Br HeartJ 1994;72:452-456

Effect of intracardiac repair on biosynthesis ofthromboxane A2 and prostacyclin in children witha left to right shunt

Ian Adatia, Susan E Barrow, Paula D Stratton, James M Ritter, Sheila G Haworth

AbstractObjective-To investigate the effect ofintracardiac repair on the abnormalbiosynthesis of prostacyclin (PGI,) andthromboxane A, (TXA,) in children withcongenital heart disease and increasedpulmonary blood flow.Design-A prospective study withimmunoaffinity chromatography and gaschromatography-mass spectrometry tomeasure the urinary excretion productsof PGI, (2,3-dinor-6-oxo-prostaglandin(PG) F,8 (2,3-dinor-6-oxo-PGF,.)) andTXA, (2,3-dinor-TXB,) before operation,in the first 12-24 h after operation, and atdischarge from hospital.Setting-A supraregional referral centrefor patients with congenital heart disease.Patients-15 patients aged 2 to 60 months(median 7 months) with a left to rightshunt who underwent intracardiacrepair.Results-The preoperative 2,3-dinor-TXB, excretion rate was greater than thatfound previously in a control group of 16healthy children with a median (range)age of 24 (6-36) months (1159(201) v592(122) nglg creatinine in controls, P =0.006). The excretion rate rose after oper-ation to 9600(3832) nglg creatinine (P =0'01) and decreased before discharge to1071(191) ng/g creatinine (NS), butremained greater than that of the controlgroup (P = 0-014). Before operation 2,3-dinor-6-oxo-PGF,. excretion rates weresimilar to those of the healthy children(482(68) v 589(95) ng/g creatinine in con-trols) but increased after operation to19 668(11 162) nglcreatinine (P = 0-002)and fell at discharge to 1621(245) nglgcreatinine although this was higher thanboth preoperative and control rates (P =0-005 and P = 0-0002 respectively). Thepreoperative ratio of 2,3-dinor-TXB, to2,3-dinor-6-oxo-PGF1. excretion wasgreater than that of the control group(3.2(0.8) v 1.3(0-22) in controls, (P =0.005)), decreased significantly afteroperation to 0.9(0.13) (P = 0-016), andchanged little, to 0-7(0-12), before dis-charge. The last two ratios were similarto those in normal children and signifi-cantly lower than those before operation(P = 0.004).Conclusion-In children with a left toright shunt the ratio of the excretionrates ofthe metabolites ofTXA, and PGI,was abnormal before operation, which

favoured vasoconstriction and plateletaggregation, but had decreased at dis-charge from hospital. The increase inexcretion of PGI, metabolites over TXA,metabolite after intracardiac repairaugurs well for pulmonary vascularrecovery.

(Br Heart J 1994;72:452-456)

The pulmonary circulation is exposed to ahigh shear stress from birth in children withcongenital heart disease and a left to rightshunt. Structural abnormalities are known tobe present by 2 months of age but endothelialdysfunction precedes morphological dam-age. 1-3 Recent studies have shown that themetabolism of several vasoactive mediators isabnormal in children with pulmonary hyper-tensive congenital heart disease.45 An imbal-ance in eicosanoid biosynthesis with an excessof thromboxane A2 (TXA2) over prostacyclin(PGI,) may occur,4 as in pulmonary hyperten-sion of other aetiologies.6 The vasoconstrictorTXA2 is a proaggregatory substance whereasPGI2 acts as a physiological antagonist toTXA2, relaxing vascular smooth muscle andinhibiting platelet aggregation.7-9 Thus beforeoperation, children with pulmonary hyperten-sive congenital heart disease have an imbal-ance in favour of vasoconstriction and plateletaggregation.

Cardiopulmonary bypass damages the pul-monary endothelium in all patients and thosewith pulmonary hypertension are particularlyvulnerable.'10-14 Pulmonary hypertension mightbe mediated by an increase in a vasoconstrictoror decrease in a vasodilator substance. Thecirculating concentration of the vasoactivesubstance derived from the endothelium,endothelin, is high in such children,5 but therelease of nitric oxide in response to acetyl-choline may be impaired.'5 We hypothesisedthat if the abnormal ratio of metabolites ofTXA2: PGI2 that may be present in childrenwith an increased pulmonary blood flowreverted to normal after surgical closure of theshunt this should reduce the dominant effectof TXA2 after the operation and might pro-vide evidence for concomitant endothelial cellrecovery. Persistent abnormalities ofeicosanoid biosynthesis might indicate resid-ual haemodynamic disturbance. Therefore weinvestigated the biosynthesis of TXA2 andPGI2 in a group of children with a left to rightshunt and pulmonary hypertension before,during, and after intracardiac repair.

Vascular Biology andPharmacology Unit,Institute of ChildHealth, LondonI AdatiaS G HaworthDepartment ofClinicalPharmacology, UnitedMedical and DentalSchools ofGuy's andSt Thomas'sHospitals, LondonS E BarrowP D StrattonJ M RitterCorrespondence to:Professor S G Haworth,Division of Physiology andPharmacology, Institute ofChild Health, 30 GuildfordStreet, LondonWC1N 1EH.Accepted for publication1 June 1994

452

EffectLof intracardiac repair on biosynthesis of thromboxane A2 and prostacyclin in children with a left to right shunt

Both PGI2 and TXA, have short half livesin the circulation and hydrolyse sponta-neously to 6-oxo-prostaglandin (PG) F,,, (6-oxo-PGF,,) and TXB,. Previous investigatorshave reported transient increases in plasmaconcentrations of both eicosanoids.'6 17 Bloodsampling may cause local endothelial traumaand platelet activation that can result in arti-factual increases of both eicosanoids in bloodsamples.1820 A non-invasive assessment of thebiosynthesis of PGI2 and TXA2 can be madeby measuring eicosanoid metabolites in urine.Excretion of 6-oxo-PGF,,, and TXB2 largelyreflect renal biosynthesis of eicosanoids2" 22

whereas 2,3-dinor-6-oxo-PGF,a and 2,3-dinor-TXB2 reflect biosynthesis of PGI2 andTXA2 outside the kidney.2324 We measuredthe urinary excretion of these 2,3-dinormetabolites before operation during the first12-24 hours after the operation, and immedi-ately before discharge from hospital in a group

of pulmonary hypertensive children with a leftto right shunt. These children were youngerthan those previously studied, which reflectsthe trend towards earlier intracardiac repair.'6 17

Patients and methodsPATIENTSThe 12 hour urinary excretion of 2,3-dinor-6-oxo-PGFI,, and 2,3-dinor-TXB2 was mea-

sured in 15 children aged 2-60 (median 7)months (table). Preoperative and dischargeurine samples were collected overnight, as

were the control samples. Twelve of thesechildren were studied before operation, 12between 12 and 24 hours after operation, and11 immediately before discharge at 6-26(median 12) days after operation. Only sixchildren could be studied serially. No childhad renal insufficiency, before or after opera-

tion. The parents of all patients gave informedconsent.

Before the operation, all the children had a

left to right shunt and a chest radiographshowing cardiomegaly and pulmonaryplethora. Fourteen children were treated withfrusemide and digoxin. Also 10 children

received spironolactone and two received cap-topril. Most of the children underwent intra-cardiac repair after a cross sectionalechocardiographic and Doppler assessment,without preoperative cardiac catheterisation(table). Of these cases, eight had a reliablenon-invasive estimation of right ventricularpressure that varied between 45 and 90 (mean61) mm Hg. The pulmonary arterial pressurewas high in all catheterised patients.Estimation of pulmonary artery pressure was

made within the week before surgery. Surgerywas entirely successful in 12 children butthree had a difficult postoperative course. Onechild had severe pulmonary hypertensivecrises and two had residual left to right shuntsand remained in heart failure. One of theserequired reoperation. All 15 patients sur-

vived.

METHODSAnalysis of eicosanoid metabolitesUrinary excretion rates of 2,3-dinor-6-oxo-PGF,0 and 2,3-dinor-TXB, were measuredwith immunoaffinity chromatography andgas chromatography-mass spectrometry as

described previously.25 Briefly, a well mixedsample of 30-50 ml was stored at -20°C untilassayed. Urine samples (10 ml) were diluted1:1 by volume with buffer at pH 8-0 andinternal standards of 2H4 2,3-dinor-TXB2 and2H4 2,3-dinor-6-oxo-PGFIa (5 ng each) were

added. Eicosanoids were extracted withcyanogen bromide activated sepharosecolumns containing immobilised antibodiesthat had been raised against 6-oxo-PGF,aand TXB2 and that cross reacted with theirrespective 2,3-dinor metabolites. Urine sam-

ples were applied under vacuum to thecolumns, which were washed with water (10ml). Eicosanoids were eluted by addition of0-5 ml acetone:water (95:5) and rotation ofthe columns for 15 min. Samples were takento dryness (N2 stream) and were derivatised as

3,5-bis-trifluoromethylbenzyl esters andtrimethylsilyl ethers. They were analysed with a

VG 70-SEQ gas chromatograph-mass spec-trometer in the electron capture mode with

Clinical and eicosanoid data

Intensive care unit 12-24 hBefore operation after operation Discharge

Preoperative DN- DN- DN- DN- DN- DN-Age PAp TXB2 6-oxo TXB2 6-oxo TXB2 6-oxo

Patient (months) Sex Diagnosis or RVp (ng/creat) (nglcreat) Ratio (ng/g creat) (nglg creat) Ratio (ng/g creat) (ng/g creat) Ratio

1 2 F VSD 70 2285 736 3-10 12720 11776 1-08 - - -2 3 F AVSD Tri 50 931 88 10-58 1326 842 1-58 2247 1968 1-143 3 M AVSD Tri - 1110 609 1-82 37182 103428 0-36 904 1295 0 704 3 F VSD 60 2606 640 4 07 7814 10593 0-74 1342 2333 0-585 4 F AVSD Tri 50 - - - 1743 2019 0-86 690 2661 0-266 7 M VSD 45 1146 219 5-24 2930 3211 0-91 - - -7 14 F AVSD 49/15 734 605 1-21 - - - - - -8 18 F VSD 73/18 - - - 14705 38052 0 39 1002 711 1-419 24 M AVSD - 376 515 0-73 7161 6353 1-13 764 1277 0-6010 30 F VSD - 1132 410 2-76 - - - 1562 2395 0-6511 36 M ASD - 504 350 1-44 822 738 1 11 826 1209 0-6812 60 F VSD 47 398 412 0-97 - - - 298 736 0 4013 4 M DORV 90 1504 922 1-63 5744 9545 0-60 1807 465 3-89 R14 4 M VSD 80 - - - 61404 60531 1-01 1124 440 2-56 R15 10 M AVSD Tri 74/24 1183 283 4-18 2172 541 4-01 - - PHCControl values 592 (122) 589 (95) 1-3 (0 22)

AVSD, atrioventricular septal defect; creat, creatinine; DN-TXB,, 2, 3-dinor-TXB,; DN-6-oxo, 2, 3-dinor-6-oxo-PGF,,0 DORV, double outlet right ventricle;PHC, pulmonary hypertensive crises; PAp, pulmonary arterial pressure; PDA, patent ductus arteriosus; R, residual VSD; RVp, right ventricular systolic pressure;Tri, trisomy 21; VSD, ventricular septal defect.

453

Adatia, Barrow, Stratton, Ritter, Haworth

methane as reagent gas. Carboxylate anions atmass:charge (m:z) ratio 557 were monitoredfor 2,3-dinor-6-oxo-PGFia and 2,3-dinor-TXB, and at m:z 561 for the deuterated inter-nal standards. The detection limit for eacheicosanoid was 5 pg/ml. Urinary creatinineconcentrations were measured with standardbiochemical methods. No patient took aspirinor a related substance and all urine sampleswere found to be free of salicyluric acid.26

ANALYSIS OF DATAThe results were expressed as mean (SEM) ngeicosanoid/g urinary creatinine to allow forchanges in renal function and urine volume.All data were transformed logarithmically toobtain normally distributed data before analy-sis. Urinary eicosanoid excretion rates werecompared with those found previously in acontrol group of 16 normal children aged6-36 (median 24) months with the unpairedStudent's t test.4 Differences were consideredsignificant at P < 0 05. For comparison ofpreoperative, intensive care unit (ICU), anddischarge values, only paired urine samplesfrom the same patients were studied. A oneway analysis of variance (ANOVA) forrepeated measures was used to detect differ-ences in the excretion rates of eicosanoidsbefore operation, on the intensive care unit,and at discharge. When significant differenceswere found (P < 0-05) the Bonferroni proce-dure for multiple comparisons was used totest for pairwise differences. These were con-sidered significant at P < 0-017. In the threechildren who had a complicated postoperativecourse eicosanoid biosynthesis was consideredto be different from the group without postop-erative complications if their values fell aboveor below the upper and lower 95% confidenceinterval (95% CI) of that group.

ResultsThe mean preoperative excretion rate of 2,3-dinor-TXB2 was 1159(201) ng/g creatinine,significantly higher than the excretion rate of592(122) ng/g creatinine in the healthy con-trol group (P = 0 006). Excluding the threechildren who had a complicated postoperativecourse, the rate of 2,3-dinor-TXB2 excretionincreased from the preoperative rate of1159(697) to 9600(3832) ng/g creatinine inthe intensive care unit (P = 0-014), and was1071(191) ng/g creatinine immediately beforedischarge P = 0 035 (NS). Its rate of excre-tion on discharge from hospital was not signif-icantly different from the preoperative rate,but was significantly greater than in the con-trol group (P = 00 14).

Before operation, the mean urinary excre-tion rate of 2,3-dinor-6-oxo-PGFi, was482(68) ng/g creatinine, similar to the controlgroup (589(95) ng/g creatinine). At 12-24hours after operation, in the intensive careunit, the rate rose to 19 668(11 162) ng/g cre-atinine (P = 0.002), and before discharge, ithad fallen to 1621(245) ng/g creatinine. Thisvalue was higher than both the preoperativebaseline values (P = 0 005) and the control

group (P = 0 0002). The ratio of 2,3-dinor-TXB2: 2,3-dinor-6-oxo-PGF,a excretionbefore operation was 3-2(0 8), compared witha control ratio of 1-3(0-22) (P = 0 005). At12-24 hours after operation, the ratiodecreased to 0-9(0 13) (P = 0-016). This ratiowas similar at the time of discharge(0-7(0- 12)) and less than the preoperativevalue (P = 0 004). The ratio at discharge wasnot significantly different from the controlvalue.

Concerning the three children who had acomplicated postoperative course, in the twowho had a residual left to right shunt, theexcretion of 2,3-dinor-6-oxo-PGFia at thetime of discharge was below the lower 95% CI(440 and 465 compared with upper and lower95% CIs of 1056 and 2185 ng/g creatinine)for the rest of the patients, although excretionof the 2,3-dinor-TXB2 was similar. The ratiosof 2,3-dinor-TXB2: 2,3-dinor-6-oxo-PGFj, atdischarge were above the 95% CI of the otherpatients (2-6 and 3*9 compared with lowerand upper 95% CIs of 0-4-1-0). One of thesechildren had a postoperative peak right ven-tricular pressure of 50 mm Hg and a pul-monary:systemic flow ratio of 2:1. Hesubsequently underwent a second and suc-cessful intracardiac repair. On that occasionthe excretion of the 2,3-dinor-6-oxo-PGFia atthe time of discharge was greater than thatseen in any other child and the ratio was 0 5.The child who had postoperative pul-

monary hypertensive crises had a lower excre-tion rate of 2,3-dinor-6-oxo-PGFia at 12-24hours after surgery, during the crises, than anyother child in the group. During the time thechild was ill in the intensive care unit theeicosanoid metabolite excretion ratio was 4 0,well above the 95% CI (06-1 0) for the post-operative group of patients with an uncompli-cated course.

DiscussionBefore operation, the 12 hour excretion rateof 2,3-dinor-TXB2 was high whereas that of2,3-dinor-6-oxo-PGFia was at the lower limitof the control group. This significantlyincreased the ratio. These findings are consis-tent with those in our previous study in whichwe found high 2,3-dinor-TXB, excretion inchildren with congenital heart disease and ahigh pulmonary blood flow, compared withthe same control group.4 In the present study,after operation excretion of both eicosanoidmetabolites was noticeably high for longerthan has previously been reported.'617 Otherinvestigators reported an increase in theplasma concentration of 6-oxo-PGF,, andTXB, in children during cardiopulmonarybypass that quickly returned to the preopera-tive concentration in the recovery room.'6 17 Inpatients undergoing palliative cardiac surgerywithout cardiopulmonary bypass the plasmaconcentration of TXB2 did not changewhereas that of 6-oxo-PGF,a increased,'7 pos-sibly attributable to intravascular manipula-tion. In our study, the pronounced increase inboth 2,3-dinor-TXB, and 2,3-dinor-6-oxo-

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Effect of intracardiac repair on biosynthesis of thromboxane A2 and prostacyclin in children with a left to right shunt

PGF,a immediately after operation may like-wise reflect intravascular manipulation. The

increase in 2,3-dinor-TXB2 after operationmight be regarded as an exaggeration of thepreoperative state, as cardiopulmonary bypassis known to activate platelets and damage thepulmonary endothelium, which is alreadyinjured in pulmonary hypertensive childrenbefore bypass.'2 1127 The circulating concen-

tration of another vasoactive substance,endothelin, also increases immediately aftercardiopulmonary bypass although unlikeTXA2, the circulating concentration of thismediator is normal before operation.28Cardiopulmonary bypass damages allendothelia, and the proportion of TXA2 andendothelin produced by the systemic and pul-monary vascular beds is unknown.Irrespective of source, both TXA2 andendothelin might be expected to have a

greater effect on the more reactive damagedpulmonary vascular bed of these children.'2The immediate postoperative increase inPGI2, a vasodilator and antiaggregatory sub-stance, might be expected to cause an increasein blood loss.'4 Prostacyclin does not inhibitplatelet adhesion to damaged blood vessels atconcentrations that prevent platelet aggrega-tion and its therapeutic use has been free ofbleeding complications, although not systemichypotension.29-31 The function of PGI2released locally at sites of incision may there-fore be to prevent the thrombus extendingfrom the sites of injury and haemostasis. Itssystemic action may help to oppose the exces-

sive vasoconstriction and the hypercoagulablestate associated with operation and trauma.

It has been suggested that because of theopposing effects of TXA2 and PGI2 on bloodvessels and platelets, the ratio of biosynthesisofTXA2 to PGI2 may have greater physiologi-cal relevance than changes in the biosynthesisof either eicosanoid alone.820 The ratio isabnormal in several different vascular dis-eases.6 20 23 32 33 In our study, the ratio of excre-

tion of 2,3-dinor-TXB2: 2,3-dinor-6-oxo-PGFia was similar to the control group

between 12 and 24 hours after cardiopul-monary bypass on account of the increase in

PGI2. One exceptional case had pulmonaryhypertensive crises and in this child theeicosanoid metabolite excretion ratio wasabove the upper 95% CI of the other children.These findings raise the possibility that theunopposed action of TXA2 may contribute tothe pathogenesis of pulmonary hypertensivecrises, and might help to explain why an infu-sion of PGI2 is generally more helpful in treat-ing crises than other intravenousvasodilators.34 Thromboxane receptor antago-nism prevents pulmonary hypertension andimproves right ventricular function after com-plement induced pulmonary hypertension indogs and may prove to be a useful adjunct in

the treatment ofpulmonary hypertension aftercardiopulmonary bypass in humans.35 Othervasoactive mediators, such as endothelin, maycontribute to postoperative pulmonary vascularreactivity, and nitric oxide release may beimpaired.5 '5 Nitric oxide has been shown to

be an effective pulmonary vasodilator aftersurgery for congenital heart disease in chil-dren."5 Nevertheless, in our study the rise inthe 2,3-dinor-TXB2: 2,3-dinor-6-oxo-PGF,aexcretion ratio during a hypertensive crisissuggests an aetiological role for this vasocon-strictor in pulmonary hypertensive crises.By the time the patients were discharged

from hospital, at a median of 12 days afteroperation, the excretion rate of 2,3-dinor-TXB2 was still high. Whether this indicatespersistent platelet dysfunction after cardiopul-monary bypass or chronic endothelial injury isunknown. The excretion rate of 2,3-dinor-6-oxo-PGF,a in those who had undergone a suc-cessful repair was greater than in the controlgroup. The mechanism of this change is notunderstood, although surgery is known tostimulate PGI2 production. 14 Excretion of2,3-dinor-6-oxo-PGFia was low, however,after operation in the two patients with a largeresidual ventricular septal defect. Para-doxically, in vitro an increase in shear stressincreases the release of PGI2.'6 Also, for thosepatients with a low rate of 2,3-dinor-6-oxo-PGFia excretion, it was not possible in ourstudy to discriminate between the direct effectof the increased pulmonary blood flow afteroperation and its probable consequences, leftatrial hypertension, and cardiac dysfunctionafter bypass.The postoperative increase in excretion of

2,3-dinor-6-oxo-PGF,a after a successfulrepair meant that when the patients were dis-charged from hospital the ratio of excretion ofthe metabolites was similar to that in the con-trol group. A previous study has shown thatone year after a successful repair the excretionrates of TXB2 and PGFia are normal, indi-cating recovery of endothelial function.4Endothelial dependent relaxation in responseto acetylcholine is preserved in young childrenwith an increase in pulmonary blood flow, butis impaired in older patients with fixed pul-monary vascular disease.'5 37 Progressiveendothelial dysfunction may be evident as animbalance in eicosanoid biosynthesis thatmight contribute to the pathogenesis of pul-monary vascular disease. Although the num-bers in our study are small, the return tonormal of the ratio soon after intracardiacrepair indicates rapid restoration of the bal-ance between vasoconstrictor and dilatormediators and augurs well for the reversibilityof the structural abnormalities.

We thank Professor A I Mallet (Director, UMDS MassSpectrometry Unit, St Thomas's Campus) for use of massspectrometry facilities, Dr R Ersser for salicyluric acid analysis,and the nursing staff of Richard Bonham Carter Ward, GreatOrmond Street Hospital for help with the urine collections.The authors also thank Dr K Gavreau ScD of the HarvardSchool of Public Health for statistical advice. This work wassupported by the British Heart Foundation.

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