Free and sulfoconjugated catecholamine at birth in newborn sheep KOTARO HOWARD
OYAMA, JAMES STEIN, LESLIE
Perinatal Research Laboratories, Torrance, California 90509
PADBURY, ALMA MARTINEZ, BARBARA BLOUNT, AND ELIZABETH BUHL University
CHAPPELL,
of California at Los Angeles Harbor Medical Center,
Oyama, Kotaro, James Padbury, Alma Martinez, Barbara Chappell, Howard Stein, Leslie Blount, and Elizabeth Buhl. Free and sulfoconjugated catecholamine responsesat birth in newborn sheep. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E23-E27, 1992.-There have been little data on catecholaminesulfoconjugation in developing animals or humans. We studied the differences in free and sulfoconjugatedcatecholaminesat birth in newborn sheep.Baseline concentrations of sulfoconjugatednorepinephrine and epinephrine were the predominant form of circulating catecholamine, representing77 & 4 and 65 t 12% of total circulating catecholamines,respectively. At birth the free epinephrine concentration increasedlo-fold (49 & 27 to 653 & 21 pg/ml, respectively), and plasmafree norepinephrine concentration rose4-fold (307 t 92 to 1,178 & 389 pg/ml). In contrast, there was only a transient twofold increasein the sulfoconjugatedepinephrine. There was no increase in the sulfoconjugated form of norepinephrine. These data demonstrate that, while the near-term newborn sheephas a well-developedmechanismfor sulfoconjugation of circulating catecholamines,this doesnot occur rapidly. During the logarithmic increasesof circulating catecholaminesat birth, there are not commensurateincreasesin the concentration of sulfoconjugatednorepinephrine or epinephrine. Thus sulfoconjugation doesnot appear to represent a significant mechanism for inactivation of the high circulating levels of catecholamines seenat birth. catecholamines;conjugated;norepinephrine; epinephrine; perinatal CATECHOLAMINES circulate both as free or unconjugated forms and as sulfate and glucuronide conjugates. In the adult human, 7080% of total circulating norepinephrine and epinephrine are sulfate conjugated and >98% dopamine is similarly conjugated (9, 10). Glucuronide conjugation accounts for another small percentage of conjugated levels (9, 10). The physiological significance of conjugated catecholamines is unclear. In patients with pheochromocytoma and high circulating concentrations of free catecholamines, there are commensurate increases in the concentration of sulfoconjugated catecholamines (11, 12,22). Because the conjugated forms are inactive, it has been suggested that conjugation represents an important protective mechanism against high circulating catecholamines during “crises” (11, 12, 22). Similar results have been suggested for catecholamine responses during strenuous exercise (24, 28, 29). There is only a single published report of free and conjugated catecholamines in the human newborn, in which the proportion of sulfoconjugated norepinephrine was 73% and epinephrine 69 % of total circulating catecholamine concentration (24). We have previously demonstrated in chronically catheterized fetal sheep that sulfoconjugated norepinephrine and epinephrine were likewise the predominant circulating form (20). The concentrations of both PLASMA
responses
free and sulfoconjugated norepinephrine and epinephrine rose proportionally during mild fetal hypoxia. A marked increase in plasma catecholamines occurs at birth, which is critical to successful physiological adaptation (18, 19). Unlike the adult animal or human, plasma catecholamines in developing animals are not only an index of sympathoadrenal activity but, because of incomplete innervation and receptor maturation, they are vital to maintenance of physiological homeostasis (19). Whether newborn animals conjugate catecholamines at birth and whether conjugation is a mechanism for attenuation of the biological effects of the high circulating catecholamine concentrations and the rapidity of the conjugation process are not known. To develop these data we conducted studies of the free and conjugated catecholamine responses at birth in newborn sheep. METHODS Surgical
procedures and animal preparation. Six eweswith singletonpregnancieswere delivered by cesareansectionat 141 k 1 days gestation (term = 150 days). The anesthetic and surgical techniques for delivery and stabilization of newborn animals were conducted as previously described, with only the modifications outlined below (18, 21). Tracheostomy of the fetus before delivery and cord cutting wasperformed aspreviously described. To enable hemodynamic measurementsand blood samplingbefore delivery and cord cutting, the left ventricle was catheterized via the right carotid artery. The fetus was delivered onto the maternal abdomenwith care to avoid traction or trauma to the umbilical cord. The animalswere allowed to stabilize for lo-15 min, and temperature was maintained with a heating pad and lamps. Arterial blood samplesfor catecholaminesand blood gasesweredrawn at -10 and 0 min, where time 0 was denoted as the time of cord cutting. Immediately after umbilical cord cutting, the animals were placed on timecycled pressure-limitedinfant ventilators (Sechrist Industries, Anaheim, CA). The lambswere cared for under an infant radiant warmer with supplementheat lampsas necessaryto maintain rectal temperature at 39 & 1°C. An umbilical arterial catheter was inserted to the level of the midabdominal aorta for monitoring and blood sampling.Heart rate and blood pressure were measuredcontinuously and wererecordedon a multichannel Sensormedic R611 rectilinear polygraph (Sensormedics, Anaheim, CA). Blood samplesfor measurementof plasmacatecholamine levels and arterial blood gaseswere obtained from the umbilical artery catheter at 15,30,60,120,180, and 240min after cord cutting. Heart rate, blood pressure,and temperature were recordedwith eachsample.Initial ventilator settings (peak inspiratory pressure28 cmH20, positive and expiratory pressure2 cmH,O, and a rate of 30/min) were adjustedto maintain PO, between 100 and 150 mmHg and pH and PCO~within normal values. A continuous infusion of normal saline at 4 ml kg- l. h-l was administered. All arterial blood samplesfor measurementof plasma catecholamineswere added to chilled test tubes containing ethyl
0193~1849/92 $2.00 Copyright 0 1992 the American Physiological
Society
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CONJUGATED
CATECHOLAMINES
lene glycol-bis (@-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) and reducedglutathione. The sampleswerecentrifuged immediately, and plasma was separatedand stored at -70°C until catecholamineanalysiswasperformed. Blood for measurement of arterial blood gaseswasdrawn into heparinized syringes and analyzed within 3 min. Blood gaseswere measuredon a BMS3 MKII Radiometer (Copenhagen,Denmark) calibrated at 39°C. Approximately 4 ml of blood were removed for each sample and replacedwith heparinized maternal blood. Analytic techniques. Plasma free and sulfoconjugatedcatecholamineconcentrations weredetermined by a radioenzymatic assaysensitive to lo-20 pg/ml norepinephrine and epinephrine and 5-10 pg/ml dopamineaspreviously described(20). Plasma total catecholamines,representingfree plus sulfoconjugatedcatecholamines,were determined by the addition of 100 mU sulfatase(arylsulfataseS-1629;Sigma Chemical)to the incubation mixture (7). This enzyme preparation is free of demonstrable glucuronidaseat pH 7.0. Preliminary studies in fetal and adult sheepblood were conducted to ascertain the maximal total catecholamineconcentrations obtained by the addition of sulfatase activity to eachincubation tube. No additional increasein total catecholamineconcentration was noted during the inclusion of glucuronidase,suggestingthat little or no glucuronide conjugatesexist in sheepplasma. Sensitivity of the total catecholamine assaywas 20-50 pg/ml for norepinephrine and epinephrine and 50-80 pg/ml for dopamine. Data analysis. Serial responsesfor all catecholamine and hemodynamic data were analyzed by analysis of variance for repeated measuresand were compared with baseline values usingDunnett’s test. To achievehomogenousvariances,plasma free and sulfoconjugatedcatecholamineconcentrations were log transformedbefore statistical comparison.All resultsare shown asmeans* SE. A value of P < 0.05 was consideredstatistically significant.
AT BIRTH
.. .. 100-
O=
.
.
.
.
.
,
.
.
Of -30
0
30
60
a0
120
180
la0
210
.
RESULTS
Baseline arterial PO, (29 t 1 mmHg), PCO~ (39 t 2 mmHg), pH (7.42 t 0.03), heart rate (170 t 6 beats/min), and left ventricular systolic pressure (64 t 5 mmHg) were all within normal values for healthy animals at comparable gestational ages (15, 18-21). Consistent with ventilatory goals, PO, was maintained at -150 mmHg, PCO~ at 30-40 mmHg, and pH within normal limits after delivery (Fig. 1). Heart rate and blood pressure both increased significantly after delivery, with peak heart rate rising to 198 t 8 beats/min and blood pressure to 107 t 7 mmHg, P < 0.01 (Fig. 2). Serial changes in plasma free and sulfoconjugated norepinephrine are shown in Fig. 3A. The baseline free norepinephrine concentration of 307 t 92 pg/ml increased almost fourfold after delivery to a maximum of 1,178 t 389 pg/ml at 30 min of age. Free norepinephrine concentration remained elevated for 3 h after delivery and then returned to concentrations closer to the baseline value (537 t 85 pg/ml) by 4 h of age. Baseline sulfoconjugated norepinephrine concentration was 920 t 187 pg/ml. This represented 77 t 4% of the total circulating norepinephrine. After delivery, when free norepinephrine concentration was rising rapidly, there was no significant change in sulfoconjugated norepinephrine. Consequently, the proportion of sulfoconjugated norepinephrine decreased to as low as 55 t 4% of total norepinephrine at 120 min. These results are illustrated in Fig. 4. Serial changes in plasma free and sulfoconjugated epinephrine are shown in Fig. 3B. Baseline free epinephrine
24Q
TIME min Fig. 1. sheep. means partial
Serial arterial blood gases and pH at birth in term newborn Time of umbilical cord cutting is designated as time 0. Values are t SE. Pace,, arterial partial presske of CO,; Pao,, arterial pressure of Oz.
(49 t 27 pg/ml) was similar to values in past studies (15, 18, 19, 21). After delivery and cord cutting, plasma free epinephrine concentration increased MO-fold to 653 t 212 pg/ml and remained significantly elevated for the remainder of the study (P < 0.05). Similar to sulfoconjugated norepinephrine, sulfoconjugated epinephrine represented the majority of total circulating epinephrine. Baseline sulfoconjugated epinephrine (145 t 21 pg/ml) was 65 t 12% of total epinephrine. After cord cutting, sulfoconjugated epinephrine increased transiently to a maximum level of 296 t 47 at 15 min of age. Because there were logarithmic increases in free epinephrine, the proportion of sulfoconjugated epinephrine decreased significantly (Fig. 4). This proportion reached a nadir of 25-30% of total epinephrine by 3-4 h of age. DISCUSSION
These are the first data to describe free and sulfoconjugated catecholamine levels and their responses to delivery in newborn animals at birth. Sulfoconjugated norepinephrin e and epinephrine represe nted the predominant form of C irculating catecholamine in fetal plasma before
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CONJUGATED
CATECHOLAMINES
E25
AT BIRTH
5ocbov
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4 4
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0 FREE l conjugated
250
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200
1 F d 8 s
150
loo
so
25!
-30
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30
60
90 TIME
120
150
180
210
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Fig. 2. Serial left ventricular (LV) systolic pressure and heart rate at birth in term newborn sheep. Time 0, umbilical cord cutting. Values are means k SE. * Significant differences from baseline values before cord cutting (P < 0.01).
delivery and cord cutting seen in this study. In previous studies of free and conjugated plasma catecholamines in fetal sheep, we observed very low concentrations of conjugated dopamine. This was true for both fetal and adult sheep (20). Also, the concentrations of free dopamine changed very little in fetal and newborn sheep during a variety of experimental paradigms we have investigated in the past (unpublished observations). Therefore we did not attempt to measure free or sulfoconjugated dopamine in the present study. After birth and severance of the umbilical-placental circulation, there were rapid logarithmic increases in the concentration of free norepinephrine and epinephrine, which rose 4- and lo-fold, respectively. These plasma concentrations of free catecholamines were similar to our previous reports (18, 19, 21). By contrast, the concentration of sulfoconjugated epinephrine rose only transiently above fetal levels and then remained unchanged thereafter. Sulfoconjugated norepinephrine did not rise after birth. The physiological and biological significance of catecholamine conjugation is unclear. Sulfoconjugation is catalyzed by phenol sulfotransferase (PST; EC 2.8.2.1). The tissue distribution of this enzyme is ubiquitous, including significant conjugation activity localized in brain (5), liver (9, lo), lung (26), and in platelets (6, 25, 31). Some investigators have suggested that circulating platelets are the predominant site for sulfoconjugation
I
1 0
-10
240
1 15
I 30 TIME
1 60
I 120
I 180
I 240
min
Fig. 3. Serial changes in free and sulfoconjugated norepinephrine (A) and free and sulfoconjugated epinephrine (B) at birth in newborn sheep. Time 0, umbilical cord cutting. Values are geometric means of: SE. * Significant difference from baseline concentration before cord cutting (P c 0.05). 100
Oa-
1
o!
I
-10
I
0
1
15
0 NOREPINEPHRINE 0 EPINEPHRINE
1
1
30
60
TIME
I
120
I
180
I
240
mln
Fig. 4. Proportion (%) of sulfoconjugated norepinephrine and epinephrine at birth. Time 0, umbilical cord cutting. Values are means k SE. * Significant difference from baseline values before cord cutting (P < 0.05).
(10). However, in patients with low platelet counts, there is no alteration in the proportion of sulfoconjugated catecholamines (13). Also, the affinity of platelet PST for each of the potential catecholamine substrates is highly variable (25). Therefore it remains unclear what the relative contribution of tissue having PST activity is to
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E26
CONJUGATED
CATECHOLAMINES
circulating sulfoconjugated catecholamines (13, 16, 25). Conjugated catecholamines appear to lack biological activity. In radioligand binding assays, epinephrine sulfate and norepinephrine sulfate are only weakly competitive for ,& and aZ-receptor ligands (32). The dissociation constants for both norepinephrine and epinephrine sulfate are lOO-1,000 times higher than for the free amines in a human mononuclear cell ,&receptor assay, and they do not inhibit binding at all of the a,-antagonist [“H]yohimbine to platelets (32). In vitro studies in isolated pulmonary artery demonstrate significant potentiation of electrically induced contractions with addition of norepinephrine but no response to the addition of norepinephrine sulfate (26). In the isolated perfused rat heart dopamine sulfate has no effect on developed tension (14). Last, infusion of sulfate conjugates in vivo does not result in a significant biological effect even at extremely high dosages (1). Conjugated catecholamines are not good substrates for subsequent biological modification involving the majority of pathways for endogenous biogenic amines. Dopamine fl-hydroxylase and phenylethanolamine-N-methyltransferase do not modify dopamine sulfate or norepinephrine sulfate in vitro (4). By contrast, small doses of [ l*C]dopamine sulfate administered in vivo to guinea pigs or rats is excreted mainly as 3,4-dihydroxyphenylacetic acid and homovanillic acid (17). This indicates that desulfation of the dopamine sulfate had taken place. A single report in vitro, using the isolated perfused heart, suggested that cardiac atria were capable of deconjugation of dopamine sulfate and release of free dopamine (14). Increased intrarenal and urinary [3H]norepinephrine has been reported in adult adrenalectomized rats after [ 3H]dopamine sulfate administration in vivo (2). Nonetheless, the majority of investigators do not consider conjugated catecholamines as representing a pool from which increases in the levels of circulating free catecholamines are derived. Some investigators interpret the high concentrations of circulating conjugated catecholamines as representing an “integrated index” of overall sympathoadrenal system activity (23). In situations associated with prolonged sympathoadrenal activation, there are parallel increases in free and conjugated catecholamines (10). Conjugated forms of each of the endogenous catecholamines are elevated significantly in patients with pheochromocytoma, where conjugation is felt to represent a form of protection from the high levels of free amines (11, 12). Adrenal venous effluent of pheochromocytoma patients is predominantly free amines (>99%), whereas the percentage of free amines in samples of peripheral blood from the same patients is only 45% (11). In critically ill patients, there is proportional elevation in free and conjugated norepinephrine, epinephrine, and dopamine (33). Exercise is a profound stimulus to catecholamine secretion in healthy adults and has been used to study the physiology and kinetics of catecholamine conjugation. It has been reported that both plasma free and conjugated norepinephrine rise with strenuous exercise (28, 29). In contrast, moderate exercise increased free catecholamine levels but actually decreased sulfoconjugated amines (3,8,
AT BIRTH
23). These authors suggested that the sulfoconjugated pools might be a source of free amines during vigorous exercise (23). However, no kinetic or radiolabeled tracer data exist to support this interpretation. Any alteration in catecholamine sulfate with exercise may not be linked to free circulating catecholamines but may be a reflection of blood flow through tissues that have high PST activity. These results suggest that the nature of the stressor may be an important factor in the sulfoconjugation of catecholamines. We have previously demonstrated that both free and sulfoconjugated plasma norepinephrine and epinephrine concentrations rise promptly in chronically catheterized fetal sheep in response to hypoxia (20). Our present results differ in that the rise in free catecholamine concentrations was much greater at birth than during the mild fetal hypoxia previously employed (20). It has been reported that chronic hypoxia increases sulfoconjugated norepinephrine in the dog pulmonary artery (27). In the present study, we did not observe a rapid increase in sulfoconjugated epinephrine or norepinephrine despite substantial increases in free amines. Differences in the nature of physiological stress between the two conditions may account for these discrepancies. An alternative explanation for the lack of parallel increase in conjugated catecholamines could be the removal of the placental contribution to catecholamine clearance and/or conjugation (30) In’ summary, we demonstrated that, despite logarithmic increases in the concentrations of circulating plasma free norepinephrine and epinephrine, there is little or no change in the concentration of the sulfoconjugated forms of either endogenous amine. Further studies of the mechanisms involved, which determine the relative proportions of free and sulfoconjugated catecholamines, should consider, among other factors, free catecholamine levels, the use of tracer methodologies, blood flow and its distribution, and relative tissue PST activities. This work was supported in part by National Heart, Lung, and Blood Institute Grants HD-18014 and HD-07013 and by an Investigative Group Fellowship Award from the American Heart Association, Los Angeles Affiliate. Address for reprint requests: J. F. Padbury, Harbor-UCLA Medical Center, UCLA School of Medicine, 1000 W. Carson St., RBl, Torrance, CA 90509. Received 31 October 1991; accepted in final form 17 February 1992. REFERENCES 1. Ackerman, D. M., J. P. Hieble, H. M. Sarau, and T. C. Jain. Pharmacological characterization of dopamine-4O-sulfate. Arch.
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OYAMA, JAMES STEIN, LESLIE
Perinatal Research Laboratories, Torrance, California 90509
PADBURY, ALMA MARTINEZ, BARBARA BLOUNT, AND ELIZABETH BUHL University
CHAPPELL,
of California at Los Angeles Harbor Medical Center,
Oyama, Kotaro, James Padbury, Alma Martinez, Barbara Chappell, Howard Stein, Leslie Blount, and Elizabeth Buhl. Free and sulfoconjugated catecholamine responsesat birth in newborn sheep. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E23-E27, 1992.-There have been little data on catecholaminesulfoconjugation in developing animals or humans. We studied the differences in free and sulfoconjugatedcatecholaminesat birth in newborn sheep.Baseline concentrations of sulfoconjugatednorepinephrine and epinephrine were the predominant form of circulating catecholamine, representing77 & 4 and 65 t 12% of total circulating catecholamines,respectively. At birth the free epinephrine concentration increasedlo-fold (49 & 27 to 653 & 21 pg/ml, respectively), and plasmafree norepinephrine concentration rose4-fold (307 t 92 to 1,178 & 389 pg/ml). In contrast, there was only a transient twofold increasein the sulfoconjugatedepinephrine. There was no increase in the sulfoconjugated form of norepinephrine. These data demonstrate that, while the near-term newborn sheephas a well-developedmechanismfor sulfoconjugation of circulating catecholamines,this doesnot occur rapidly. During the logarithmic increasesof circulating catecholaminesat birth, there are not commensurateincreasesin the concentration of sulfoconjugatednorepinephrine or epinephrine. Thus sulfoconjugation doesnot appear to represent a significant mechanism for inactivation of the high circulating levels of catecholamines seenat birth. catecholamines;conjugated;norepinephrine; epinephrine; perinatal CATECHOLAMINES circulate both as free or unconjugated forms and as sulfate and glucuronide conjugates. In the adult human, 7080% of total circulating norepinephrine and epinephrine are sulfate conjugated and >98% dopamine is similarly conjugated (9, 10). Glucuronide conjugation accounts for another small percentage of conjugated levels (9, 10). The physiological significance of conjugated catecholamines is unclear. In patients with pheochromocytoma and high circulating concentrations of free catecholamines, there are commensurate increases in the concentration of sulfoconjugated catecholamines (11, 12,22). Because the conjugated forms are inactive, it has been suggested that conjugation represents an important protective mechanism against high circulating catecholamines during “crises” (11, 12, 22). Similar results have been suggested for catecholamine responses during strenuous exercise (24, 28, 29). There is only a single published report of free and conjugated catecholamines in the human newborn, in which the proportion of sulfoconjugated norepinephrine was 73% and epinephrine 69 % of total circulating catecholamine concentration (24). We have previously demonstrated in chronically catheterized fetal sheep that sulfoconjugated norepinephrine and epinephrine were likewise the predominant circulating form (20). The concentrations of both PLASMA
responses
free and sulfoconjugated norepinephrine and epinephrine rose proportionally during mild fetal hypoxia. A marked increase in plasma catecholamines occurs at birth, which is critical to successful physiological adaptation (18, 19). Unlike the adult animal or human, plasma catecholamines in developing animals are not only an index of sympathoadrenal activity but, because of incomplete innervation and receptor maturation, they are vital to maintenance of physiological homeostasis (19). Whether newborn animals conjugate catecholamines at birth and whether conjugation is a mechanism for attenuation of the biological effects of the high circulating catecholamine concentrations and the rapidity of the conjugation process are not known. To develop these data we conducted studies of the free and conjugated catecholamine responses at birth in newborn sheep. METHODS Surgical
procedures and animal preparation. Six eweswith singletonpregnancieswere delivered by cesareansectionat 141 k 1 days gestation (term = 150 days). The anesthetic and surgical techniques for delivery and stabilization of newborn animals were conducted as previously described, with only the modifications outlined below (18, 21). Tracheostomy of the fetus before delivery and cord cutting wasperformed aspreviously described. To enable hemodynamic measurementsand blood samplingbefore delivery and cord cutting, the left ventricle was catheterized via the right carotid artery. The fetus was delivered onto the maternal abdomenwith care to avoid traction or trauma to the umbilical cord. The animalswere allowed to stabilize for lo-15 min, and temperature was maintained with a heating pad and lamps. Arterial blood samplesfor catecholaminesand blood gasesweredrawn at -10 and 0 min, where time 0 was denoted as the time of cord cutting. Immediately after umbilical cord cutting, the animals were placed on timecycled pressure-limitedinfant ventilators (Sechrist Industries, Anaheim, CA). The lambswere cared for under an infant radiant warmer with supplementheat lampsas necessaryto maintain rectal temperature at 39 & 1°C. An umbilical arterial catheter was inserted to the level of the midabdominal aorta for monitoring and blood sampling.Heart rate and blood pressure were measuredcontinuously and wererecordedon a multichannel Sensormedic R611 rectilinear polygraph (Sensormedics, Anaheim, CA). Blood samplesfor measurementof plasmacatecholamine levels and arterial blood gaseswere obtained from the umbilical artery catheter at 15,30,60,120,180, and 240min after cord cutting. Heart rate, blood pressure,and temperature were recordedwith eachsample.Initial ventilator settings (peak inspiratory pressure28 cmH20, positive and expiratory pressure2 cmH,O, and a rate of 30/min) were adjustedto maintain PO, between 100 and 150 mmHg and pH and PCO~within normal values. A continuous infusion of normal saline at 4 ml kg- l. h-l was administered. All arterial blood samplesfor measurementof plasma catecholamineswere added to chilled test tubes containing ethyl
0193~1849/92 $2.00 Copyright 0 1992 the American Physiological
Society
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CONJUGATED
CATECHOLAMINES
lene glycol-bis (@-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) and reducedglutathione. The sampleswerecentrifuged immediately, and plasma was separatedand stored at -70°C until catecholamineanalysiswasperformed. Blood for measurement of arterial blood gaseswasdrawn into heparinized syringes and analyzed within 3 min. Blood gaseswere measuredon a BMS3 MKII Radiometer (Copenhagen,Denmark) calibrated at 39°C. Approximately 4 ml of blood were removed for each sample and replacedwith heparinized maternal blood. Analytic techniques. Plasma free and sulfoconjugatedcatecholamineconcentrations weredetermined by a radioenzymatic assaysensitive to lo-20 pg/ml norepinephrine and epinephrine and 5-10 pg/ml dopamineaspreviously described(20). Plasma total catecholamines,representingfree plus sulfoconjugatedcatecholamines,were determined by the addition of 100 mU sulfatase(arylsulfataseS-1629;Sigma Chemical)to the incubation mixture (7). This enzyme preparation is free of demonstrable glucuronidaseat pH 7.0. Preliminary studies in fetal and adult sheepblood were conducted to ascertain the maximal total catecholamineconcentrations obtained by the addition of sulfatase activity to eachincubation tube. No additional increasein total catecholamineconcentration was noted during the inclusion of glucuronidase,suggestingthat little or no glucuronide conjugatesexist in sheepplasma. Sensitivity of the total catecholamine assaywas 20-50 pg/ml for norepinephrine and epinephrine and 50-80 pg/ml for dopamine. Data analysis. Serial responsesfor all catecholamine and hemodynamic data were analyzed by analysis of variance for repeated measuresand were compared with baseline values usingDunnett’s test. To achievehomogenousvariances,plasma free and sulfoconjugatedcatecholamineconcentrations were log transformedbefore statistical comparison.All resultsare shown asmeans* SE. A value of P < 0.05 was consideredstatistically significant.
AT BIRTH
.. .. 100-
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.
.
.
.
.
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.
.
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0
30
60
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120
180
la0
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.
RESULTS
Baseline arterial PO, (29 t 1 mmHg), PCO~ (39 t 2 mmHg), pH (7.42 t 0.03), heart rate (170 t 6 beats/min), and left ventricular systolic pressure (64 t 5 mmHg) were all within normal values for healthy animals at comparable gestational ages (15, 18-21). Consistent with ventilatory goals, PO, was maintained at -150 mmHg, PCO~ at 30-40 mmHg, and pH within normal limits after delivery (Fig. 1). Heart rate and blood pressure both increased significantly after delivery, with peak heart rate rising to 198 t 8 beats/min and blood pressure to 107 t 7 mmHg, P < 0.01 (Fig. 2). Serial changes in plasma free and sulfoconjugated norepinephrine are shown in Fig. 3A. The baseline free norepinephrine concentration of 307 t 92 pg/ml increased almost fourfold after delivery to a maximum of 1,178 t 389 pg/ml at 30 min of age. Free norepinephrine concentration remained elevated for 3 h after delivery and then returned to concentrations closer to the baseline value (537 t 85 pg/ml) by 4 h of age. Baseline sulfoconjugated norepinephrine concentration was 920 t 187 pg/ml. This represented 77 t 4% of the total circulating norepinephrine. After delivery, when free norepinephrine concentration was rising rapidly, there was no significant change in sulfoconjugated norepinephrine. Consequently, the proportion of sulfoconjugated norepinephrine decreased to as low as 55 t 4% of total norepinephrine at 120 min. These results are illustrated in Fig. 4. Serial changes in plasma free and sulfoconjugated epinephrine are shown in Fig. 3B. Baseline free epinephrine
24Q
TIME min Fig. 1. sheep. means partial
Serial arterial blood gases and pH at birth in term newborn Time of umbilical cord cutting is designated as time 0. Values are t SE. Pace,, arterial partial presske of CO,; Pao,, arterial pressure of Oz.
(49 t 27 pg/ml) was similar to values in past studies (15, 18, 19, 21). After delivery and cord cutting, plasma free epinephrine concentration increased MO-fold to 653 t 212 pg/ml and remained significantly elevated for the remainder of the study (P < 0.05). Similar to sulfoconjugated norepinephrine, sulfoconjugated epinephrine represented the majority of total circulating epinephrine. Baseline sulfoconjugated epinephrine (145 t 21 pg/ml) was 65 t 12% of total epinephrine. After cord cutting, sulfoconjugated epinephrine increased transiently to a maximum level of 296 t 47 at 15 min of age. Because there were logarithmic increases in free epinephrine, the proportion of sulfoconjugated epinephrine decreased significantly (Fig. 4). This proportion reached a nadir of 25-30% of total epinephrine by 3-4 h of age. DISCUSSION
These are the first data to describe free and sulfoconjugated catecholamine levels and their responses to delivery in newborn animals at birth. Sulfoconjugated norepinephrin e and epinephrine represe nted the predominant form of C irculating catecholamine in fetal plasma before
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CONJUGATED
CATECHOLAMINES
E25
AT BIRTH
5ocbov
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0 FREE l conjugated
250
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200
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150
loo
so
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30
60
90 TIME
120
150
180
210
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Fig. 2. Serial left ventricular (LV) systolic pressure and heart rate at birth in term newborn sheep. Time 0, umbilical cord cutting. Values are means k SE. * Significant differences from baseline values before cord cutting (P < 0.01).
delivery and cord cutting seen in this study. In previous studies of free and conjugated plasma catecholamines in fetal sheep, we observed very low concentrations of conjugated dopamine. This was true for both fetal and adult sheep (20). Also, the concentrations of free dopamine changed very little in fetal and newborn sheep during a variety of experimental paradigms we have investigated in the past (unpublished observations). Therefore we did not attempt to measure free or sulfoconjugated dopamine in the present study. After birth and severance of the umbilical-placental circulation, there were rapid logarithmic increases in the concentration of free norepinephrine and epinephrine, which rose 4- and lo-fold, respectively. These plasma concentrations of free catecholamines were similar to our previous reports (18, 19, 21). By contrast, the concentration of sulfoconjugated epinephrine rose only transiently above fetal levels and then remained unchanged thereafter. Sulfoconjugated norepinephrine did not rise after birth. The physiological and biological significance of catecholamine conjugation is unclear. Sulfoconjugation is catalyzed by phenol sulfotransferase (PST; EC 2.8.2.1). The tissue distribution of this enzyme is ubiquitous, including significant conjugation activity localized in brain (5), liver (9, lo), lung (26), and in platelets (6, 25, 31). Some investigators have suggested that circulating platelets are the predominant site for sulfoconjugation
I
1 0
-10
240
1 15
I 30 TIME
1 60
I 120
I 180
I 240
min
Fig. 3. Serial changes in free and sulfoconjugated norepinephrine (A) and free and sulfoconjugated epinephrine (B) at birth in newborn sheep. Time 0, umbilical cord cutting. Values are geometric means of: SE. * Significant difference from baseline concentration before cord cutting (P c 0.05). 100
Oa-
1
o!
I
-10
I
0
1
15
0 NOREPINEPHRINE 0 EPINEPHRINE
1
1
30
60
TIME
I
120
I
180
I
240
mln
Fig. 4. Proportion (%) of sulfoconjugated norepinephrine and epinephrine at birth. Time 0, umbilical cord cutting. Values are means k SE. * Significant difference from baseline values before cord cutting (P < 0.05).
(10). However, in patients with low platelet counts, there is no alteration in the proportion of sulfoconjugated catecholamines (13). Also, the affinity of platelet PST for each of the potential catecholamine substrates is highly variable (25). Therefore it remains unclear what the relative contribution of tissue having PST activity is to
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E26
CONJUGATED
CATECHOLAMINES
circulating sulfoconjugated catecholamines (13, 16, 25). Conjugated catecholamines appear to lack biological activity. In radioligand binding assays, epinephrine sulfate and norepinephrine sulfate are only weakly competitive for ,& and aZ-receptor ligands (32). The dissociation constants for both norepinephrine and epinephrine sulfate are lOO-1,000 times higher than for the free amines in a human mononuclear cell ,&receptor assay, and they do not inhibit binding at all of the a,-antagonist [“H]yohimbine to platelets (32). In vitro studies in isolated pulmonary artery demonstrate significant potentiation of electrically induced contractions with addition of norepinephrine but no response to the addition of norepinephrine sulfate (26). In the isolated perfused rat heart dopamine sulfate has no effect on developed tension (14). Last, infusion of sulfate conjugates in vivo does not result in a significant biological effect even at extremely high dosages (1). Conjugated catecholamines are not good substrates for subsequent biological modification involving the majority of pathways for endogenous biogenic amines. Dopamine fl-hydroxylase and phenylethanolamine-N-methyltransferase do not modify dopamine sulfate or norepinephrine sulfate in vitro (4). By contrast, small doses of [ l*C]dopamine sulfate administered in vivo to guinea pigs or rats is excreted mainly as 3,4-dihydroxyphenylacetic acid and homovanillic acid (17). This indicates that desulfation of the dopamine sulfate had taken place. A single report in vitro, using the isolated perfused heart, suggested that cardiac atria were capable of deconjugation of dopamine sulfate and release of free dopamine (14). Increased intrarenal and urinary [3H]norepinephrine has been reported in adult adrenalectomized rats after [ 3H]dopamine sulfate administration in vivo (2). Nonetheless, the majority of investigators do not consider conjugated catecholamines as representing a pool from which increases in the levels of circulating free catecholamines are derived. Some investigators interpret the high concentrations of circulating conjugated catecholamines as representing an “integrated index” of overall sympathoadrenal system activity (23). In situations associated with prolonged sympathoadrenal activation, there are parallel increases in free and conjugated catecholamines (10). Conjugated forms of each of the endogenous catecholamines are elevated significantly in patients with pheochromocytoma, where conjugation is felt to represent a form of protection from the high levels of free amines (11, 12). Adrenal venous effluent of pheochromocytoma patients is predominantly free amines (>99%), whereas the percentage of free amines in samples of peripheral blood from the same patients is only 45% (11). In critically ill patients, there is proportional elevation in free and conjugated norepinephrine, epinephrine, and dopamine (33). Exercise is a profound stimulus to catecholamine secretion in healthy adults and has been used to study the physiology and kinetics of catecholamine conjugation. It has been reported that both plasma free and conjugated norepinephrine rise with strenuous exercise (28, 29). In contrast, moderate exercise increased free catecholamine levels but actually decreased sulfoconjugated amines (3,8,
AT BIRTH
23). These authors suggested that the sulfoconjugated pools might be a source of free amines during vigorous exercise (23). However, no kinetic or radiolabeled tracer data exist to support this interpretation. Any alteration in catecholamine sulfate with exercise may not be linked to free circulating catecholamines but may be a reflection of blood flow through tissues that have high PST activity. These results suggest that the nature of the stressor may be an important factor in the sulfoconjugation of catecholamines. We have previously demonstrated that both free and sulfoconjugated plasma norepinephrine and epinephrine concentrations rise promptly in chronically catheterized fetal sheep in response to hypoxia (20). Our present results differ in that the rise in free catecholamine concentrations was much greater at birth than during the mild fetal hypoxia previously employed (20). It has been reported that chronic hypoxia increases sulfoconjugated norepinephrine in the dog pulmonary artery (27). In the present study, we did not observe a rapid increase in sulfoconjugated epinephrine or norepinephrine despite substantial increases in free amines. Differences in the nature of physiological stress between the two conditions may account for these discrepancies. An alternative explanation for the lack of parallel increase in conjugated catecholamines could be the removal of the placental contribution to catecholamine clearance and/or conjugation (30) In’ summary, we demonstrated that, despite logarithmic increases in the concentrations of circulating plasma free norepinephrine and epinephrine, there is little or no change in the concentration of the sulfoconjugated forms of either endogenous amine. Further studies of the mechanisms involved, which determine the relative proportions of free and sulfoconjugated catecholamines, should consider, among other factors, free catecholamine levels, the use of tracer methodologies, blood flow and its distribution, and relative tissue PST activities. This work was supported in part by National Heart, Lung, and Blood Institute Grants HD-18014 and HD-07013 and by an Investigative Group Fellowship Award from the American Heart Association, Los Angeles Affiliate. Address for reprint requests: J. F. Padbury, Harbor-UCLA Medical Center, UCLA School of Medicine, 1000 W. Carson St., RBl, Torrance, CA 90509. Received 31 October 1991; accepted in final form 17 February 1992. REFERENCES 1. Ackerman, D. M., J. P. Hieble, H. M. Sarau, and T. C. Jain. Pharmacological characterization of dopamine-4O-sulfate. Arch.
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