Biol Neonate 1991;60:102-107
Plasma Methionine Enkephalin Levels in the Human Newborn at Birth Alma M. Martinez, James F. Padbnry, Elizabeth E. Burnell, Siang L. Thio Department o f Pediatrics. Harbor-UCLA Medical Center. UCLA School o f Medicine. Torrance. Calif.. USA
Key Words. Methionine enkephalin • Catecholamine
Introduction The opiate peptides (both enkephalins and endorphins) are postulated to have phys iologic effects in the newborn and perinatal period in ventilatory control and cardiovas cular responses to the stress of birth [1-3]. The enkephlins are found in the peripheral nervous system localized in sympathetic ganglia [4] and stored with catecholamines in adrenal medullary cells [5]. The enkepha lins are stored as low-molecular-weight pentapeptides (met-enkephalin and leu-enkephalin) and as higher-molecular-weight forms. The larger forms include carboxy-extended
forms of met-enkephalin and preoenkephalin A [6]. In vitro studies show that all forms of enkephalins are cosecreted with cat echolamines in response to a variety of stim uli [6]. Previous assays of plasma met-enkepha lin have suffered from poor reproducibility and erratic recovery. We recently tested and validated a radioimmunoassay system for enkephalin pentapeptides and the high-mo lecular-weight forms of enkephalin in plas ma. These studies were designed to deter mine the plasma enkephalin peptide levels in a selected group of healthy term newborn infants.
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Abstract. Plasma met-enkephalin immunoreactivity (MET-ENKi) and catecholamine lev els were measured in umbilibal cord blood from 46 healthy newborn infants. Clinical data including Apgar scores, birth weight, gestational age, route of delivery, fetal heart tracings and arterial blood gas values were also obtained. Thirty-nine infants were delivered by the vaginal route. All but 1 infant delivered by cesarian section had undergone a trial of labor. Plasma MET-ENKi in the newborn infants was markedly greater than levels found in healthy adult volunteers; 360 ± 25 versus 25 ± 2 pg/ml, respectively. MET-ENKi levels were similar in umbilical arterial and umbilical venous blood, and in infants delivered vaginally or by cesarian section.
Plasma MET-ENKi Levels in the Human Newborn at Birth
Patient Selection and Study Protocol Institutional review board approval was obtained to collect umbilical cord blood from healthy, term infants born at Harbor-UCLA Medical Center. Um bilical cord blood was drawn from the placenta imme diately after cord clamping. Clinical data were ob tained from the mothers’ medical record and in cluded: Apgar scores, fetal heart rate tracings, birth weight, gestational age and route o f delivery. Forty-six infants had plasma met-cnkcphalin lev els measured from umbilical cord blood. Umbilical cord blood gases were also measured. Comparisons o f met-enkephalin levels from arterial versus venous ori gin were made in 18 cases to determine if there was an umbilical arterial-to-venous gradient. Arterial plasma norepinephrine and epinephrine were measured with met-enkephalin levels in 28 infants. Plasma metenkephalin levels were also determined in 11 healthy, nonpregnant adult volunteers for comparison with the newborn levels. The blood for met-enkephalin analysis was placed into chilled test tubes containing sodium citrate (0.5 mM . final concentration), Trasylol (500U /m l), and EDTA (3 m V/). Blood for catecholamine mea surement was placed into tubes containing reduced glutathione (3 m .l/) and ethyleneglycol-bis(P-aminoethyl ether)-N.N.N'N'-tetraacelic acid (4 rnM ). The samples were centrifuged immediately, and plasma was separated and frozen at - 7 0 ° C for later analy sis. Laboratory A nalysis Plasma for measurement of met-enkephalin was first extracted through polystyrene columns and then measured by radioimmunoassay using commercial antiserum (Immunonuclear, Stillwater. Minn.. USA). This antiserum cross-reacts 2.8% with leu-enkephalin. Our data therefore represents met-enkephalin immunoreactivity (MET-ENKi). Plasma for measure ment of the high-molecular-weight enkephalin pep tides (total MET-ENKi) first underwent sequential proteolytic digestion with trypsin and carboxypepti dase B as previously described [6], The plasma was then extracted and measured by radioimmunoassay as described above. The assay is sensitive to 2 pg. The inter- and intra-assay variabilities were 13 and 9%. respectively. Recovery of met-enkephalin using this assay was determined to be greater than 90% after
1,000. 500 or 200 pg/ml additions to unextracted plasma. To characterize the identity o f the plasma immunoreactivity, plasma was subjected to gel filtra tion using a Sephadex G-75 column (0.9 X 57 cm). The plasma immunoreactivity (MET-ENKi) coeluted precisely with met-enkephalin standard. Plasma first separated by gel filtration and then subjected to tryp sin and carboxypeptidase B demonstrated that sub strates of a molecular weight o f 16,000-90,000 yielded MET-ENKi following proteolytic degrada tion. Catecholamines were measured by a radioenzymatic assay sensitive to 1-2 pg o f norepinephrine and epinephrine as previously described [7], Data Analysis The data are presented as means and standard error o f the mean (SEM). Because catecholamine val ues are not normally distributed, the data were log transformed prior to statistical analysis and are pre sented as the geometric means and the SEM. Stu dent’s t test was used to compare met-enkephalin lev els between the different groups, p < 0.05 was consid ered statistically significant.
Results Forty-six infants, with a mean gestational age of 39 weeks and birth weight 3.3 kg were studied. As can be seen from table 1, this study was carried out on a group of healthy, vigorous infants. The mean arterial blood pH was 7.26, and the median Apgar scores were 8 at 1 min and 9 at 5 min. The arterial plasma catecholamine levels were elevated with a geometric mean norepinephrine level of 2,010 ± 613 pg/ml and an epinephrine level of 226 ± 68 pg/ml. Thirty-nine infants were delivered by the vaginal route. All but 1 infant delivered via cesarian section had un dergone a trial of labor. Indications for ce sarian section included cephalopelvic dis proportion (n = 3), breech presentation (n = 1) and suspicious fetal monitor tracing (n = 3). There were no statistically significant dif
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Materials and Methods
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Martinez/Padbury/Burnell/Thio
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Discussion This study was undertaken to determine the umbilical plasma levels of met-enkepha lin in healthy newborn infants. We found the mean levels of MET-ENKi to be signifi cantly elevated above those in adult volun teers. The levels of the large-molecularweight forms of enkephalin (total METENKi) were similar in the newborn when compared to adult levels. The plasma METENKi levels were also similar in umbilical
Table I. Patient characteristics Mean±SEM
Range
pH (arterial)
7.26 ± 0.01
7.02-7.38
Apgar (1 min)1
8
4 -9
Apgar (5 min)1
9
10
Body weight, kg
3.3 ±0.1
2.5-4.65
Gestational age, weeks
39.1 ± 0.4
36-42
Norepinephrine, pg/ml
2,010 ± 613
147-17,143
Epinephrine, pg/ml
226 ±68
17-2.088
Route o f delivery Vaginal Cesarian section
39 7
1
Median.
arterial and venous blood. This finding sug gests no significant placental uptake or deg radation of either met-enkephalin or the high-molecular-weight peptide forms. We and others have shown that catechol amine levels are elevated in human umbili cal cord blood [7, 8]. We have previously shown a significant correlation between um bilical arterial catecholamines, umbilical ar terial O2 pressure, arterial pH and duration of labor [7]. Moreover, by multiple-regres sion analysis we demonstrated that umbili cal cord arterial pH was the most important determinant of catecholamine elevation at birth. Our present descriptive study docu ments peptide levels in a group of healthy newborn infants. As indicated by the Apgar scores and initial blood gases, these infants underwent minimal perinatal ‘stress’. In or der to describe any similar correlation be tween plasma met-enkephalin peptides and these traditional markers of perinatal stress, a group of ill or asphyxiated neonates would need to be studied.
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ferences in the arterial pH values, Apgar scores or gestational ages between infants delivered vaginally or via cesarian section. The plasma met-enkephalin levels in these infants are shown in figure 1. For com parison, plasma met-enkephalin levels of a group of adult volunteers are also shown in the figure. As can be seen, the MET-ENKi levels were significantly greater in the new born infants in comparison with the adult plasma levels, with newborn levels of 360 ± 25 versus adult levels of 25 ± 2 pg/ml. There was no statistical difference in the newborns’ total MET-ENKi levels (the high-molecularweight forms of the met-enkephalin peptide) when compared to the adult levels, 57 versus 70 ng/ml, respectively. A comparison of umbilical arterial plasma levels with umbilical venous levels of both MET-ENKi and total MET-ENKi from 18 infants is shown in figure 2. There were no differences in either MET-ENKi or total MET-ENKi when arterial and venous levels were compared. Plasma MET-ENKi and total MET-ENKi from infants born vaginally or by cesarian section were also similar with levels of 380 ± 30 versus 280 ± 60 pg/ml and 60 ± 10 versus 50 ± 10 ng/ml, respectively.
105
Fig. 1. Comparison of plasma met-enkephalin immunoreactivity in newborn umbilical cord blood ver sus adult venous levels. Values shown as means ± SEM. * p < 0.01 (Student's t test).
Fig. 2. Plasma met-enkephalin immunoreactivity in arterial versus venous umbilical cord blood. Values shown as means ± SEM.
Enkephalin peptides are found in brain stem nuclei involved in autonomic and car diovascular control [9], In the peripheral nervous system they are located in sympa thetic neurons and ganglia [4] and stored in large amounts with catecholamines in adre nal medullary cells [5]. They are stored as pentapeptides (met- and leu-enkephalin) and as larger forms, including carboxy-extended forms of met-enkephalin and the prohor mone, proenkephalin A. In vitro studies show that all forms of enkephalins are se creted with catecholamines in response to physiologic stimuli [6]. In the limited in vivo studies available, the nature of the enkepha lin peptide released appears to be dependent on the type of stimulus used to evoke a response. In dogs exposed to hypoglycemia, increased levels of the high-molecularweight peptides are described with no change noted in the pentapeptide levels [10]. In fetal sheep exposed to hypoxia, we have noted a significant elevation of the highmolecular-weight form of circulating met-
enkephalin associated with elevated plasma catecholamine levels with no change in the pentapeptide levels [11]. In the present study, we found significantly higher levels of plasma met-enkephalin at birth in neonates when compared to adults. It is unclear from the present data, or from previous in vivo studies, whether there is differential release of high- or low-molecular-weight forms of met-enkephalin peptides during periods of stress, whether there are major differences in the metabolism or plasma clearance rates of the different peptides and whether there are important species differences in responses. Another major class of opiate peptides are endorphins. Circulating plasma endor phins are known to be elevated in pregnancy and at delivery in humans and fetal sheep [12-15], Numerous clinical studies have also shown significantly elevated levels of circu lating P-endorphins in human neonates with hypoxia and perinatal asphyxia [16, 17]. The endogenous opiate peptides are pos tulated to have unique physiologic effects in
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Acknowledgements The authors gratefully acknowledge the assistance of the Perinatal Research Staff at Harbor-UCLA Medical Center including Christine Mori. RN. Sarah Alvarez. RN, and Artemcsa McCullough. RN. This work was supported in part by grants from the USPHS HD-22003. HD-18014 and MOI RR-00425.
References 1 Chernick C: Endorphins and ventilatory control. N Engl J Med 1981.20:1227-1228. 2 De Boeck C. Van Reempts P. Rigatto H. et al: Naloxone reduces the decrease in ventilation in duced by hypoxia in newborn infants. J Appl Physiol 1984:56:1507-1511. 3 Lewis AB, Ferry DA. Sadeghi M: Fetal cardiovas cular and breathing movement responses to en dogenous opiates. Biol Neonate 1986:50:278— 287. 4 Schultzbcrg M. Hokfelt T. Lundberg JM. et al: Enkephalin-like medullary immunoreaclivity in nerve terminals in sympathetic ganglia and adre nal medulla and in adrenal medullary gland cells. Acta Physiol Scand 1978:103:475-477. 5 Vivcros OH. Diliberto EJ Jr. Hazum E. et al: Opiate-like materials in the adrenal medulla: Evi dence for storage and secretion with catechol amines. Mol Pharmacol 1979:16:1101-1108. 6 Chaminade M. Foutz AS. Rossicr J: Co-release of enkephalins and precursors with catecholamines by the perfused cat adrenal in-situ. J Physiol 1984: 353:157-169. 7 Padburv JF . R oberm an B. O ddie T H . et al: Fetal
8
9
10
11
catecholamine release in response to labor and delivery. Obstet Gynecol 1982:60:607-611. Langerkratz H. Bistoletti P: Catecholamine re lease in the newborn infant at birth. Pediatr Res 1977:11:889-893. Holaday JW: Cardiovascular effects of endoge nous opiate systems. Annu Rev Pharmacol Toxi col 1983:23:541-594. Medbak S. Mason DFJ. Rees LH: Plasma metenkephalin and catecholamine responses to insu lin-induced hypoglycaemia in greyhounds. J En docrinol 1987:1 14:81—87. Martinez AM. Padbury JF, et al: The effects of
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the newborn and perinatal period in cardio vascular and respiratory control mecha nisms. Both central and peripheral infusions of met-enkephalin in fetal sheep are shown to have significant effects on heart rate, blood pressure and fetal breathing move ments [3, 18]. These effects are eliminated by autonomic blockade and blunted by nal oxone. an opiate peptide antagonist. Nalox one has been shown to decrease the duration of apnea in asphyxiated newborn rabbits [19] as well as reverse the depression in res pirations, muscle tone and activity level [20] . Naloxone has also been shown to re duce the hypoventilation induced by hyp oxia in newborn infants [2], The physiologic significance of the ele vated levels of met-enkephalin at birth is unclear. Numerous investigators have sug gested that a variety of neuropeptides, in cluding the opiate peptides, neuropeptide Y, substance P. as well as adenosine and prosta glandins, are elevated at the time of birth and function as modulators of the sympatho adrenal system and affect respiratory, vascu lar and sleep states in the immature organism [9, 21-23], The opiate peptides inhibit in vitro norepinephrine release in sympathetic ganglia [24] as well as adrenal catecholamine release in response to cholinergic stimulation [25], We have shown that catecholamine re lease at birth is vital for successful postnatal adaptation [26]. We have also shown that opiate receptor blockade at birth signifi cantly augments catecholamine release and cardiovascular stability [27]. Whether the el evated met-enkephalin plasma levels at birth described herein are a response to the stress of labor and delivery or are necessary for the organism’s adaptation during the transition from fetal to extrauterine life is unclear and deserves further research.
107
Plasma MET-ENKJ Levels in the Human Newborn at Birth
13
14
15
16
17
18
19
20
21 Langercrantz H: Neuromodulators and respira tory control in the infant. Clin Pcrinatol 1987:14: 683-695. 22 Moss IR. Runold M. Dahlin I. Fredholm BB, Nyberg F. Langercrantz H: Respiratory and neu roendocrine responses of piglets to hypoxia during postnatal development. Acta Physiol Scand 1987; 131:533-541. 23 Moss IR. Inman JG: Neurochemicals and respira tory control during development J Appl Physiol 1989:67:1-13. 24 Konishi S, Tsunoo A. Otsuka M: Enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia. Nature 1979:282:515-516. 25 Kumakura K, Karoum F, Costa E: Modulation of nicotinic receptors by opiate receptor agonists in cultured adrenal chromaffin cells. Nature 1980: 83:489-492. 26 Padbury JF, Martinez AM: Sympathoadrenal sys tem aclivity at birth: Integration o f postnatal ad aptation. Semin Perinatol 1988:12:163-172. 27 Padbury JF, Agata Y. Polk DH. et al: Neonatal adaptation: Naloxone increases the catecholamine surge at birth. Pediatr Res 1987;21:590-593.
Alma M. Martinez. MD Department of Pediatrics Harbor-UCLA Medical Center UCLA School of Medicine 1000 West Carson Street. RB I Torrance. CA 90509 (USA)
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12
hypoxia on enkephalin and catecholamine release in fetal sheep. Clin Res 1988;36( 1):222A. Wardlaw SL. Stark Rl. Baxi L. et al: Plasma pendorphin and P-lipotropin in the human fetus at delivery: Correlation with arterial pH and pCK J Clin Endocrinol Metab 1979:49:888-891. Moss IR. Conner H. Yce WFH. ct al: Human Pendorphin-like immunoreactivity in the perinatal/ neonatal period. J Pediatr 1983:101:443-446. Padbury JF. Agata Y. Polk DH. et al: Neonatal adaptation: Naloxone increases the catecholamine surge at burth. Dev Pharmacol Ther 1988:1 1:4450. Wardlaw SL. Stark Rl. Daniel S. et al: Effects of hypoxia on P-endorphin and p-lipotropin release in fetal, newborn, and maternal sheep. Endocri nology 1981:108:1710-1715. Hindmarsh KW, Sankaran K. Watson VG: Plasma beta-endorphin concentrations in neo nates associated with acute stress. Dev Pharmacol Ther 1984:7:198-204. Davidson S. Gil-Ad I. Rogovin H. et al: Cardio respiratory depression and plasma P-endorphin levels in low-birth weight infants during the first day of life. Am J Dis Child 1987:141:145-148. Lagamma EF. Itskovitz J. Rudolph AM: Matura tion o f circulatory responses to methionine-en kephalin. Pediatr Res 1983:17:162-167. Chernick V. Madansky DL. Lawson EE: Nalox one decreases the duration of primary apnea with neonatal asphyxia. Pediatr Res 1980; 14:357— 359. Chernick V. Craig RG: Naloxone reverses neona tal depression caused by fetal asphyxia. Science 1982:216:1252-1253.
Plasma Methionine Enkephalin Levels in the Human Newborn at Birth Alma M. Martinez, James F. Padbnry, Elizabeth E. Burnell, Siang L. Thio Department o f Pediatrics. Harbor-UCLA Medical Center. UCLA School o f Medicine. Torrance. Calif.. USA
Key Words. Methionine enkephalin • Catecholamine
Introduction The opiate peptides (both enkephalins and endorphins) are postulated to have phys iologic effects in the newborn and perinatal period in ventilatory control and cardiovas cular responses to the stress of birth [1-3]. The enkephlins are found in the peripheral nervous system localized in sympathetic ganglia [4] and stored with catecholamines in adrenal medullary cells [5]. The enkepha lins are stored as low-molecular-weight pentapeptides (met-enkephalin and leu-enkephalin) and as higher-molecular-weight forms. The larger forms include carboxy-extended
forms of met-enkephalin and preoenkephalin A [6]. In vitro studies show that all forms of enkephalins are cosecreted with cat echolamines in response to a variety of stim uli [6]. Previous assays of plasma met-enkepha lin have suffered from poor reproducibility and erratic recovery. We recently tested and validated a radioimmunoassay system for enkephalin pentapeptides and the high-mo lecular-weight forms of enkephalin in plas ma. These studies were designed to deter mine the plasma enkephalin peptide levels in a selected group of healthy term newborn infants.
Downloaded by: University of Leeds 129.11.21.2 - 10/25/2017 10:08:22 AM
Abstract. Plasma met-enkephalin immunoreactivity (MET-ENKi) and catecholamine lev els were measured in umbilibal cord blood from 46 healthy newborn infants. Clinical data including Apgar scores, birth weight, gestational age, route of delivery, fetal heart tracings and arterial blood gas values were also obtained. Thirty-nine infants were delivered by the vaginal route. All but 1 infant delivered by cesarian section had undergone a trial of labor. Plasma MET-ENKi in the newborn infants was markedly greater than levels found in healthy adult volunteers; 360 ± 25 versus 25 ± 2 pg/ml, respectively. MET-ENKi levels were similar in umbilical arterial and umbilical venous blood, and in infants delivered vaginally or by cesarian section.
Plasma MET-ENKi Levels in the Human Newborn at Birth
Patient Selection and Study Protocol Institutional review board approval was obtained to collect umbilical cord blood from healthy, term infants born at Harbor-UCLA Medical Center. Um bilical cord blood was drawn from the placenta imme diately after cord clamping. Clinical data were ob tained from the mothers’ medical record and in cluded: Apgar scores, fetal heart rate tracings, birth weight, gestational age and route o f delivery. Forty-six infants had plasma met-cnkcphalin lev els measured from umbilical cord blood. Umbilical cord blood gases were also measured. Comparisons o f met-enkephalin levels from arterial versus venous ori gin were made in 18 cases to determine if there was an umbilical arterial-to-venous gradient. Arterial plasma norepinephrine and epinephrine were measured with met-enkephalin levels in 28 infants. Plasma metenkephalin levels were also determined in 11 healthy, nonpregnant adult volunteers for comparison with the newborn levels. The blood for met-enkephalin analysis was placed into chilled test tubes containing sodium citrate (0.5 mM . final concentration), Trasylol (500U /m l), and EDTA (3 m V/). Blood for catecholamine mea surement was placed into tubes containing reduced glutathione (3 m .l/) and ethyleneglycol-bis(P-aminoethyl ether)-N.N.N'N'-tetraacelic acid (4 rnM ). The samples were centrifuged immediately, and plasma was separated and frozen at - 7 0 ° C for later analy sis. Laboratory A nalysis Plasma for measurement of met-enkephalin was first extracted through polystyrene columns and then measured by radioimmunoassay using commercial antiserum (Immunonuclear, Stillwater. Minn.. USA). This antiserum cross-reacts 2.8% with leu-enkephalin. Our data therefore represents met-enkephalin immunoreactivity (MET-ENKi). Plasma for measure ment of the high-molecular-weight enkephalin pep tides (total MET-ENKi) first underwent sequential proteolytic digestion with trypsin and carboxypepti dase B as previously described [6], The plasma was then extracted and measured by radioimmunoassay as described above. The assay is sensitive to 2 pg. The inter- and intra-assay variabilities were 13 and 9%. respectively. Recovery of met-enkephalin using this assay was determined to be greater than 90% after
1,000. 500 or 200 pg/ml additions to unextracted plasma. To characterize the identity o f the plasma immunoreactivity, plasma was subjected to gel filtra tion using a Sephadex G-75 column (0.9 X 57 cm). The plasma immunoreactivity (MET-ENKi) coeluted precisely with met-enkephalin standard. Plasma first separated by gel filtration and then subjected to tryp sin and carboxypeptidase B demonstrated that sub strates of a molecular weight o f 16,000-90,000 yielded MET-ENKi following proteolytic degrada tion. Catecholamines were measured by a radioenzymatic assay sensitive to 1-2 pg o f norepinephrine and epinephrine as previously described [7], Data Analysis The data are presented as means and standard error o f the mean (SEM). Because catecholamine val ues are not normally distributed, the data were log transformed prior to statistical analysis and are pre sented as the geometric means and the SEM. Stu dent’s t test was used to compare met-enkephalin lev els between the different groups, p < 0.05 was consid ered statistically significant.
Results Forty-six infants, with a mean gestational age of 39 weeks and birth weight 3.3 kg were studied. As can be seen from table 1, this study was carried out on a group of healthy, vigorous infants. The mean arterial blood pH was 7.26, and the median Apgar scores were 8 at 1 min and 9 at 5 min. The arterial plasma catecholamine levels were elevated with a geometric mean norepinephrine level of 2,010 ± 613 pg/ml and an epinephrine level of 226 ± 68 pg/ml. Thirty-nine infants were delivered by the vaginal route. All but 1 infant delivered via cesarian section had un dergone a trial of labor. Indications for ce sarian section included cephalopelvic dis proportion (n = 3), breech presentation (n = 1) and suspicious fetal monitor tracing (n = 3). There were no statistically significant dif
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Materials and Methods
103
Martinez/Padbury/Burnell/Thio
104
Discussion This study was undertaken to determine the umbilical plasma levels of met-enkepha lin in healthy newborn infants. We found the mean levels of MET-ENKi to be signifi cantly elevated above those in adult volun teers. The levels of the large-molecularweight forms of enkephalin (total METENKi) were similar in the newborn when compared to adult levels. The plasma METENKi levels were also similar in umbilical
Table I. Patient characteristics Mean±SEM
Range
pH (arterial)
7.26 ± 0.01
7.02-7.38
Apgar (1 min)1
8
4 -9
Apgar (5 min)1
9
10
Body weight, kg
3.3 ±0.1
2.5-4.65
Gestational age, weeks
39.1 ± 0.4
36-42
Norepinephrine, pg/ml
2,010 ± 613
147-17,143
Epinephrine, pg/ml
226 ±68
17-2.088
Route o f delivery Vaginal Cesarian section
39 7
1
Median.
arterial and venous blood. This finding sug gests no significant placental uptake or deg radation of either met-enkephalin or the high-molecular-weight peptide forms. We and others have shown that catechol amine levels are elevated in human umbili cal cord blood [7, 8]. We have previously shown a significant correlation between um bilical arterial catecholamines, umbilical ar terial O2 pressure, arterial pH and duration of labor [7]. Moreover, by multiple-regres sion analysis we demonstrated that umbili cal cord arterial pH was the most important determinant of catecholamine elevation at birth. Our present descriptive study docu ments peptide levels in a group of healthy newborn infants. As indicated by the Apgar scores and initial blood gases, these infants underwent minimal perinatal ‘stress’. In or der to describe any similar correlation be tween plasma met-enkephalin peptides and these traditional markers of perinatal stress, a group of ill or asphyxiated neonates would need to be studied.
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ferences in the arterial pH values, Apgar scores or gestational ages between infants delivered vaginally or via cesarian section. The plasma met-enkephalin levels in these infants are shown in figure 1. For com parison, plasma met-enkephalin levels of a group of adult volunteers are also shown in the figure. As can be seen, the MET-ENKi levels were significantly greater in the new born infants in comparison with the adult plasma levels, with newborn levels of 360 ± 25 versus adult levels of 25 ± 2 pg/ml. There was no statistical difference in the newborns’ total MET-ENKi levels (the high-molecularweight forms of the met-enkephalin peptide) when compared to the adult levels, 57 versus 70 ng/ml, respectively. A comparison of umbilical arterial plasma levels with umbilical venous levels of both MET-ENKi and total MET-ENKi from 18 infants is shown in figure 2. There were no differences in either MET-ENKi or total MET-ENKi when arterial and venous levels were compared. Plasma MET-ENKi and total MET-ENKi from infants born vaginally or by cesarian section were also similar with levels of 380 ± 30 versus 280 ± 60 pg/ml and 60 ± 10 versus 50 ± 10 ng/ml, respectively.
105
Fig. 1. Comparison of plasma met-enkephalin immunoreactivity in newborn umbilical cord blood ver sus adult venous levels. Values shown as means ± SEM. * p < 0.01 (Student's t test).
Fig. 2. Plasma met-enkephalin immunoreactivity in arterial versus venous umbilical cord blood. Values shown as means ± SEM.
Enkephalin peptides are found in brain stem nuclei involved in autonomic and car diovascular control [9], In the peripheral nervous system they are located in sympa thetic neurons and ganglia [4] and stored in large amounts with catecholamines in adre nal medullary cells [5]. They are stored as pentapeptides (met- and leu-enkephalin) and as larger forms, including carboxy-extended forms of met-enkephalin and the prohor mone, proenkephalin A. In vitro studies show that all forms of enkephalins are se creted with catecholamines in response to physiologic stimuli [6]. In the limited in vivo studies available, the nature of the enkepha lin peptide released appears to be dependent on the type of stimulus used to evoke a response. In dogs exposed to hypoglycemia, increased levels of the high-molecularweight peptides are described with no change noted in the pentapeptide levels [10]. In fetal sheep exposed to hypoxia, we have noted a significant elevation of the highmolecular-weight form of circulating met-
enkephalin associated with elevated plasma catecholamine levels with no change in the pentapeptide levels [11]. In the present study, we found significantly higher levels of plasma met-enkephalin at birth in neonates when compared to adults. It is unclear from the present data, or from previous in vivo studies, whether there is differential release of high- or low-molecular-weight forms of met-enkephalin peptides during periods of stress, whether there are major differences in the metabolism or plasma clearance rates of the different peptides and whether there are important species differences in responses. Another major class of opiate peptides are endorphins. Circulating plasma endor phins are known to be elevated in pregnancy and at delivery in humans and fetal sheep [12-15], Numerous clinical studies have also shown significantly elevated levels of circu lating P-endorphins in human neonates with hypoxia and perinatal asphyxia [16, 17]. The endogenous opiate peptides are pos tulated to have unique physiologic effects in
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Plasma MET-ENKi Levels in the Human Newborn at Birth
Martinez/Padbury/Burnell/Thio
106
Acknowledgements The authors gratefully acknowledge the assistance of the Perinatal Research Staff at Harbor-UCLA Medical Center including Christine Mori. RN. Sarah Alvarez. RN, and Artemcsa McCullough. RN. This work was supported in part by grants from the USPHS HD-22003. HD-18014 and MOI RR-00425.
References 1 Chernick C: Endorphins and ventilatory control. N Engl J Med 1981.20:1227-1228. 2 De Boeck C. Van Reempts P. Rigatto H. et al: Naloxone reduces the decrease in ventilation in duced by hypoxia in newborn infants. J Appl Physiol 1984:56:1507-1511. 3 Lewis AB, Ferry DA. Sadeghi M: Fetal cardiovas cular and breathing movement responses to en dogenous opiates. Biol Neonate 1986:50:278— 287. 4 Schultzbcrg M. Hokfelt T. Lundberg JM. et al: Enkephalin-like medullary immunoreaclivity in nerve terminals in sympathetic ganglia and adre nal medulla and in adrenal medullary gland cells. Acta Physiol Scand 1978:103:475-477. 5 Vivcros OH. Diliberto EJ Jr. Hazum E. et al: Opiate-like materials in the adrenal medulla: Evi dence for storage and secretion with catechol amines. Mol Pharmacol 1979:16:1101-1108. 6 Chaminade M. Foutz AS. Rossicr J: Co-release of enkephalins and precursors with catecholamines by the perfused cat adrenal in-situ. J Physiol 1984: 353:157-169. 7 Padburv JF . R oberm an B. O ddie T H . et al: Fetal
8
9
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catecholamine release in response to labor and delivery. Obstet Gynecol 1982:60:607-611. Langerkratz H. Bistoletti P: Catecholamine re lease in the newborn infant at birth. Pediatr Res 1977:11:889-893. Holaday JW: Cardiovascular effects of endoge nous opiate systems. Annu Rev Pharmacol Toxi col 1983:23:541-594. Medbak S. Mason DFJ. Rees LH: Plasma metenkephalin and catecholamine responses to insu lin-induced hypoglycaemia in greyhounds. J En docrinol 1987:1 14:81—87. Martinez AM. Padbury JF, et al: The effects of
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the newborn and perinatal period in cardio vascular and respiratory control mecha nisms. Both central and peripheral infusions of met-enkephalin in fetal sheep are shown to have significant effects on heart rate, blood pressure and fetal breathing move ments [3, 18]. These effects are eliminated by autonomic blockade and blunted by nal oxone. an opiate peptide antagonist. Nalox one has been shown to decrease the duration of apnea in asphyxiated newborn rabbits [19] as well as reverse the depression in res pirations, muscle tone and activity level [20] . Naloxone has also been shown to re duce the hypoventilation induced by hyp oxia in newborn infants [2], The physiologic significance of the ele vated levels of met-enkephalin at birth is unclear. Numerous investigators have sug gested that a variety of neuropeptides, in cluding the opiate peptides, neuropeptide Y, substance P. as well as adenosine and prosta glandins, are elevated at the time of birth and function as modulators of the sympatho adrenal system and affect respiratory, vascu lar and sleep states in the immature organism [9, 21-23], The opiate peptides inhibit in vitro norepinephrine release in sympathetic ganglia [24] as well as adrenal catecholamine release in response to cholinergic stimulation [25], We have shown that catecholamine re lease at birth is vital for successful postnatal adaptation [26]. We have also shown that opiate receptor blockade at birth signifi cantly augments catecholamine release and cardiovascular stability [27]. Whether the el evated met-enkephalin plasma levels at birth described herein are a response to the stress of labor and delivery or are necessary for the organism’s adaptation during the transition from fetal to extrauterine life is unclear and deserves further research.
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Plasma MET-ENKJ Levels in the Human Newborn at Birth
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21 Langercrantz H: Neuromodulators and respira tory control in the infant. Clin Pcrinatol 1987:14: 683-695. 22 Moss IR. Runold M. Dahlin I. Fredholm BB, Nyberg F. Langercrantz H: Respiratory and neu roendocrine responses of piglets to hypoxia during postnatal development. Acta Physiol Scand 1987; 131:533-541. 23 Moss IR. Inman JG: Neurochemicals and respira tory control during development J Appl Physiol 1989:67:1-13. 24 Konishi S, Tsunoo A. Otsuka M: Enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia. Nature 1979:282:515-516. 25 Kumakura K, Karoum F, Costa E: Modulation of nicotinic receptors by opiate receptor agonists in cultured adrenal chromaffin cells. Nature 1980: 83:489-492. 26 Padbury JF, Martinez AM: Sympathoadrenal sys tem aclivity at birth: Integration o f postnatal ad aptation. Semin Perinatol 1988:12:163-172. 27 Padbury JF, Agata Y. Polk DH. et al: Neonatal adaptation: Naloxone increases the catecholamine surge at birth. Pediatr Res 1987;21:590-593.
Alma M. Martinez. MD Department of Pediatrics Harbor-UCLA Medical Center UCLA School of Medicine 1000 West Carson Street. RB I Torrance. CA 90509 (USA)
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hypoxia on enkephalin and catecholamine release in fetal sheep. Clin Res 1988;36( 1):222A. Wardlaw SL. Stark Rl. Baxi L. et al: Plasma pendorphin and P-lipotropin in the human fetus at delivery: Correlation with arterial pH and pCK J Clin Endocrinol Metab 1979:49:888-891. Moss IR. Conner H. Yce WFH. ct al: Human Pendorphin-like immunoreactivity in the perinatal/ neonatal period. J Pediatr 1983:101:443-446. Padbury JF. Agata Y. Polk DH. et al: Neonatal adaptation: Naloxone increases the catecholamine surge at burth. Dev Pharmacol Ther 1988:1 1:4450. Wardlaw SL. Stark Rl. Daniel S. et al: Effects of hypoxia on P-endorphin and p-lipotropin release in fetal, newborn, and maternal sheep. Endocri nology 1981:108:1710-1715. Hindmarsh KW, Sankaran K. Watson VG: Plasma beta-endorphin concentrations in neo nates associated with acute stress. Dev Pharmacol Ther 1984:7:198-204. Davidson S. Gil-Ad I. Rogovin H. et al: Cardio respiratory depression and plasma P-endorphin levels in low-birth weight infants during the first day of life. Am J Dis Child 1987:141:145-148. Lagamma EF. Itskovitz J. Rudolph AM: Matura tion o f circulatory responses to methionine-en kephalin. Pediatr Res 1983:17:162-167. Chernick V. Madansky DL. Lawson EE: Nalox one decreases the duration of primary apnea with neonatal asphyxia. Pediatr Res 1980; 14:357— 359. Chernick V. Craig RG: Naloxone reverses neona tal depression caused by fetal asphyxia. Science 1982:216:1252-1253.
