Urinary Excretion of Catecholamines and Their
Metabolites in Relation to Circulating Catecholamines Six-Hour Infusion of Peter
Epinephrine and Norepinephrine in Healthy Volunteers
Moleman, PhD; Joke Arie J. Man in
Tulen, MSc; Peter J. Blankestijn, PhD; 't Veld, PhD; Frans Boomsma, PhD H. M.
depressed patients have been shown to excrete abnormal amounts of catecholamines and their metabolites in urine. Some studies suggest that hypersecretion of epinephrine by the adrenals and of norepinephrine by the peripheral sympathetic system cause increased excretion of urinary catecholamines and their metabolites in a subgroup of patients. To evaluate the effect of increased catecholamine levels in the peripheral circulation on urinary catecholamine and metabolite levels, we infused healthy volunteers during 6 hours with epinephrine, norepinephrine, or pla\s=b\ Some
cebo, respectively,
in a three-period, double-blind, crossdesign. The results indicate that (1) urinary epinephrine and norepinephrine levels were the most sensitive indicators of increased circulating epinephrine and norepinephrine levels, respectively; (2) changes in circulating epinephrine or norepinephrine levels were not readily reflected in changes in urinary vanillylmandelic acid or 3-methoxy-4-hydroxyphenylglycol levels; and (3) increased normetanephrine excretion was not only induced by infusion of norepinephrine but also by epinephrine. This last finding may be due to activation of the sympathetic nervous system by circulating epinephrine. These results may help to explain the mechanism of adrenal epinephrine and sympathetic nervous system norepinephrine hypersecretion observed in subgroups of depressed patients. (Arch Gen Psychiatry. 1992;49:568-572) over
1965, Schildkraut1 suggested that depression may be In caused by functional deficit of norepinephrine (NE). different of catecholamine metabolism a
Many
measures
have been scrutinized to distinguish depressed patients from healthy controls or to differentiate among subtypes of depressive disorders.2"16 Early studies reported that
Accepted for publication July 31, 1991. From the Departments of Psychiatry (Dr Moleman and Mr Tulen) and Internal Medicine (Drs Blankestijn, Man in 't Veld, and Boomsma), University Hospital Rotterdam-Dijkzigt and the Medical Faculty of Erasmus University Rotterdam (the Netherlands), and
Moleman Research, Rotterdam (Dr Moleman). Reprint requests to Department of Psychiatry, AZR-Dijkzigt, Dr Molewaterplein 40, 3015 GD Rotterdam, the Netherlands (Dr
Tulen).
some depressed patients excreted low amounts of urinary 3-methoxy-4-hydroxyphenylglycol (MHPG), the metabo¬
lite that was believed to reflect metabolism of NE in the central nervous system.2'6-711 More recently, the focus has been more on dysfunctions of peripheral noradrenergic systems. A number of authors have reported increased plasma NE levels or spillover in subgroups of patients with affective disorders.8'91214 Also, urinary excretion patterns of catecholamines and their main metabolites have been reported in large groups of depressed patients and healthy controls.101315'16 Maas et al13 and Davis et al15 identified depressed patients excreting high levels of epinephrine (E), metanephrine (M), NE, and normetanephrine (NM). These and studies of Schildkraut et al4-5 and Schatzberg et al16 suggest that abnormalities in excretion of urinary cate¬ cholamines and a number of their metabolites are related. This is in line with high correlations between urinary cat¬ echolamines and their major metabolites found in healthy controls101718 and depressed patients.10161819 However, the origin of the abnormalities in metabolites in patients remains obscure. Changes in urinary excretion patterns could be due to changes in the central or peripheral metabolism of cate¬ cholamines. 3-Methoxy-4-hydroxyphenylglycol is the main central metabolite of NE, but urinary excretion of MHPG is a weak indicator of central metabolism, because MHPG is also formed in the periphery and because a considerable proportion of MHPG of both origins is con¬ verted to vanillylmandelic acid (VMA) in the periphery. The other metabolites, M, NM, and VMA, as well as the catecholamines NE and E, cannot cross the blood-brain barrier, and excretions in urine are therefore of peripheral origin. Another distinction can be made between extraneuronal and intraneuronal metabolism. It appears that urinary NE and its metabolite NM reflect to a certain ex¬ tent extraneuronal metabolism and are sensitive to changes in sympathetic neuronal activity. Vanillylman¬ delic acid, the main metabolite of NE, is formed intraneuronally and probably reflects more closely the basal me¬ tabolism of NE.13-2022 Attention has been focused mainly on NE and the sympathetic nervous system, but abnor-
Downloaded From: http://archpsyc.jamanetwork.com/ by a Michigan State University User on 06/08/2015
during this period and were kept awake when they dozed epinephrine (82 pinol-kg^-min"1), nor¬ epinephrine (178 pmolkg"1min^1), or a physiological saline solu¬ tion was infused into the dominant arm at a rate of 5.4 mL/h. Epi¬ nephrine and norepinephrine were diluted in physiological saline just before the infusion started. All three infusions were adminis¬ tered to each subject in a three-period, double-blind, randomized, crossover design. Infusions were at least 10 days apart. The total amount infused during the 6-hour period was 2.31 ±0.21 µ of epinephrine and 5.00±0.45 µ of norepinephrine, respectively. or smoke
off. From 10 AM to 4 PM,
2.5-
|2.0 E
°-
Q.
5-
oí 1.5 e
§
1.0
.
0
0.5
Sample Collection
0J
Every hour from 10 AM until 5 PM, a 10-mL venous and a 10-mL arterial blood sample, respectively, were obtained from the
r
nondominant < 1.5
tti.o
arm
(infusions
were
into the dominant
arm).
Samples were collected in chilled tubes containing 19 mg of ethylene glycol-bis[beta-aminoethyl ether] N^N^NMetraacetic acid and 12 mg of glutathione.25 Plasma was prepared within 30 minutes and stored at 70°C until assayed for catecholamines. Urine was collected in three portions: urine sample 1 collected during the infusion from 8 am to 6 PM; urine sample 2 collected during the next night from 6 PM to 8 am, which always included the morning voiding; and urine sample 3 collected during the next day from 8 AM to 6 PM. Urine was collected in polyethylene —
10 11 12 13 14 15 16 17
Infusion
Period,
Infusion Period, h
h
Mean and SEM values of arterial (top) and venous (bottom) plasma epinephrine and norepinephrine levels before, during, and after infusion of epinephrine (open circles), norepinephrine (closed cir¬
cles),
or
placebo (closed triangles).
malities in E and adrenal activity are at least as important because of prominent abnormalities in some depressed
patients.1315 The present study was designed to evaluate which changes in urinary excretion can be explained by in¬ creased concentrations of catecholamines in the periph¬ eral circulation and to distinguish the role of circulating E and NE in such
changes.
SUBJECTS AND METHODS
Subjects
healthy male volunteers (mean age [±SD], 24±3 years; height, 182±9 cm; weight, 78.3±7.0 kg). They gave written informed consent to participate in the study. The protocol was approved by the Medical Ethical Committee of the University Hospital Rotterdam-Dijkzigt, the Netherlands. The subjects received a medical examination, including labora¬ tory screening and electrocardiogram, and a psychiatric exami¬ nation to ensure physical and mental health. Also, Dutch versions of the Minnesota Multiphasic Personality Inventory23 and the State-Trait Anxiety Inventory24 revealed no abnormal values. Six subjects were nonsmokers, and none reported excessive drinking habits. The
subjects
were
10
Procedure
Subjects had to keep a regular sleep-wake cycle and avoid ab¬ normal physical or psychic activities. Before each experimental session, the subjects had a standardized light breakfast without tea or coffee at 8 am in the hospital, and they voided urine. At 8:15 AM, catheters were inserted into the antecubital veins of both The brachial artery of the nondominant arm was cannulated with a Teflon catheter, introduced by the Seldinger tech¬ nique, which was connected to a miniature transducer-perfusion device. The intra-arterial pressure signal and electrocardiogram were monitored continuously and recorded analogue on an in¬ strumentation recorder. From 9 AM until 5 PM, the subjects rested in a supine position and received a 5% dextrose solution, infused intravenously at a rate of 2 mL/min into the dominant arm. They did not eat, drink,
arms.
containers over 0.5 g of sodium edetic acid and 0.5 g of sodium metabisulfite and kept at 4°C in the dark.26 After the collection 80°C. was complete, aliquote were transferred to a freezer at —
Biochemical
Assays
were assayed by highperformance liquid Chromatographie techniques, combined with electrochemical detection for determination of plasma catecho¬
Catecholamines and metabolites
lamines,25 urinary
total MHPG,27 free VMA,28 and total M values and combined with fluorescence detection and precolumn derivatization for determination of urinary free cat¬
echolamines.29
Statistics Differences between the infusion conditions in the urinary ex¬ cretions of catecholamines and their metabolite? -vere analyzed with repeated-measures analysis of variance, with infusion con¬ dition as the within-subjects factor. Where appropriate, a square root transformation was applied to obtain normal distributions. The analyses were done separately for urine samples 1, 2, and 3 and the 24-hour excretion rates, which were computed by tak¬ ing the sum of urine samples 1 and 2. Differences in plasma catecholamine concentrations were analyzed with repeatedmeasures analysis of variance, with time and infusion condition as within-subject factors.
RESULTS Infusion of epinephrine resulted in a 10-fold increase in arte¬ rial and venous concentrations of E in the contralateral forearm (Figure). These increases are in the high physiological range and similar to those observed during public speaking30 or maximal exercise.31·32 The physiological reactions were moderate, consist¬ ing of a decrease of mean arterial blood pressure of 6.9% and an increase in heart rate of . .33·34 Infusion of norepinephrine re¬ sulted in a fivefold increase in arterial and a 3.5-fold increase in venous concentrations of NE (Figure). The physiological effects of this infusion were also moderate, consisting of an increase in mean arterial blood pressure of 6.6% and a decrease in heart rate of 3.9%. No significant effect on NE plasma concentrations was ob¬ served during epinephrine infusion. Infusion of norepinephrine caused a slight but significant decrease in venous and arterial plasma E levels (both P
Metabolites in Relation to Circulating Catecholamines Six-Hour Infusion of Peter
Epinephrine and Norepinephrine in Healthy Volunteers
Moleman, PhD; Joke Arie J. Man in
Tulen, MSc; Peter J. Blankestijn, PhD; 't Veld, PhD; Frans Boomsma, PhD H. M.
depressed patients have been shown to excrete abnormal amounts of catecholamines and their metabolites in urine. Some studies suggest that hypersecretion of epinephrine by the adrenals and of norepinephrine by the peripheral sympathetic system cause increased excretion of urinary catecholamines and their metabolites in a subgroup of patients. To evaluate the effect of increased catecholamine levels in the peripheral circulation on urinary catecholamine and metabolite levels, we infused healthy volunteers during 6 hours with epinephrine, norepinephrine, or pla\s=b\ Some
cebo, respectively,
in a three-period, double-blind, crossdesign. The results indicate that (1) urinary epinephrine and norepinephrine levels were the most sensitive indicators of increased circulating epinephrine and norepinephrine levels, respectively; (2) changes in circulating epinephrine or norepinephrine levels were not readily reflected in changes in urinary vanillylmandelic acid or 3-methoxy-4-hydroxyphenylglycol levels; and (3) increased normetanephrine excretion was not only induced by infusion of norepinephrine but also by epinephrine. This last finding may be due to activation of the sympathetic nervous system by circulating epinephrine. These results may help to explain the mechanism of adrenal epinephrine and sympathetic nervous system norepinephrine hypersecretion observed in subgroups of depressed patients. (Arch Gen Psychiatry. 1992;49:568-572) over
1965, Schildkraut1 suggested that depression may be In caused by functional deficit of norepinephrine (NE). different of catecholamine metabolism a
Many
measures
have been scrutinized to distinguish depressed patients from healthy controls or to differentiate among subtypes of depressive disorders.2"16 Early studies reported that
Accepted for publication July 31, 1991. From the Departments of Psychiatry (Dr Moleman and Mr Tulen) and Internal Medicine (Drs Blankestijn, Man in 't Veld, and Boomsma), University Hospital Rotterdam-Dijkzigt and the Medical Faculty of Erasmus University Rotterdam (the Netherlands), and
Moleman Research, Rotterdam (Dr Moleman). Reprint requests to Department of Psychiatry, AZR-Dijkzigt, Dr Molewaterplein 40, 3015 GD Rotterdam, the Netherlands (Dr
Tulen).
some depressed patients excreted low amounts of urinary 3-methoxy-4-hydroxyphenylglycol (MHPG), the metabo¬
lite that was believed to reflect metabolism of NE in the central nervous system.2'6-711 More recently, the focus has been more on dysfunctions of peripheral noradrenergic systems. A number of authors have reported increased plasma NE levels or spillover in subgroups of patients with affective disorders.8'91214 Also, urinary excretion patterns of catecholamines and their main metabolites have been reported in large groups of depressed patients and healthy controls.101315'16 Maas et al13 and Davis et al15 identified depressed patients excreting high levels of epinephrine (E), metanephrine (M), NE, and normetanephrine (NM). These and studies of Schildkraut et al4-5 and Schatzberg et al16 suggest that abnormalities in excretion of urinary cate¬ cholamines and a number of their metabolites are related. This is in line with high correlations between urinary cat¬ echolamines and their major metabolites found in healthy controls101718 and depressed patients.10161819 However, the origin of the abnormalities in metabolites in patients remains obscure. Changes in urinary excretion patterns could be due to changes in the central or peripheral metabolism of cate¬ cholamines. 3-Methoxy-4-hydroxyphenylglycol is the main central metabolite of NE, but urinary excretion of MHPG is a weak indicator of central metabolism, because MHPG is also formed in the periphery and because a considerable proportion of MHPG of both origins is con¬ verted to vanillylmandelic acid (VMA) in the periphery. The other metabolites, M, NM, and VMA, as well as the catecholamines NE and E, cannot cross the blood-brain barrier, and excretions in urine are therefore of peripheral origin. Another distinction can be made between extraneuronal and intraneuronal metabolism. It appears that urinary NE and its metabolite NM reflect to a certain ex¬ tent extraneuronal metabolism and are sensitive to changes in sympathetic neuronal activity. Vanillylman¬ delic acid, the main metabolite of NE, is formed intraneuronally and probably reflects more closely the basal me¬ tabolism of NE.13-2022 Attention has been focused mainly on NE and the sympathetic nervous system, but abnor-
Downloaded From: http://archpsyc.jamanetwork.com/ by a Michigan State University User on 06/08/2015
during this period and were kept awake when they dozed epinephrine (82 pinol-kg^-min"1), nor¬ epinephrine (178 pmolkg"1min^1), or a physiological saline solu¬ tion was infused into the dominant arm at a rate of 5.4 mL/h. Epi¬ nephrine and norepinephrine were diluted in physiological saline just before the infusion started. All three infusions were adminis¬ tered to each subject in a three-period, double-blind, randomized, crossover design. Infusions were at least 10 days apart. The total amount infused during the 6-hour period was 2.31 ±0.21 µ of epinephrine and 5.00±0.45 µ of norepinephrine, respectively. or smoke
off. From 10 AM to 4 PM,
2.5-
|2.0 E
°-
Q.
5-
oí 1.5 e
§
1.0
.
0
0.5
Sample Collection
0J
Every hour from 10 AM until 5 PM, a 10-mL venous and a 10-mL arterial blood sample, respectively, were obtained from the
r
nondominant < 1.5
tti.o
arm
(infusions
were
into the dominant
arm).
Samples were collected in chilled tubes containing 19 mg of ethylene glycol-bis[beta-aminoethyl ether] N^N^NMetraacetic acid and 12 mg of glutathione.25 Plasma was prepared within 30 minutes and stored at 70°C until assayed for catecholamines. Urine was collected in three portions: urine sample 1 collected during the infusion from 8 am to 6 PM; urine sample 2 collected during the next night from 6 PM to 8 am, which always included the morning voiding; and urine sample 3 collected during the next day from 8 AM to 6 PM. Urine was collected in polyethylene —
10 11 12 13 14 15 16 17
Infusion
Period,
Infusion Period, h
h
Mean and SEM values of arterial (top) and venous (bottom) plasma epinephrine and norepinephrine levels before, during, and after infusion of epinephrine (open circles), norepinephrine (closed cir¬
cles),
or
placebo (closed triangles).
malities in E and adrenal activity are at least as important because of prominent abnormalities in some depressed
patients.1315 The present study was designed to evaluate which changes in urinary excretion can be explained by in¬ creased concentrations of catecholamines in the periph¬ eral circulation and to distinguish the role of circulating E and NE in such
changes.
SUBJECTS AND METHODS
Subjects
healthy male volunteers (mean age [±SD], 24±3 years; height, 182±9 cm; weight, 78.3±7.0 kg). They gave written informed consent to participate in the study. The protocol was approved by the Medical Ethical Committee of the University Hospital Rotterdam-Dijkzigt, the Netherlands. The subjects received a medical examination, including labora¬ tory screening and electrocardiogram, and a psychiatric exami¬ nation to ensure physical and mental health. Also, Dutch versions of the Minnesota Multiphasic Personality Inventory23 and the State-Trait Anxiety Inventory24 revealed no abnormal values. Six subjects were nonsmokers, and none reported excessive drinking habits. The
subjects
were
10
Procedure
Subjects had to keep a regular sleep-wake cycle and avoid ab¬ normal physical or psychic activities. Before each experimental session, the subjects had a standardized light breakfast without tea or coffee at 8 am in the hospital, and they voided urine. At 8:15 AM, catheters were inserted into the antecubital veins of both The brachial artery of the nondominant arm was cannulated with a Teflon catheter, introduced by the Seldinger tech¬ nique, which was connected to a miniature transducer-perfusion device. The intra-arterial pressure signal and electrocardiogram were monitored continuously and recorded analogue on an in¬ strumentation recorder. From 9 AM until 5 PM, the subjects rested in a supine position and received a 5% dextrose solution, infused intravenously at a rate of 2 mL/min into the dominant arm. They did not eat, drink,
arms.
containers over 0.5 g of sodium edetic acid and 0.5 g of sodium metabisulfite and kept at 4°C in the dark.26 After the collection 80°C. was complete, aliquote were transferred to a freezer at —
Biochemical
Assays
were assayed by highperformance liquid Chromatographie techniques, combined with electrochemical detection for determination of plasma catecho¬
Catecholamines and metabolites
lamines,25 urinary
total MHPG,27 free VMA,28 and total M values and combined with fluorescence detection and precolumn derivatization for determination of urinary free cat¬
echolamines.29
Statistics Differences between the infusion conditions in the urinary ex¬ cretions of catecholamines and their metabolite? -vere analyzed with repeated-measures analysis of variance, with infusion con¬ dition as the within-subjects factor. Where appropriate, a square root transformation was applied to obtain normal distributions. The analyses were done separately for urine samples 1, 2, and 3 and the 24-hour excretion rates, which were computed by tak¬ ing the sum of urine samples 1 and 2. Differences in plasma catecholamine concentrations were analyzed with repeatedmeasures analysis of variance, with time and infusion condition as within-subject factors.
RESULTS Infusion of epinephrine resulted in a 10-fold increase in arte¬ rial and venous concentrations of E in the contralateral forearm (Figure). These increases are in the high physiological range and similar to those observed during public speaking30 or maximal exercise.31·32 The physiological reactions were moderate, consist¬ ing of a decrease of mean arterial blood pressure of 6.9% and an increase in heart rate of . .33·34 Infusion of norepinephrine re¬ sulted in a fivefold increase in arterial and a 3.5-fold increase in venous concentrations of NE (Figure). The physiological effects of this infusion were also moderate, consisting of an increase in mean arterial blood pressure of 6.6% and a decrease in heart rate of 3.9%. No significant effect on NE plasma concentrations was ob¬ served during epinephrine infusion. Infusion of norepinephrine caused a slight but significant decrease in venous and arterial plasma E levels (both P
