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Metabolic fate of 3,4-dihydroxyphenylethyleneglycol (DHPG) in mouse brain
m. NeuroPsychopharmacol b Biol. Psychiat 1983. Vol. 7. pp. 751-754 Printed in Great Brttatn Au rtghb reserved.
METABOLIC
Copyright
FATE OF 3,4-DIHYDRO xypHwryLBTHyLwlBGLYCOL IN MOUSE BRAIN
0
0278-5846m $0.00 + .50 1083 Per&mn~ Ress Ltd.
@MPG)
PETEK P. LI, JERRY J. WABSH AND DAMODAK D. GODSE
Section of Biochemical Psychiatry Clarke Institute of Psychiatry Toronto, Ontario Canada
(Final form, June 1983)
Abstract
Li, Peter P., Jerry J. Warsh and Damodar D. Godse: Metabolic fate of 3,4-dihydroxyphenylethyleneglycol (DHPG) in mouse brain. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1983, L (4-6):751-754. 1.
2.
3. 4.
Mouse brain DHPG and MHPG turnover rates were estimated by determining their initial rates of d&appearance or accumulation following MAO and/or COMT inhibition. Similar turnover estimates of brain DHPG were obtained following MAO or COMT inhibition, which were comparable to the estimated NE turnover obtained from its initial accumulation following MAO plus COMT inhibition. It was estimated that negligible amounts of DHPG were eliminated directly from brain, the majority being cleared through O-methylation. These findings indicate that mouse brain NE is primarily cleared through DHPG formation followed by O-methylation,and also suggest that brain DHPG turnover is more indicative of NE turnover.
3-methoxy-4-hydroxyphenylethyleneglycol, 3,4-dihydroxyphenylethyleneglycol, inhibition, mouse brain, turnover rates
Abbreviations: catechol-O-methyltransferase (COMT); 3,4-dihydroxyphenylethyleneglycol (DHPG); fractional rate constant of elimination (k); half life (8%); monoamine oxidase (MAO); 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG); norepinephrine (NE)
Introduction Although DHPG and MHPG are the major metabolitee of brain NE (Wareh et al., 1981a,b), the metabolic interrelationship between these metabolites has not been completely clarified. Thus, it is still uncertain to what extent NE is dearinated to DHPG and subsequently 0-methylated to MHPG or whether MHPG is formed exclusively from normetanephrine (NMN). Indirect evidence from radioisotopic studies suggested that under basal conditions, most of the released NE is taken up into preeynaptic terminals and deaminated to DHPG with subsequent 0-methylation to MHPG. It is thought that under conditions of increased NE release, MHPG is formed primarily from NMN (DeMet and Halarie, 1979). The dynamics of this system may become more complicated in species in which these glycol metabolites occur in both free and conjugated form.
751
752
P.P. Li et al.
To clarify these issues, a series of kinetic studies were undertaken to evaluate the degree of conversion of DHPG to MHPG and the amount of DHPG eliminated unchanged from the brain. Studies were undertaken in the mouse, a species in which these brain metabolites occur predominantly in free form, as in humans. Brain DHPG and MHPG turnover rates were estimated from the disappearance curves of these metabolites following synthesis inhibition. Tropolone was employed as an inhibitor of COMT, whereas clorgyline and psrgyline were used as inhibitors of MAO-A and MAO-B, respectively.
Methods
Animals. Male Swiss Hanschka mice (CD-l, Charles River, Montreal) weighing 30-40 g were housed five per cage in a temperature (21 + L'C) and light (lights on 0800-2000 h) controlled environment for two weeks before use. Animals had free access to food and water.
Druge. Clorgyline-HCl (May and Baker Ltd., Dagenham, U.K.), pargyline-HCl (Regis Chemical Co., Morton Grove, IL) and tropolone (Aldrtch Chemical Co., Milwaukee, WI) were dissolved in saline and administered intraperitoneallyin a volume of 10 mL/kg.
Experimental Procedures. Although brain NE is a preferential substrate for MAO-A, preliminary experiments had shown that a small but significant amount of brain NE is deaminated by MAO-B when MAO-A and COMT are comoletely inhibited. To achieve comolete MAO inhibition, a combination of clorgyline and pargyline-was used.
To estimate brain DHPG and MHPG turnover rates, the animals were treated with the following drug regimens: (A) clorgyline (37 nmol/kg) and pargyline (471 nmol/kg), (B) tropolone (614 nmol/kg) and (C) clorgyline (37 nmol/kg), pargyline (471 nmol/kg) and tropolone (614 nmol/kg). Groups of 4-6 mice were then killed by decapitation at selected time interval8 following drug administration. The brains were rapidly excised, frozen and stored at -70 C. Brain free DHPG and MHPG were determined simultaneously by mass frsgmentography as their respective acetyl-trifluoroacylderivatives using 2H2-DHPG and 2H3MHPG as internal standards (Warsh et al., 1981a).
Data were analyzed by univariate analysis of variance followed by Newman Keul's test. Disappearance curves were derived by calculating best-fitting lines to the data points by least square linear regression analysis (Winer, 1971).
Results Brain DHPG levels increased curvilinearly with respect to time over a 60 min interval following tropolone administration. Based on the linear regression analysis of the data from the first 30 min, the accumulation rate of DHPG was estimated to be 1000 pmol/g/h (data not shown). Following administrationof clorgyllne plus pargyline, there was an immediate and exponential decline of brain DHPG levels (Fig. 1). The fractional rate constant of disappearance (k) was estimated by the least square method (k - -2.303 x slope) to be 9.9 kO.6 h-1 (correspondingto a half life t% - 4.2 min); the turnover of brain DHPG was calculated to be 913 pmol/g/h. In contrast, brain MWPG levels remained unchanged during the first 10 min and only declined thereafter (data not shown). In mice receiving a combination of clorgyline, pargyline and tropolone, the disappearance of brain MHPG became exponential (Pig. 2). The MHPG turnover was calculated to be 663 pmol/g/h (k = 2.0 f 0.1 h-l; t%= 20 min). Concurrently, there was only a slight decrease of DHPG levels over a 30 min Interval (data not shown).
753
Mouse brain DHPG, MHPG and NE turnover
IO
1.0
I
2
4 TIME
6
8
0
10
(min)
5
IO
15
20
25
30
Time (mid
Fig. 1. The semflogarithmicdecline of mouse brain DHPG following administrationof clorgyline (37 nmol/kg) in combination with pargyline (471 nmolfkg). Each point is the mean * S.E.M. for groups of 4-6 animals.
The semilogarithmicdecline Fig. 2. of mouse brain MHPG following administration of clorgyline (37 pmol/kg) in combination with pargyline (471 pmol/kg) and tropolone (614 nmol/kg). Each point is the the mean f S.E.M. for 4-6 animals.
Discussion
The findings that comparable turnover rates of brain DHPG were obtained following either MAO or COMT inhibition indicated that mouse brain DHPG is primarily cleared through 0-methylation. This is in marked contrast to the clearance of rat brain DHPG which proceeds through sulfo-conjugationas vell as O-methylation with subsequent elimination via an active transport process (Gale and Maas, 1977; Li et al., 1981). These findings emphasize marked species differences In the metabolic clearance of brain DHPG which may, in part, be dependent upon the conjugating capacity for these glycols.
One minute after an injection of clorgyline plus pargyline, there was an almost complete (97-100X) inhibition of MAO-A and MAO-B activities (data not shown). This immediate inhibition of MAO together with the exponential decline of DHPG following clorgyllne plus pargyline suggests that brain MAO-A and MAO-B are completely and instantaneouslyinhibited following these drugs. The turnover rate of DHPG calculated by this method (913 pmol/g/h) was similar to the NE turnover value (944 pmol/g/h) estimated from its Initial accumulation after the degradattve enzymes had been inhibited (unpublished data). These findings support the notion that deamination of NE to DHPG is the primary route of brain NE metabolism. It also suggests that DHPG and not MHPG turnover provides a more accurate Index of brain NE turnover. Similar observations have been made in the rat (Li et al., 1981).
Unlike DHPG, brain MHPG levels did not decline exponentially after clorgylfne plus pargyline. However, the decline of MHPG appeared to occur 10 min after the drug Furthermore, the injection, at a time when DHPG levels were almost depleted. disappearance of MHPG became exponential when COMT, in addition to MAO-A and -B, was also inhibited (Fig. 2). These observations suggest that the initial slower rate of
754
P.P. Li et at.
disappearance of MHPG after MAD-A and MA&B inhibition is due to the ongoing 0-methylation of DHPG to MHPG during that interval, and support the notion that DHPG is primarily cleared through 0-methylation in this species.
Based on the foregoing turnover studies, we have estimated that 913-1000 pmol/g/h DHPG is formed and subsequently O-methylated in the brain with minimal direct elimination from the brain. The finding that the estimate of MHPG turnover (663 pmol/g/h) was lower than that determined for DHPG may reflect one of two possibilities. Firstly, there may be multiple co-existing MHPG pools which behave in a kinetically different manner, such that the smaller pool(s) with high turnover are neglected in the overall turnover estimates. Alternatively, DAPG may also be metabolized to other, as yet unidentified, methylated products.
Assuming the dynamics and disposition of DHPG and MHPG are similar in mouse and human brain, the above observations suggest that in humans, plasma and urinary DHPG are likely derived from peripheral sources and more likely provide an index of peripheral sympathetic function.
Acknowledgements
We wish to thank Mrs Susan McNally for her preparation of the manuscript, Mr. Andrew Chiu and Ms. Cathy Spegg for data analysis. This work was supported in part by MRC Canada #MA-7300.
References
DEMET, E.M. and HALARIS, A.E. (1979) Origin and distribution of 3-methoxy-4-hydroxyphenylglycol in body fluids. Biochem. Pharmacol. -28: 3043-3050. GALE, St. W. and MAAS, J.W. (1977) A study of the formation and metabolic disposition of 3,4-dihydroxyphenylethyleneglycol in whole rat brain. J. Neural Transm. -41: 59-72. LI, P.P., WARS& J.J. and GODSE, D.D. (1981) 3,4-Dihydroxypbenylethyleneglycol (DHPG) formation : the major route of rat brain norepinephrinemetabolism. Prog. Neuro-Psychopharmacol. 2: 531-535. WARSH, J.J., GODSE, D.D., CHEUNG, S.W. and LI, P.P. (1981a) Rat brain and plasrllanorepinephrine glycol metabolites determined by gas chromatography-massfragmentography. J. Neurochem. -36: 893-901. WARSH, J.J., LI, P.P., GODSE, D.D. and CHRUNG, S. (1981b) Brain noradrenergic neuronal Life Sci. -29: activity affects 3,4-dihydroxyphenylethyleneglycol (DHPG) levels. 1303-1307. WINER, B.J. (1971) Statistical Principles in Experimental Design. McGraw-Hill, New York.
Inquiries and reprint requests should be addressed to:
Mr. Peter Li Section of Biochemical Psychiatry Clarke Institute of Psychiatry 250 College Street Toronto, ONT., Canada M5T lR8
METABOLIC
Copyright
FATE OF 3,4-DIHYDRO xypHwryLBTHyLwlBGLYCOL IN MOUSE BRAIN
0
0278-5846m $0.00 + .50 1083 Per&mn~ Ress Ltd.
@MPG)
PETEK P. LI, JERRY J. WABSH AND DAMODAK D. GODSE
Section of Biochemical Psychiatry Clarke Institute of Psychiatry Toronto, Ontario Canada
(Final form, June 1983)
Abstract
Li, Peter P., Jerry J. Warsh and Damodar D. Godse: Metabolic fate of 3,4-dihydroxyphenylethyleneglycol (DHPG) in mouse brain. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1983, L (4-6):751-754. 1.
2.
3. 4.
Mouse brain DHPG and MHPG turnover rates were estimated by determining their initial rates of d&appearance or accumulation following MAO and/or COMT inhibition. Similar turnover estimates of brain DHPG were obtained following MAO or COMT inhibition, which were comparable to the estimated NE turnover obtained from its initial accumulation following MAO plus COMT inhibition. It was estimated that negligible amounts of DHPG were eliminated directly from brain, the majority being cleared through O-methylation. These findings indicate that mouse brain NE is primarily cleared through DHPG formation followed by O-methylation,and also suggest that brain DHPG turnover is more indicative of NE turnover.
3-methoxy-4-hydroxyphenylethyleneglycol, 3,4-dihydroxyphenylethyleneglycol, inhibition, mouse brain, turnover rates
Abbreviations: catechol-O-methyltransferase (COMT); 3,4-dihydroxyphenylethyleneglycol (DHPG); fractional rate constant of elimination (k); half life (8%); monoamine oxidase (MAO); 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG); norepinephrine (NE)
Introduction Although DHPG and MHPG are the major metabolitee of brain NE (Wareh et al., 1981a,b), the metabolic interrelationship between these metabolites has not been completely clarified. Thus, it is still uncertain to what extent NE is dearinated to DHPG and subsequently 0-methylated to MHPG or whether MHPG is formed exclusively from normetanephrine (NMN). Indirect evidence from radioisotopic studies suggested that under basal conditions, most of the released NE is taken up into preeynaptic terminals and deaminated to DHPG with subsequent 0-methylation to MHPG. It is thought that under conditions of increased NE release, MHPG is formed primarily from NMN (DeMet and Halarie, 1979). The dynamics of this system may become more complicated in species in which these glycol metabolites occur in both free and conjugated form.
751
752
P.P. Li et al.
To clarify these issues, a series of kinetic studies were undertaken to evaluate the degree of conversion of DHPG to MHPG and the amount of DHPG eliminated unchanged from the brain. Studies were undertaken in the mouse, a species in which these brain metabolites occur predominantly in free form, as in humans. Brain DHPG and MHPG turnover rates were estimated from the disappearance curves of these metabolites following synthesis inhibition. Tropolone was employed as an inhibitor of COMT, whereas clorgyline and psrgyline were used as inhibitors of MAO-A and MAO-B, respectively.
Methods
Animals. Male Swiss Hanschka mice (CD-l, Charles River, Montreal) weighing 30-40 g were housed five per cage in a temperature (21 + L'C) and light (lights on 0800-2000 h) controlled environment for two weeks before use. Animals had free access to food and water.
Druge. Clorgyline-HCl (May and Baker Ltd., Dagenham, U.K.), pargyline-HCl (Regis Chemical Co., Morton Grove, IL) and tropolone (Aldrtch Chemical Co., Milwaukee, WI) were dissolved in saline and administered intraperitoneallyin a volume of 10 mL/kg.
Experimental Procedures. Although brain NE is a preferential substrate for MAO-A, preliminary experiments had shown that a small but significant amount of brain NE is deaminated by MAO-B when MAO-A and COMT are comoletely inhibited. To achieve comolete MAO inhibition, a combination of clorgyline and pargyline-was used.
To estimate brain DHPG and MHPG turnover rates, the animals were treated with the following drug regimens: (A) clorgyline (37 nmol/kg) and pargyline (471 nmol/kg), (B) tropolone (614 nmol/kg) and (C) clorgyline (37 nmol/kg), pargyline (471 nmol/kg) and tropolone (614 nmol/kg). Groups of 4-6 mice were then killed by decapitation at selected time interval8 following drug administration. The brains were rapidly excised, frozen and stored at -70 C. Brain free DHPG and MHPG were determined simultaneously by mass frsgmentography as their respective acetyl-trifluoroacylderivatives using 2H2-DHPG and 2H3MHPG as internal standards (Warsh et al., 1981a).
Data were analyzed by univariate analysis of variance followed by Newman Keul's test. Disappearance curves were derived by calculating best-fitting lines to the data points by least square linear regression analysis (Winer, 1971).
Results Brain DHPG levels increased curvilinearly with respect to time over a 60 min interval following tropolone administration. Based on the linear regression analysis of the data from the first 30 min, the accumulation rate of DHPG was estimated to be 1000 pmol/g/h (data not shown). Following administrationof clorgyllne plus pargyline, there was an immediate and exponential decline of brain DHPG levels (Fig. 1). The fractional rate constant of disappearance (k) was estimated by the least square method (k - -2.303 x slope) to be 9.9 kO.6 h-1 (correspondingto a half life t% - 4.2 min); the turnover of brain DHPG was calculated to be 913 pmol/g/h. In contrast, brain MWPG levels remained unchanged during the first 10 min and only declined thereafter (data not shown). In mice receiving a combination of clorgyline, pargyline and tropolone, the disappearance of brain MHPG became exponential (Pig. 2). The MHPG turnover was calculated to be 663 pmol/g/h (k = 2.0 f 0.1 h-l; t%= 20 min). Concurrently, there was only a slight decrease of DHPG levels over a 30 min Interval (data not shown).
753
Mouse brain DHPG, MHPG and NE turnover
IO
1.0
I
2
4 TIME
6
8
0
10
(min)
5
IO
15
20
25
30
Time (mid
Fig. 1. The semflogarithmicdecline of mouse brain DHPG following administrationof clorgyline (37 nmol/kg) in combination with pargyline (471 nmolfkg). Each point is the mean * S.E.M. for groups of 4-6 animals.
The semilogarithmicdecline Fig. 2. of mouse brain MHPG following administration of clorgyline (37 pmol/kg) in combination with pargyline (471 pmol/kg) and tropolone (614 nmol/kg). Each point is the the mean f S.E.M. for 4-6 animals.
Discussion
The findings that comparable turnover rates of brain DHPG were obtained following either MAO or COMT inhibition indicated that mouse brain DHPG is primarily cleared through 0-methylation. This is in marked contrast to the clearance of rat brain DHPG which proceeds through sulfo-conjugationas vell as O-methylation with subsequent elimination via an active transport process (Gale and Maas, 1977; Li et al., 1981). These findings emphasize marked species differences In the metabolic clearance of brain DHPG which may, in part, be dependent upon the conjugating capacity for these glycols.
One minute after an injection of clorgyline plus pargyline, there was an almost complete (97-100X) inhibition of MAO-A and MAO-B activities (data not shown). This immediate inhibition of MAO together with the exponential decline of DHPG following clorgyllne plus pargyline suggests that brain MAO-A and MAO-B are completely and instantaneouslyinhibited following these drugs. The turnover rate of DHPG calculated by this method (913 pmol/g/h) was similar to the NE turnover value (944 pmol/g/h) estimated from its Initial accumulation after the degradattve enzymes had been inhibited (unpublished data). These findings support the notion that deamination of NE to DHPG is the primary route of brain NE metabolism. It also suggests that DHPG and not MHPG turnover provides a more accurate Index of brain NE turnover. Similar observations have been made in the rat (Li et al., 1981).
Unlike DHPG, brain MHPG levels did not decline exponentially after clorgylfne plus pargyline. However, the decline of MHPG appeared to occur 10 min after the drug Furthermore, the injection, at a time when DHPG levels were almost depleted. disappearance of MHPG became exponential when COMT, in addition to MAO-A and -B, was also inhibited (Fig. 2). These observations suggest that the initial slower rate of
754
P.P. Li et at.
disappearance of MHPG after MAD-A and MA&B inhibition is due to the ongoing 0-methylation of DHPG to MHPG during that interval, and support the notion that DHPG is primarily cleared through 0-methylation in this species.
Based on the foregoing turnover studies, we have estimated that 913-1000 pmol/g/h DHPG is formed and subsequently O-methylated in the brain with minimal direct elimination from the brain. The finding that the estimate of MHPG turnover (663 pmol/g/h) was lower than that determined for DHPG may reflect one of two possibilities. Firstly, there may be multiple co-existing MHPG pools which behave in a kinetically different manner, such that the smaller pool(s) with high turnover are neglected in the overall turnover estimates. Alternatively, DAPG may also be metabolized to other, as yet unidentified, methylated products.
Assuming the dynamics and disposition of DHPG and MHPG are similar in mouse and human brain, the above observations suggest that in humans, plasma and urinary DHPG are likely derived from peripheral sources and more likely provide an index of peripheral sympathetic function.
Acknowledgements
We wish to thank Mrs Susan McNally for her preparation of the manuscript, Mr. Andrew Chiu and Ms. Cathy Spegg for data analysis. This work was supported in part by MRC Canada #MA-7300.
References
DEMET, E.M. and HALARIS, A.E. (1979) Origin and distribution of 3-methoxy-4-hydroxyphenylglycol in body fluids. Biochem. Pharmacol. -28: 3043-3050. GALE, St. W. and MAAS, J.W. (1977) A study of the formation and metabolic disposition of 3,4-dihydroxyphenylethyleneglycol in whole rat brain. J. Neural Transm. -41: 59-72. LI, P.P., WARS& J.J. and GODSE, D.D. (1981) 3,4-Dihydroxypbenylethyleneglycol (DHPG) formation : the major route of rat brain norepinephrinemetabolism. Prog. Neuro-Psychopharmacol. 2: 531-535. WARSH, J.J., GODSE, D.D., CHEUNG, S.W. and LI, P.P. (1981a) Rat brain and plasrllanorepinephrine glycol metabolites determined by gas chromatography-massfragmentography. J. Neurochem. -36: 893-901. WARSH, J.J., LI, P.P., GODSE, D.D. and CHRUNG, S. (1981b) Brain noradrenergic neuronal Life Sci. -29: activity affects 3,4-dihydroxyphenylethyleneglycol (DHPG) levels. 1303-1307. WINER, B.J. (1971) Statistical Principles in Experimental Design. McGraw-Hill, New York.
Inquiries and reprint requests should be addressed to:
Mr. Peter Li Section of Biochemical Psychiatry Clarke Institute of Psychiatry 250 College Street Toronto, ONT., Canada M5T lR8