Plasma 6-keto prostaglandin F1α and thromboxane B2 in sick preterm neonates

Prostaglandins Leukotrienes and Medicine 18: 163-181, 1985

PLASMA C-KETO PROSTAGLANDIN F1 AND THROHBOXANE B2 IN SICK PRETEM NEONATES Alastair A. Hutchison,* Martin L. Ogletree,** Charlotte J. H. Palme* Bruno P. Leheup,* Jeffrey M. Barrett,*** Arthur C. Fleischer,**** Mildred T. Stahlman,* Kenneth L. Brigham** Division of Neonatoloqy, Department of Pediatrics,* Division of Neonatology, University of Florida, College of Medicine, Department of Pediatrics, Box J-296 JHMHC, Gainesville, FL 32610, Pulmonary Circulation Center, Department of Medicine,** Division of Maternal/Fetal Medicine, Department of Obstetrics and Gynecology,*** Ultrasound Section, Department of Radiology and Radiological Sciences,**** School of Medicine, Vanderbilt University, Nashville, TN 37232 (reprint requests to AAH) To determine if vascular abnormalities in preterm neoABSTRACT: nates might be related to vasoactive prostaglandins, stable and prostacyclin (6-KPGF o) thromboxane A2 (TxB 1 metabolites in arteriat blood were measured at < 6 hours a3ter birth and at 24, 48, and 72 hours using a rZdioimmunoassay. Neonates of ~32 weeks gestation (N=26) were diagnosed as having either the idiopathic respiratory distress syndrome (IRDS, N=15) or pulmonary edema (PE, N=ll), and were also grouped according to the presence or absence of intracranial hemorrhage (ICH, N=ll) or patent ductus arteriosus (PDA, ~=io). Initial plasma 6-KPGFl, was greater in neonates with ICH (0.23 + 0.04 ng/ml, mean + SE) than without ICH (0.11 + 0.04, p <--0.05). Neonates with both ICH and IRDS (N=8)-had significantly elevated TxB2 at all sampling times compared to neonates with IRDS and no ICH (N=7). Both TxB2 and 6-KPGFl, increased with time in those with major ICH. Among neonates without ICH, 7 with IRDS had higher initial 6-KPGFl, (0.19 + 0.07 ng/ml) and lower TxB2 (0.15 + 0.04 ng/ml) than 8%ith PE (0.04 + 0.01 and 0.37 + O.Oq ng/ml, respectively). The initial 6-KPGFl, (0.02c + 0.003 ng/ml), measured in neonates with PE and without PDA or ICH (N=6), was significantly less than the corresponding value in the other neonates (0.201 + 0.036 ng/ ml) (N=20).

163

Prostacyclin (PGI2) and thromboxane A (TxA2) are among the most potent naturally occurring vasoactive su2bstances Cl]. The vasoconstrictive action of TxA2 has been linked to vasospasm in the cerebral and coronary circulations [2,3]. Prostacyclin, a vasodilator, may antagonize these and other actions of TxA2. Prostaglandins may play a role in certain physiological and pathological perinatal events. In man, raised plasma concentrations have been noted in maternal and neonatal plasma during term and preterm labor [41. In sheep and goats the onset of ventilation with air at birth has been associated with the pulmonary production of the pulmonary vasodilator PGI [5]. In the fetal and neonatal lamb, PG12 has been reported to 21ower pulmonary vascular resistance [61, whereas prenatal administration of inhibitors of prostaglandin synthesis has been associated with the development of raised pulmonary vascular resistance [73. However, the physiological influence of PGI may primarily affect the gradual, pulmonary vasodilation of the earz y postnatal period, since inhibitors of prostaglandin synthesis attenuate only this phase rather than the pronounced pulmonary vasodilation which accompanies the onset of ventilation [8]. Prostaglandin synthesis inhibitor studies suggest a role for prostaglandins in the control of fetal lung maturation E91, tracheal surfactant flux [lo], fetal breathing [ill and in the maintenance of patency of the fetal ductus arteriosus C7,121. Raised plasma prostaglandin concentrations have been noted in association with symptomatic patent ductus arteriosus (PDA) [13], and the idiopathic respiratory distress syndrome (IRDS) [14,15,161. Prostaglandin synthesis inhibitors are used to contrict the symptomatic patent ductus arteriosus, whereas prostaglandin E is employed to prolong ductal patency in certain forms of cyanotic congenital heart disease [17l. Prostacyclin has been used in the treatment of neonatal persistent pulmonary hypertension C181. Since neonatal platelets demonstrate increased release of arachidonic acid [19], and since hypoxia and ischemia have been reported to stimulate prostaglandin and thromboxane synthesis [3,201, insults such as perinatal asphyxia might promote their release and cosequently produce circulatory derangements. This might be one explanation of the common association of asphyxia and intracranial hemorrhage (ICH), IRDS, and PDA. It was hypothesized that vasoactive prostaglandins might be involved in the pathogenesis of intracranial, respiratory, and vascular problems of the preterm neonate. The object of this study was, therefore, to determine whether ICH, IRDS, and PDA were associated with consistent alterations in the plasma concentrations of 6-KPGFl, and TxB2 in preterm neonates.

164

METHODS

Inclusion

Criteria

Inborn preterm neonates with a gestational age of thirty two weeks None of their mothers had or less were included in the study. pregnancy-induced hypertension, nor had been induced with syntocinon, and none had received salicylate, indomethacin or corticosteroid therapy prior to delivery. All of the study patients had an arterial catheter in situ. Prior to inclusion in the study parental consent was obtained. The study was approved by the committee for the Protection of Human Subjects-Vanderbilt University Institutional Review. Gestational age was determined from maternal dates and gestational age assessment using the Dubowitz score. If a discrepancy between the maternal dates and the gestational age assessment score exceeded two The weeks then the score was recorded as the true gestational age. birthweights of the newborn infants were determined to be appropriate, small or large for gestational age. Diagnosis and Definition of Terms Intracranial hemorrhage was diagnosed on the basis of ultra sound and/or postmortem examinations. Intracranial hemorrhage was classified as follows [21]: Type 1.

SEH

subependymal hemorrhage only.

Type 2.

SEH/IVH-

SEH associated with a minor intraventricular hemorrhage (IVH).

Type 3.

IVH-

major intraventricular hemorrhage occupying greater than 50% of the ventricular lumen.

Type 4.

IPH-

intraparenchymal hemorrhage with or without porencephaly or hemorrhagic leukomalacia.

Of the above, types 1 and 2 were considered to be minor and types 3 and 4 to be major ICH. Idiopathic Respiratory Distress Syndrome (IRDS) was defined by the following criteria C221: a.

Retraction and diminished breath sounds during spontaneous respiratory effort.

b.

FI02 > 0.3 to maintain a Pa02 > 60 Torr beginning during the first 24 hours of life and lasting at least 60 hours with continuous positive airway pressure (CPAP) or 72 hours without CPAP.

165

C.

A maximum FI02 requirement

d.

Chest x-ray findings of diffuse, finely granular or reticular infiltrates persisting at least until the third day of life.

e.

Respiratory

Pulmonary

of > 0.4.

distress not attributable

to other causes.

edema (PE) was defined by the following criteria:

a.

Tachypnea and retractions with or without diminished breath sounds during spontaneous respiratory effort in the first 24 hours of life.

b.

A progressive decrease in the requirement for intermittent mandatory ventilation (IMV), the FIO being < 0.25 by 48 hours of life and the majority of af Pected neonates not requiring IMV by that postnatal age. An exception to this course may be seen in infants whose improvement is slowed or ceased as a result of the appearance of a left to right shunt, secondary to a PDA.

C.

Chest x-ray findings on the first day of life of clear lung fields, or diffuse interstitial edema, or radiological findings interpreted as alveolar flooding which progressively clear during the first 48 hours of life.

d.

Respiratory

distress not attributable

to other causes.

Symptomatic patent ductus arteriosus was diagnosed on the basis of previously published criteria [22], with echocardiographic evidence of left atria1 and left ventricular dilatation to support the diagnosis of a left to right shunt.

Collection and Assay of 6-KPGFl, and TxB2 A one milliliter blood sample was obtained at a mean (+ SE) of 2.4 + 0.2 hours (range 0.7 to 6.0 hours) after birth (time 7) hours) and again at 24, 48 and 72 hours postnatally. All blood samples were collected from an indwelling arterial catheter, the vast majority from a non-heparinized umbilical arterial catheter, located in the aorta at the level of the fourth lumbar vertebra, and occasionally from a radial If the catheter was removed, no further samples were artery catheter. The blood was drawn into an indomethacin coated syringe, collected. and then directly transferred to a cooled plastic tube containing EDTA which was immediately placed in ice. Within 15 minutes, the sample was centrifuged at 3,000 rpm for 20 minutes in a refrigerated (4°C) Beckman The plasma was separated and stored at Model 568 centrifuge C231. -20°C. Rabbit anti-TxB2 and anti-6-KPGFI, antibodies were obtained from Dr. J. Bryan Smith of the Cardeza Foundation, Philadelphia. had a less than 1% cross reaction with The anti-TxB2 antibodies and a less than 3% cross reaction PGF2o and 6-KPGF a PGE2, antibodies had a less than 1% The anti- k -KPGFI, with PGD2. cross reaction with PGE2, PGD2 and TxB2 and a less than 3% cross

166

Authentic prostaglandins and TxB2 were reaction with PGF2,. generously supplied by Dr. John Pike of the Upjohn Company, Kalamazoo, Michigan. Radiolabeled (5,6,8,9,11,12, 14,15-3H)TxB2 and (5,8,9, were purchased from New England Nucle11,12,14,15-3H)-6-KPGF The radiolabeled ligand, about 20,000 ar, Boston, Massachus!?ts. c.p.m. per tube, was first mixed with bovine gammaglobulin (Sigma 10 mg/ml in Trizma pH 7.4). To 100 microliter (mcl) aliquots of this mixture were added 100 mcl ali,quotsof sample or unlabeled standard dilutions. The binding reaction was initiated by the addition of 100 mcl of antibody diluted to produce 60% binding or radiolabeled ligand in the absence of unlabeled ligand. After a 60 minute period of incubation at 37"C, the binding reaction was terminated by precipitation of the immune complexes with ammonium sulfate at a final concentration of 50% of saturation. After centrifugation, at 2,500g at 4°C for 10 minutes, the radioactivity of 300 mcl of the supernatant was counted in aquasol (New England Nuclear, Boston, MA). Each sample was assayed in triplicate. The intra-assay variability was less than 10% and the inter-assay variability was between 12-14%. The detection limit of both assays was 20 picograms per milliter. Statistical Analysis The differences in the 6-KPGFI, and TxB2 concentrations measured at the different times, in the presence or absence of ICH, IRDS, and PDA, were analyzed using the two-tailed Mann Whitney rank test. After appropriate corrections were applied for tied ranks, a normal approximation of the Mann Whitney Test was calculated. A probability of < 0.05 was taken as significant. Plasma 6-KPGFI, and TxB2 concentrations at time D were correlated to perinatal factors by calculating the Spearman's rank correlation coefficients (rs). Differences between the concentrations of 6-KPGFI, and TxB2 at time 0 in neonates delivered vaginally or by cesarean section were analyzed using the two-tailed Mann Whitney rank test. The 72 hour 6-KPGFI, and TxB2 concentrations, measured in one neonate who received indomethacin at 55 hours, were not included in that analysis. The factors presented in the tables were analyzed by the two-tailed Mann Whitney rank test and Fisher's exact test. All data were expressed as mean + standard error of the mean. RESULTS Study Population Twenty-six inborn preterm neonates were studied. Their mean gestational age was 29.2 + 0.4 weeks (range 25 - 32 weeks) and their mean birthweight was 1176 F65 grams (range 560 - 1760 grams). There were 14 males and 12 femaTes. Twenty-five of the 26 neonates had birthweights appropriate for gestational age, and 1 was small for gestational age. There were 5 black and 21 Caucasian neonates. The mean Apgar scores, at 1 and 5 minutes respectively, were 4 + 0.4 and 6 t 0.3. The mean admission temperature was 36.4 + O.l"C and ?he mean admission mean blood pressure was 35 -t 2 nnnHg. -

167

TABLE I Comparison of perinatal factors in study neonates with and without intracranial hemorrhage. FACTOR

INTRACRANIAL HEMORRHAGE

NO INTRACRANIAL HEMORRHAGE

11

15

7:4

7:8

Gestational age (wks)

28.6 + 0.8

29.6 2 0.5

Birthweight (g)

1141 + 114

1202 -+ 78

Birthweight: Gestational Age

11 AGA *

14 AGA 1 SGA**

5

0+

Number Male:Female

Died

11

14

Vaginal Delivery

6

7

Cesarean Section

5

8

Premature Labor

Apgar 1'

3 + 0.6

4 -+ 0.5

Apgar 5'

6 -+ 0.3

7 -+ 0.5

36.4 + 0.2

36.5 + 0.1

9.1 + 0.2

8.3 -+ 0.2

45 -+ 4

39 -+ 3

Admission Temperature ("C) Admission Mean Airway Pressure (cmH20) Admission P,CO2 . . Admission Pa02

78 -+ 10

143 -+ 26

7.23 -+ 0.04

7.26 + 0.03

Admission Mean Blood Pressure (mn Hg)

34 -+ 3

35 -+ 3

Admission Hematocrit

50 -+ 12

46 + 2

172 -+ 11

194 -+ 10

IRDS

8

7

PE

3

8

PDA

3

7

Admission pH

Admission Platelets (xlOS/mnS)

* Appropriate for gestational age ** Small for gestational age No significant differences were seen, except for mortality? (p < O-05). 168

A comparison of perinatal factors in those neonates with and without ICH, is shown in Table I. Prostaglandin Concentratfons a.

Perinatal

The plasma concentration of 6-KPGFI, at time 0 hours in neonates delivered vaginally was 0.139 + 0.033 ng/ml while that of neonates delivered by cesarean section wax 0.181 + 0.052 ng/ml. The values were not significantly different. The plasma concentration of TxB2 at time 0 hours in neonates delivered vaginally was 0.477 + 0.094 ng/ml and was significantly greater than that of neonates delivered by cesarean section which was 0.272 + 0.092 ng/ml. (p < 0.05). The birthweight of those babies delivered by cesarean section was significantly greater than that of those delivered by the vaginal route (1312 + 749 versus 1041 + 969). There were no other differences between the groups.* The Spearman rank correlation coefficients (rss) for plasma 6-KPGFI, and TxB2 concentrations at time 0 hours and the following factors were not significant ones: birthweight; gestational age; Apgar at 1 minute; admission mean airway pressure; admission mean arterial blood pressure; admission hematocrit; admission pH; admission P co2; admission base excess; admission temperature; admission pPatelet count; cord pH; cord P,CO2; cord P 02; and cord base excess.** Cord blood gas correlations were basec! on samples obtained from seventeen of the study population. Only the Apgar at 5 minutes and the admission Pa02 were significantly correlated to the plasma concentrations, the rss being -0.44 (p=O.O32) and -0.43 ;;E$a, The corresponding TxB2 concentrations were respectively. not significantly correlated. No neonate received a blood transfusion prior to the withdrawal of the first blood sample. In the subsequent three days, seventeen neonates received blood transfusions, fifteen of these on two or three consecutive days. In this latter group, there was no significant correlation between the volume of blood transfusion in the twenty-four hour period prior to the time of blood sampling and the corresponding 6-KPGF Q and TxB plasma concentrations, rs values being 0.21 and 0.!! 1 respective z y. b.

Intracranial Hemorrhage

Compared to newborns without ICH, those with ICH had a significantly higher concentration of 6-KPGFI, at time 0, and a significantly higher concentration of TxB2 at 24 and 48 hours. The differences at zero and 24 hours were significant in those with major or minor ICH. *A table containing the comparisons is available from the author upon request. **The correlation coefficients are available from the author upon request.

169

At 48 hours however only the major ICH group showed signficantly increased concentrations of both 6-KPGFI, and TxB (Figure 1). Intracranial hemorrhage was associated with significantfy elevated concentrations of 6-KPGFI, and TxB2 in neonates with both IRDS (Figure 2) and PE (Figure 3). Among babies with IRDS, initial TxB concentrations were significantly higher in those who developed ICb compared to those who did not. Concentrations of 6-KPGFI, and TxB2 increased with time in neonates with major ICH, but in those with minor ICH, 6-KPGFI, concentrations decreased with time. C.

Comparison of IRDS and PE

When all neonates with IRDS were compared with all those with PE, regardless of whether they had an ICH, no differences in the absolute values of 6-KPGFI, or TxB2 were seen at 0, 24, 48 or 72 hours. However, the ratio of the concentration of 6-KPGF o:TxB2 was greater in neonates with IRDS at 0 and 48 hours (Figure h 1. In neonates without ICH, those with IRDS had initial 6-KPGF1, concentrations significantly greater and initial TxB2 concentrations significantly less than those with PE (Figure 5). The initial ratio of ;;KPGFI,:TxB2 was significantly less than those with PE (Figure The initial ratio of 6-KPGFIp:TxB2 was significantly greater'in those with IRDS. The only significant difference between the two groups was a higher mean admission Pa02 in those with PE.* d.

Uncomplicated PE

Circulating concentrations of 6-KGPF o at 0 hours were very low (0.024 + 0.003 ng/ml) in 6 neonates witt PE who did not develop a PDA or ICH.- Catheters were removed from most of these babies before 24 hour samples could be taken, so further trends could not be establishInitial 6-KGPFI concentrations in the other 20 neonates were ed. significantly higher b.201 -+ 0.036 ng/ml). e.

Patent Ductus Arteriosus

When neonates with PDA in the first postnatal week were compared with those without PDA, the concentration of 6-KPGF o at 48 hours A. At 0 hours was greater in the PDA group than in those without a P!J the ratio of 6-KPGFI,:TxB2 was also significantly greater in the PDA group (Figure 6). Among the neonates without an ICH, a comparison of those with a PDA in the first postnatal week and those without a PDA revealed a significant difference only at 0 hours, when the concentration of 6-KPGFI, and the ratio of the 6-KPGFI,:TxB2 concentrations were greater in the PDA group (Figure 7). There were no other significant differences between the two groups.* Individual neonates showed marked increases in the concentrations of 6-KPGFI, and TxB2 concurrent with the clinical appearance of a symptomatic PDA. After indomethacin therapy it was noted in one neonate that the 6-KPGFI, concentration fell to a minimum whilst that of TxB2 increased. *A table containing the comparisons is available from the author upon request. 170

* T

0

MAJOR ICH

I

TOTAL ICH

09 2

E

08

d

07

:-

06

g

05

\

l-7 5

??

T

:u)

04

m

03

4 0

0.2

N&

*

0.1 0

0

48

24

72

hrs

TIME

0

36 T

3.2

‘D C

2.8

N z c-

MAJOR ICH

i

2.4 2.0

08 04 0

24

48

72

hrs

TIME

F ig.1.

The relationship of plasma 6-KPGFla and TxBp concentrations with ICH during the first three postnatal days. The number of patients in each of the neonatal study groups 1s documented at the top of each bar. A significant difference (p < 0.05) in the absolute prostaglandin concentrations between the groups with ICH and the no ICH group is shown by *.

171

I.0

09

I

0.3

0.

3+

0

0.1

0.2

0 24

:

48

cl ICH

MAJOR

72 tlr*

TOTAL ICH

ICH

MAJOR

??

El

Fig.2: The relationship of 6-KPGFIQ and TxB2 concentrations with ICH during the first three postnatal days in neonates with IRDS. The number of patients in each of the neonatal study groups is documented at the top of each bar. A significant difference (p < 0.05) in the absolute prostaglandin concentrations between the groups with ICH and the no ICH group is shown by *.

c

ZO L?

E

O’

-a 04

:: s

D

5

$ OS

^

0

0

8

8

k

iI 3

0

3

24

3-48

ICH

MINOR

72hrs

?N ?&

I

Fig.3: The relationship of plasma 6-KPGFI, and lXT2 concentrations with ICH during the first three postnatal days in neonates with PE. The number of patients in each of the neonatal study groups is documented at the top of each bar. A significant difference (p < 0.05) in the absolute prostaglandin concentrations between the group with ICH and the no ICH groups is shown by *.

i:

s 20'

g 0.2 (D

0 LA-

3

1I

CO3ie

z‘D

04

05

I.

I.

0

cfl

‘0

II

T

7’ T

?? PE

Fig.4. The relationship of plasma 6-KPGFI, and lXfB Concentrations with IRDS and PE during the first three postnatal days in all of the neonates studied. A significant difference (p < 0.05) in the ratio of the plasma 6-KPGFI :TxBB concentrations between the IRDS and P!! groups is shown by t.

2

0.4

1 0.

0.

+ .

15

t 1

I.6

a? ‘. x 0.8

S

:

^

1.8

%i

.

0

01

0.3-

O4-

L7

*t

0

B

The relationship of plasma 6-KPGFI, and concentrations with IRDS and PE during the first three postnatal days in neonates without A significant difference (p < 0.05) in ICH. the ratio of the plasma 6-KPGFI,:TxBz concentrations between the IRDS and PE groups is shown by t. A significant difference (p < 0.05) in the absolute prostaglandin concentrations between the IRDS and PE groups is shown by *.

p’

Y9%

D lc B

2

= E

Y

174

0

, 24 TIME (hrs)

48

72

k

0.6 .

0

1

0.1 .

0.2 .

0.3 .

0.4 .

0.5.

) 0

24 TIME

(hrs)

40

72

Figs.8 and 9. The relationship of plasma 6-KPGFla and TxB2 concentrations with clinical events and ultrasound examination results during the first three postnatal days The ultrasound results are highlighted thus: 1-1 in three neonates with ICH.

0

SEIZURES

0.8.

%

0.7 .

1.0. 0.9.

S

1.2. I.1 .

1.3.

1.4.

1.5.

1.6.

1.7.

$ t

= E . P -

1.8.

1.9.

2.0

3.0

4.0

5.0

6.0

Several factors should be considered before discussing the possible significance of plasma concentrations of 6-KPGFI, and TxB2: 1) These compounds are chemically stable products possessing little known biological activity at physiologic concentrations. 21 Although the parent compounds are potent vasoactive agents with diverse and opposing biological actions, they are unstable in aqueous solution and appear to have short circulating half lives in vivo. 3) It is not known how long 6circulate before they are excreted or metabolized KPGFI, and TxB to imnunogenicalfy inactive products. 4) Of the total measured plasma metabolite concentration, the contribution, if any, of the active parent compound in the blood at the time of sampling is also unknown. 5) Increases in concentrations of metabolite in plasma could reflect an increased rate of biosynthesis of the parent compound or a decreased rate of catabolism or excretion. With these considerations in mind, it is clear that the measurements reported here do not necessarily reflect local concentrations of PG12 and TxA2, nor their rates of biosynthesis. However, for the purposes of this discussion, it will generally be assumed that the increased circulating metabolite concentrations resulted from an increased rate of synthesis of the parent compound, although the site of biosynthesis remains unknown. Radioimnunoassay measurements of circulating 6-KPGFI, have been criticized as giving unreasonably high Since. in this study the lower limit for 6-KPGFI, convalues [241. centrations was 20 pg/ml, true values falling below this detection limit were overestimated. Nonetheless, in the control population consisting of those neonates with uncomplicated pulmonary edema, the mean arterial plasma 6-KPGFl, concentration (24 + 3 pg/ml) was less than that obtained in adult human venous plasma after extraction (233 + 42 pg/ml) or silicic acid column chromatography (60 + 20 pg/ml) and closQto the value obtained following reverse phase high-performance liquid chromatography (4.7 t 3.2 pg/mll C241. The most striking finding in this study was the association of increased plasma concentrations of 6-KPGFl, and TxB2 with ICH. If the ICH was major, the concentrations of both metabolltes increased with time. If the ICH was minor, 6-KPGFI, concentrations decreased with time and TxB2 concentrations did not rise above the values measured at 24 hours. In the only neonate who survived a major ICH, the concentrations of 6-KPGFIo and TxB2 were declining by 72 hours. The increased concentrations of prostacyclin and thromboxane metabolites in plasma from neonates developing demonstrable ICH could contribute to and/or result from cerebrovascular injury. Recent evidence in the beagle puppy model of ICH suggests a causative role for prostaglandins in the genesis of ICH. Prior treatment of the puppies with indomethacin preventIn the ed the occurrence of ICH in response to vascular stress [25]. present study, it was possible to document the clinical courses, results of serial ultrasound measurements, and measurements of 6-KPGFI, and In at TxB2 Concentrations in three neonates who died after a major ICH. least one case, a steep rise in 6-KPGFI, and TxB2 Preceded the appearance of the ICH (Figures 8 and 9).

176

Thromboxane A2 is a potent cerebral vasoconstrictor [Z]. On the other hand, PG12 opposes the known actions of TxA$, and PG121 may be synthesized by intact vascular endothelium in assoc ation with ocal It is therefore possible that release of PG12 TxA2 release [261. during ischemia could cause an inappropriate increase of perfusion in regions with fragile or injured blood vessels, thus precipitating ICH. In the neonatal lamb hypoxia is known to stimulate prostacyclfn synthesis by the lungs [27]. The significant negative correlations of the it$iaP;06-KPGFIa concentrations and Apgar at 5 minutes and admissuggests that this sequence of events could occur perinatally in 2human neonates. Although it is known that the diagnosis of ICH cannot be made reliably from clinical signs, it is possible that the "classical" signs associated with an ICH including shock, apnea, seizures, and peripheral vasoconstriction may be related to raised plasma concentrations of vasoactive prostaglandins. Vasodilator prostaglandins given therapeutically can be associated with apnea and seizures [173. In the rat the peripheral administration of PGE2 Potentfates the cerebral effects of hypoxia, in that seizures occur earlier and are of greater intensity [281. Neonates with symptomatic PDA have raised prostaglandin concentrations [13], and can present clinically with decreased peripheral perfusion, mimicking the vascular collapse seen in association with ICH. Thromboxane A2 may be the most potent vasoconstrictive metabolite among the cyclooxygenase products. Increasing plasma concentrations of vasoactive prostaglandins prior to the occurrence of an ICH could explain the observation noted in our nursery that neonates with “classical" signs of a catastrophic ICH may not have an ICH demonstrable by ultrasound examination at the time when the clinical signs present. Thus the "classical" signs of a catastrophic ICH may precede the hemorrhage and represent a result of ischemia. The ICH itself may appear in association with the re-establishment of perfusion following such an ischemic insult. Among neonates without ICH (Figure 51, initial plasma concentrations of the PGI2 metabolite were significantly lower in those with PE than in those with IRDS. In contrast, initial TxB2 concentrations were higher in those with PE than in those with IRDS. Consequently, the initial ratio of 6-KPGFIo:TxB2 was significantly greater in those with IRDS than in those with PE. Plasma C-KPGFi! concentrations moderated over the first 24 hours only in those w h IRE, and plasma TxB2 concentrations moderated over the first 24 hours only in those with PE. These findings suggest that IRDS may be associated with an initial increase in PG12 sy nthesis in the perinatal period, whereas PE may be associated with an initial increase in TxA2 synthesis. K&pa and co-workers also found significantly elevated plasma 6-KPGFIa concentrations during the first postnatal day in babies with IRDS [16]. In human neonates with IRDS, and in the lamb model of IRDS, lower than normal pulmonary artery pressures and large left to right shunts can occur C291. Rudolph et al. concluded that a "generalized lack of vasoconstrictor tone could possibly be responsible for systemic and pulmonary arterial hypotension as well as for the widely patent ductus 177

arteriosus" [29]. In neonates with IRDS, the observed combination of an increased vasodilator PG12 influence and a decreased vasoconstrictor TxA2 infuence may thus contribute to the vascular changes characteristic of the condition. However, early or preterminal vasoconstriction, both pulmonary and peripheral, may be seen in neonates with severe IRDS. It is interesting to note that the initial TxB2 concentration was high in those infants with severe IRDS which was complicated by a later ICH (Figure 2). The association of high initial TxB2 concentrations with later ICH, in neonates with IRDS, suggests that perinatal events which influence TxA2 concentrations might be related to the etiology of ICH. Of those perinatal factors analysed, the only factor significantly associated with the initial TxB2 concentrations was the mode of delivery. Although of lesser birthweight, neonates delivered vaginally had significantly higher initial TxB2 concentrations when compared to those delivered by cesarean section. In neonates with IRDS who were delivered vaginally, six of nine had an ICH, in contrast to two of six who were delivered by cesarean section. However, the incidence of severe ICH in neonates with IRDS was approximately the same in the two groups, four of nine in vaginally delivered versus two of six in cesarean section delivered neonates. The clinical picture in IRDS may vary according to the relative influences of vasodilator and vasoconstrictor prostaglandin metabolites, due to their actions upon the pulmonary, cerebral, and peripheral vasculature. Involvement of prostaglandins and related compounds in the closure of the ductus arteriosus and in the development of the PDA is supported by numerous animal studies Cl21 and by the common efficacy of cyclooxygenase inhibitors in the closure of the PDA 17,131. The relevant finding in the current study is that the initial concentration of the vasodilator metabolite, and the ratio of that metabolite relative to vasoconstrictor metabolite (i.e., 6-KPGFI :TxB2) that of the were significantly greater in neonates who developed a PDA &an in those who did not. This data can be interpreted in at least two ways. 1) PDA may be associated with high concentrations of 6-KPGFI, (Figure 71, the PG12 metabolite known to be the principle product of the ductus arteriosus of the fetal sheep 1301. Exogenous prostaglandin ;;bs;,s ;;;e p;ge;e;han. PGI2 in dilating the isolated ductus arte. ln the isolated fetal lamb ductus arteriosus PG12 synthase is the 'primary enzymatic-pathway for the metabolism of exogenous prostaglandin H2, and no pathway for intramural biosyntheProstacyclin does dilate the sis of PGE2 has been identified [31]. ductus arteriosus C7,12], and inhibition of prostacyclin synthetase by high oxygen tension within the ductus has been proposed as a mechanism Since the impact of humoral PGE2, PG12, for ductal closure C321. and TXA2 on ductal patency has been stressed [31], it was of interest that the initial plasma concentration of 6-KPGFI, showed a signifi2) Alternatively, the prescant negative correlation to the P,O2. ence of relatively high concentrations of vasodilator seen in babies who developed circulatory complications may require sufficient concentrations of a vasoconstrictor, such as TxA2, for closure of the 178

ductus arteriosus. In neonates with IRDS and no ICH and those who developed a PDA, TxB2 concentrations were consistently low at 0 hours and remained low for at least two days post-partum. However in the case of the ductus arteriosus the influence of TxA is most likely to be an indirect one since, at least in the lamb, it Eas no action on the ductus arteriosus L-71. The possibility exists that insufficient synthesis of a vasoconstrictor may contribute to the development of a PDA in the human. CONCLUSION This study has demonstrated associations between plasma 6KPGFl, and TxB2 concentrations and the occurrence of ICH, IROS, and PDA in preterm neonates with a gestational age of 32 weeks or less. The results of the study support the view that fetal development and perinatal events may alter prostaglandin and thromboxane synthesis, and the subsequent changes in vascular tone may contribute to the pathogenesis of vascular abnormalities which occur commonly in the preterm neonate. ACKNOWLEDGEMENTS The authors wish to thank Drs. Cotton, Sundell, Shenai, and Rojas and the staff of the Vanderbilt University Neonatal Intensive Care Unit, for considerable aid in the organization of this study, Howard B. Johnston, M.S., for his assistance in the use of the CLINFLO data management and analysis system, Ms. G.A. King for her assistance with the radioimnunoassay, and Ms. S.J. Crooms, Ms. J. Arnold and Ms. 0. Kater for secretarial assistance. REFERENCES 1.

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