up to 100 ng of DOPAC and HVA . Using peak areas instead of peak heights for the calculation did not improve the reliability of the assay . The method's sensitivity is largely dependent upon the performance of the electrochemical detector . The carbon paste electrode was replaced weekly to ensure a good baseline . With directly injected standards, 5 pg of DOPAC and HVA could be detected ; with standards carried through the extraction procedure, a limit sensitivity (defined as the amount giving a peak twice as high as the fluctuation of the baseline) of 50-100 pg was achieved . No special effort was made to increase the sensitivity to lower levels ; sensitivity could possibly be increased by using slower flow rates, greater buffer strength, and smaller column diameters . Table 1 lists DOPAC and HVA concentrationa .found in different rat brain regions rich in dopaminergic innervation . The concentrations are in the range of those previously reported (5-7) . TABLE 1 DOPAC and HVA Concentrations in Rat Brain Areas Rich in Dopaminergic Innervation DOPAC (ng/~J)
(ng/mg)
Substantia nigra
0 .15 + 0 .03
0 .18 + 0 .03
Corpus striatum
1 .95 + 0 .13
0 .69 + 0 .06
Nucleus accumbens + olfactory tubercle
0 .80 + 0 .12
0 .30 + 0 .03
Prefrontal cortex
0 .05 + 0 .01
0 .04 + 0 .01
HVA
Data are given as means + S .E .M . ; n = 5 or 6 .
780
HPLC Method for DOPAC and HVA
Voi . 25, No . 9, 1979
In all tested areas, only peaks corresponding to DOPAC and HVA were observed . No endogenous VA could be detected, which permitted its use as an internal standard . Endogenous VA should be tested in each new area to be analyzed . Table 2 shows a pharmacological experiment confirming the reliability of the method : as previously reported (1,2), haloperidol and probenecid increased the levels of the dopamine metabolites, whereas apomorphine reduced them . TABLE 2 Effects of Probenecid, Haloperidol, and Apomorphine on Striatal DOPAC and HVA Concentrations DOPAC (nq/mg)
HVA (ng/mg)
Saline (n = 6)
15 .8 + 1 .0
5 .1 + 0 .3
Acetic acid (n = 4)
16 .2 + 2 .6
4 .2 + 0 .8
Probenecid, 200 mg/kg, 1 hour (n = 7)
19 .6 + 1 .O a
11 .0 + 1 .5 b
Haloperidol, 5 mg/kg, 30 minutes (n = 6)
33 .8 + 2 .6 b
14 .4 + 1 .6 b
Apomorphine, 1 mg/kg, 30 minutes (n = 6)
13 .8 + 1 .2
3 .1 + 0 .3b
Haloperidol was dissolved in a few drops of concenThe trated acetic acid and then diluted with saline . vehicle was injected as control . Proteins were determined according to the method of Lowry _et _al . (15) . Data are presented as means + S .E .M . aP < 0 .05, differs from control . bP < 0 .01, differs from control . Acknowledgements The author thanks Miss C . Watkins and Dr . B . Glaeser for technical assistance and Dr . R.J . Wurtman for his support and interest . References 1 . J . KORF, L . GRASDIJK and B .H .C . WESTERINK, J. Pieurochem . _26 : 579-584 (1976) . 2 . R .H . ROTH, L.C . MURRIN and J .R . WALTERS, Eur . J . Pharmacol . _36 : 163-171 (1976) . 3 . G .F . MURPHY, D . ROBINSON and D .F . SHARMAN, Br . J . Pharmacol . 36 : 107-115 (1969) . 4. J .R . WALTERS and R.H . ROTH, Biochem . Pharmacol . 21 : 2111-2121 (1972) . 5 . B .H .C . WESTERINK and J . KORF, Eur . J . Pharmacol . _38 : 281-291 (1976) . 6 . A. ARGIOLAS, F . FADDA, E . STEFANINI and G.L . GESSA, J . Neurochem . 29 : 599-601 (1977) .
Vol . 25, No, 9, 1979
HPLC Method for DOPAC and HVA
781
7, J.W . KEBABIAN, J.M . SAAVEDRA and J . AXELROD, J. Neurochem, _28 : 795-801 (1977) . 8 . C .F, SALLER and M,J . ZIGMOND, Life Sci . 23 : 117-1130 (1978) . 9 . J .D .M . PEARSON and D .F, SHARMAN, Br . J . PTiarmacol . _53 : 143148 (1975) . 10 . E . WATSON, B . TRAVIS and S, WILK, Life Sci, _15 : 2167-2178 (1974) . 11, D .S . WALKER, P .W . DETTMAR, K . TAYLOR, G .M, SHILOCK and A . COWAN, J . Neurochem 30 : 929-931 (1978) . 12, L .J . FELICE, J.D . FELICSand P .T . KISSINGER, J . Neurochem . _31 : 1461-1465 (1978) . 13 . R . KELLER, A, OKE, I . MEFFORD and R .N . ADAMS, Life Sci . _19 : 959~1004 (1976) . 14, S . UDENFRIEND, E . TITUS and H . WEISSBACH, J, Biol . Chem . _216 : 499-512 (1955) . 15 . O .H, LOWRY, N.J . ROSEBROUGH, A. FARR and R. J . RANDALL, J . Biol, Chem . 193 : 265-275 (1951) .
