neonatal period

Volume 101 Number 3

Clinical and laboratory observations

REFERENCES 1. Welsh JW, and May JT: Breast milk and infant infection, Med J Aust 2:66, 1979. 2. Hayes K, Danks DM, Gibas J, and Jack I: Cytomegalovirus in human milk, N Engl J Med 287:177, 1972. 3 Stagno S, Reynolds DW, Pass RF, and Alford CA: Breast milk and the risk of cytomegalovirus infection, N Engl J Med 302:1073, 1980. 4. Yeager AS, Grument FC, Hafleigh EB, Arvin AM, Bradley JS, and Prober CG: Prevention of transfusion-acquired cytomegalovirus infections in newborn infants, J PEDIATR98:281, 1981. 5. Ballard RA, Drew L, Hufnagle KG, and Riedel PA: Acquired cytomegalovirus infection in preterm infants, Am J Dis Child 133:482, 1979. 6. Barness LA, Dallman PR, Anderson H, Collipp P J, Nichols

7.

8.

9.

10.

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BL Jr, Walker WA, and Woodruff CW: Human milk banking, Pediatrics 65:854, 1980. Liebhaber M, Lewiston N J, Asquith MT, Olds-Arroyo L, and Sunshine P: Alterations of lymphocytes and of antibody content of human milk after processing, J PEDIATR91:897, 1977. Bj6rksten B, Burman LG, De Chateau P, Fredrikzon B, Gothefors L, and Herneli O: Collecting and banking human milk: to heat or not to heat? Br Med J 281:765, 1980. Welsh JK, Arsenakis M, Coelen R J, and May JT: Effect of antiviral lipids, heat, and freezing on the activity of viruses in human milk, J Infect Dis 140:322, 1979. Stagno S, Pass RF, Reynolds DW, Moore MA, Nahmias A J, and Alford CA: Comparative study of diagnostic procedures for congenital cytomegalovirus infection, Pediatrics 65:251, 1980.

Human ~-endorphin-like immunoreactivity in the perinatal/neonatat period Immanuela R. Moss, M.D., Ph.D.,* Helen Conner, B.A., William F. H. Yee, M.D., Paola Iorio, M.D., and Emile M. Scarpelli, M.D., Ph.D., Bronx, N.Y.

BECAUSE of our long-standing interest in the relationship between endorphins and respiratory control, ~3 we measured ~-endorphin-like immunoreactivity in maternalfetal-neonatal compartments and compared it to that in adult subjects. We report that maternal plasma E L I is elevated during pregnancy and is highest at labor and delivery; E L I is also elevated in umbilical blood. Most importantly, we show that EL1 is elevated in the early neonatal period and declines to adult values after the first few days of postnatal life.

METHODS Subjects. Venous blood was drawn from five normal adult men and four nonpregnant women, six pregnant women not in labor (eight samples during 19 to 37 weeks' gestation), and seven pregnant women, who were both at term and in labor. Umbilical venous (n -- 8) and arterial blood ( n - - 8 ) , as well as amniotic fluid ( n - - 4 ) were

From the Pediatric Pulmonary Division, Albert Einstein College o f Medicine. Supported by research grants from the NHLBL HL 23995 and HL 07060, National Institutes o f Health. Dr. Moss is a recipient of a Research Career Development Award from the NHLBI, National Institutes of Health. *Reprint address." Pediatric Pulmonary Division, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.

0022-3476/82/090443+04500.40/0 9 1982 The C, V. Mosby Co.

collected at delivery. In addition, 43 venous or arterial samples were drawn from 18 infants (203 to 294 gestational days at birth) from < 1 to 24 days of age (Table). The infants had no disturbances of respiratory control. Abbreviations used ELI: ~3-endorphin-like immunoreactivity /3h-EP: human ~3-endorphin

Informed consent was obtained for blood sampling. In the infants and women in labor, E L I was measured in samples obtained for routine screening tests or for medical reasons. Umbilical blood was drawn from the placenta in utero immediately following cord ligation and section. Sample preparation and radioimmunoassay. Samples were collected into chilled, heparinized, silicone-coated tubes, stored on ice for about 30 minutes, and centrifuged. Cell-free plasma and amniotic fluid were collected into polypropylene vials and stored at - 1 0 ~ for subsequent assay. Thus, samples were thawed only once. ELI was measured using a human/~-endorphin kit (New England Nuclear Corp., Boston) in accordance with the suggested protocol. W e used unextracted samples because Wilkes et al 4 report that similar values are obtained both in extracted and unextracted material and that excessive extractions and separations of endorphins result in significant decrease of measured ELI.

444

Clinical and laboratory observations

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The Journal of Pediatrics September 1982

A

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G= Gravida, 19-37 wks, gestation TL= Term; labor UV= Umbilical Vein T UA=Umbilical Artery r - ' - 1 AF = Amniotic Fluid [ [ **= N0. subjects [ [ --

*=N~

I

Cgd

, ,(9)

I

B

I-~

G

TL

UV

UA

AF

(6)

(7)

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o~ ~

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(7) (12) (4) (18) (8)
H

H

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Figure. Endorphin-like immunoreactivity in control subjects and in maternal-fetal-neonatal compartments. Each bar represents the mean + SEM of ELI in a group of samples. The number of samples included in each group is shown inside the bar and the number of subjects below the bar. Statistical comparison between groups of samples was made by group analysis, because it was not always possible to obtain samples from all compartments during a single pregnancy and delivery. Abbreviations and numerical results of ELI (pg/ml, ~ _+ SEM) are as follows: A, C d o = control adult male and nonpregnant female subjects (232 _+ 10; C d = 228 _+ 16 [n = 5]; C o = 237 _+ 12 [n = 4]); G = 19- to 37-week pregnant women, not in labor (285 _+ 19); TL = women at term and in labor (350 _ 19); UV = umbilical vein (299 _+ 11); UA = umbilical artery (264 -+ 21); AF = amniotic fluid (126 _+ 19). B, d Q, < 1 to 1 days, < 1 to 4 days, 5 to 24 days, H = healthy male and female infants during the first day of life (301 _+ 21), the first 4 (294 _+ 15), and 5 through 24 days of life (214 _+ 15). The last two bars include values from ill (1) infants with a variety of disease entities (see Table) excluding disturbances of respiratory control (< 1 to 4 days: 284 + 13; 5 to 24 days: 243 + 14).

Samples and standards of 100 ul were incubated at 4 to 6 ~ for 16 to 18 hours with 100 ul antiserum containing antibodies against human/3-endorphin and 0.01 # C i / 1 0 0 #1 [1~5I] /3h-EP. After incubation, unbound [1251] /3h-EP and /3h-EP were separated from /3h-EP bound to the antibody by centrifugation with charcoal. The supernatants containing bound flh-EP were surveyed for 125I activity in a Beckman Biogamma II gamma counter. Standard curves were constructed with 5, 10, 15, 25, 35, 50, 125, and 500 pg standard/3h-EP/100 #1 buffer. Each was processed and counted in duplicate, and two separate standard curves were constructed. Samples were also processed in duplicate. The results presented in this report are based on a single fl-endorphin kit. W e evaluated the reproducibility of the standard curve in our hands against that provided by N e w England Nuclear Corp. and found that the two are virtually superimposable. Intra-assay variability was 11%. The range of sensitivity of this assay is 5-500 pg of endorphin per 100 #l sample. Cross-reactivity of the antiserum with a-endorphin, leu-enkephalin, metenkephalin, and a - M S H is nil, but that with/3-1ipotropin is 50%. Thus, our results include both /3-endorphin and fl-lipotropin.

RESULTS There was no difference in E L I between adult males and non-pregnant females. During 19 to 37 weeks of gestation, maternal plasma E L I was higher when compared with adult control values ( P < 0.05, Student t. test). This increase, however, was not progressive with advancing gestation during the time span studied, as determined by regression analysis. Labor at term was associated with further elevation of EL1 in maternal plasma, which was higher than in controls ( P < 0 . 0 0 1 ) and in pregnant women not in labor (P < 0.05). Umbilical venous and arterial EL1 were similar, and both were lower than ELI of maternal plasma during labor ( P < 0 . 0 5 ; P < 0 . 0 0 1 , respectively). Umbilical venous (but not arterial) E L I was higher than in controls (P < 0.001). The lowest E L I was obtained from amniotie fluid (Figure). The E L I of healthy neonates during the first four days of life was not different from that of cord blood and was significantly higher than control E L I (P < 0.01). However, from the fifth to the twenty-fourth day, the measured ELI was lower (P < 0.001) as compared with the first four days of life and comparable to values of adult control subjects. When results from ill infants, in whom respiratory control

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Clinical and laboratory observations

445

Table. Description of infants included in this study

No.

infants

Sex

12

10 M 2F 4M 2F

6

Gestational age at birth (days)

Presence of labor

238-294

12

203-294

3

Mode of delivery

Clinical condition

Sample span: Days of life

No. of blood samples

8 4 3 3

Healthy

< 1 through 24

26

II1"

< 1 through 20

17

Vaginal C/S Vaginal C/S

*Sepsis; pneumothorax; aspiration; respiratory distress syndrome, but no documented periodic breathing, apnea or other disturbances of respiratory regulation. M = male; F = female; C / S = cesarean section.

was clinically normal, were included with the results from healthy infants, the pattern described and the range of values were unchanged (the differences in ELI in all infants between the first four and five to 25 days of life was significant, P < 0.05). There was no correlation between Apgar scores (1 and 5 minutes) and ELI at age 1 to 4 days in healthy or ill infants. We also found no correlation between the infants' postconceptional age at birth and ELI during the first day of life. DISCUSSION In this study we have shown that levels of ELI in neonates of various postconceptional ages at birth who had no documented disturbances of respiratory regulation was increased during the first four days of postnatal life, then declined to control (adult) levels. We chose to compare our measured neonatal ELI to values in maternal-fetal compartments and control adult subjects, because those had been reported previously and summarized recently? Measurements of/3-endorphin vary greatly depending on methods. Because we performed our measurements in raw plasma we avoided the losses that can be associated with extraction of endorphins and separation of ~-endorphin from /3-1ipotropin.4 Because of the 50% cross-reactivity between the two peptides it is not surprising that our control adult values are high. However, when we take into account the cross-reactivity in our assay, the apparently stable molar ratio between/3-endorphin and/3-1ipotropin,6 and the recovery rate of added peptides after extraction and chromatography, 6 our re-calculated/3-endorphin levels fall within the range of those measured following extraction and chromatography. 6 Moreover, our control values are similar to those obtained by others who used our method. 7 The reliability of our studies is further enhanced by our use of the same kit for all measurements. Although our findings of increased ELI in maternal plasma during 19 to 37 weeks gestation is supported by Akil et al s (also in unextracted samples), Goland et aP found only an apparent, but statistically insignificant, increase above control values during the first, second, and

third trimesters. It is possible that this difference is caused by the preassay peptide extraction and chromatography of Goland et al,6 which lowers the measured endorphin values significantly.6 We could not detect a progressive increase in ELI over the gestational period under study, though Gintzler9 reported gradual increase of endorphin-related threshold to pain during pregnancy in rats. The highest ELI that we measured was found in the plasma of women who were both at term and in labor. Similar results have been reported by other investigators.46 The increase of endorphins during pregnancy and their "surge" during labor have been ascribed both to placental production I~and to maternal stress and pain during labor. H The design of the present study does not permit definition of maternal versus fetal contribution to the increased ELI in cord and neonatal blood. We may, however, conclude from the following evidence that the neonates themselves are capable of producing the elevated endorphin levels at birth and of sustaining these high levels during the first days of postnatal life: (1) /3-endorphin concentration is high in fetal brain~Z; (2) any transferred endorphin would disappear within several hours of birth because of its high turnoverS3; and (3) there is no correlation between maternal plasma and cord/3-endorphin values. 6 It is conceivable that the increased ELI that we have found in early neonatal life may influence respiratory control in the perinatal period, when such mechanisms are most labile. We thank the Division of Neonatology, the Delivery Room staff, and the volunteer blood donors at Albert Einstein College of Medicine and Bronx Municipal Hospital Center for making this study possible. REFERENCES

1. Moss IR, and Friedman E:/3-Endorphin: effects on respiratory regulation, Life Sci 23:1271, 1978. 2. Moss IR, and Scarpelli EM: /%endorphincentral depression of respiration and circulation, J Appl Physiol 50:1011, 1981. 3. Moss IR, and Scarpelli EM: Generation and regulation of

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5.

6.

7.

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Clinical and laboratory observations

breathing in utero: fetal COs response test, J Appl Physiol 47:527, 1979. Wilkes MM, Stewart RD, Bruni JF, Quigley ME, Yen SSC, Ling N, and Chr6tien M: A specific homologous radioimmunoassay for human 3-endorphin: Direct measurement in biological fluids, J Clin Endocrinol Metab 50:309, 1980. Kimball CD, Chang CM, Huang SM, and Houck JC: Immunoreactive endorphin peptides and prolactin in umbilical vein and maternal blood, Am J Obstet Gynecol 140:157, 1981. Goland RS, Wardlaw SL, Stark RI, and Frantz AG: Human plasma 3-endorphin during pregnancy, labor, and delivery, J Clin Endocrinol Metab 52:74, 1981. Riseh SC, Cohen RM, Janowsky DS, Kalin NH, and Murphy DL: Mood and behavioral effects of physostigmine on humans are accompanied by elevations in plasma 3-endorphin and cortisol, Science 209:1545, 1980. Akil H, Watson S J, Barchas JD, and Li CH: 3-endorphin immunoreactivity in rat and human blood: Radioimmunoas-

The Journal of Pediatrics September 1982

9. 10.

11. 12.

13.

say, comparative levels and physiological alterations, Life Sci 24:1659, 1979. Gintzler AR: Endorphin-mediated increases in pain threshold during pregnancy, Science 210:193, 1980. Liotta AS, and Krieger DT: In vitro biosynthesis and comparative posttranslational processing of immunoreactive precursor corticotropin/3-endorphin by human placental and pituitary cells, Endocrinology 106:1504, 1980. Terenius L: Endorphins and pain, Front Horm Res 8:162, 1981. Bayon A, Shoemaker W J, Bloom FE, Mauss A, and Guillemin R: Perinatal development of the endorphin- and enkephalin-containing systems in the rat brain, Brain Res 179:93, 1979. Foley KM, Kourides IA, Inturrisi CE, Kaiko RG, Zaroulis CG, Posner JB, Houde RW, and Li CH: 3EP: analgesic and hormonal effect in humans, Proc Natl Acad Sci USA 78:5377, 1979.

Macroscopic hemoglobinuria in a neonate with massive blood aspiration O. S. Saia, M.D.,* and G. Gasparotto, M.D., Padua, Italy

THE PURPOSE of this report is to present the clinical course of a newborn i n f a n t with massive blood aspiration at birth who, during the first hours of life, developed transient macroscopic hemoglobinuria. Electrophoresis of the urine d e m o n s t r a t e d adult hemoglobin from m a t e r n a l blood.

CASE REPORT This female infant was delivered by cesarean section because of abruptio placentae after a 38-week gestation in a primigravida, 23 years of age. The blood type of the mother was A Rh negative. The baby weighed 3,300 gin, and had blood type A Rh positive; the direct Co0mbs test was negative. Apgar scores were 2 and 5 at one and five minutes, respectively. She had massive blood aspiration, and required tracheal intubation and lavage with saline, suction, and manual ventilation with FIO2 of 0.4. She received 5 mEq of NaHCO3 and 0.5 gm of dextrose in the umbilical vein, then was transferred to the neonatal intensive care unit of the University of Padua and treated with IMV and PEEP. At admission the baby was cyanotic. The chest examination revealed diffuse fine rales. A

From the Department of Pediatrics and Clinical Medicine, Division of lmmunopathology, University of Padua, Medical School. *Reprint address: Department of Pediatrics. The University of Padua, School of Medicine, Via Giustiniani, 3, 1--35100 Padova, Italy.

radiograph of the chest demonstrated a bilateral homogeneous density. The patient had her umbilical artery cannulated; the blood gas analysis at one hour of life showed pH 7.35, Pac% 34.8 torr, Pao 2 40 torr while the baby was receiving IMV (FIo2 of 0.6). Abbreviations used FIO2: fraction of oxygen in inspired gases IMV: intermittent mandatory ventilation PEEP: positive end expiratory pressure Paco2: arterial CO2 tension, in torr Pao2: arterial 02 tension, in tort BP: blood pressure Hct: hematocrit Hgb: hemoglobin The blood pressure was 90 mm Hg by Doppler. The baby had fresh blood in the stomach and her meeonium was frankly bloody. At the fourth hour the baby passed urine which was dark red-brown and clear. There were no red blood cells on microscopic examination but the urine tests were positive for hemoglobin and slightly positive for protein and glucose. At the tenth hour the patient required muscle relaxation (pancuronium bromide) and controlled ventilation with FIo2 of 1.00 because of worsening of the blood gas analysis. The gross hemoglobinuria persisted. The BP was then 140 mm Hg. Two hours later the baby had metabolic acidosis (pH 7.14, Paco2 30 torr, Pao2 62 torr, HCO3 9 mEq/L, total CO2 9.5 mEq/L, base deficit 17.6), urea 22 mg/dl, creatinine 1.3 mg/dl, Na 143, K 2.8,

0022-3476/82/090446+02500.20/0 @ 1982 The C. V. Mosby Co.