Changes in plasma fibronectin in children after elective repair of congenital heart defects

Changes in plasma fibronectin in children after elective repair of congenital heart defects Plasma fibronectin is an attachment protein important for maintaining capillary integrity and host defense mechanisms. Depletion of plasma fibronectin has been' shown to occur in adults after septic shock, major trauma, and burns. Limited laboratory and clinical studies suggest a correlation between decreased plasma fibronectin levels and increased pulmonary capillary permeability and tissue perfusion. Mild and transient plasma fibronectin depletion has been observed in adults after cardiovascular operations. We measured plasma fibronectin by immunoturbidometric assay in 20 children (age 6 months to 12 years) undergoing repair of congenital heart defects. Plasma fibronectin levels immediately after operations and daily thereafter were compared with the preoperative values. Plasma fibronectin declined on postoperative days 1, 2, 3, 4, and 5 (p < 0.05). A nadir was reached on day 3 with a tendency toward recovery thereafter. Patients with a therapeutic intervention score of more than 35 had greater magnitude of plasma fibronectin decline than those with a score of less than 35 at 24 hours after the operation (p < 0.005). We conclude that (1) significant and prolonged plasma fibronectin depletion occurs after cardiovascular operations in children; and (2) postoperative plasma fibronectin depletion is associated with increasingly complex surgical intervention. Reduced plasma fibronectin synthesis and more extensive operations for congenital heart defects are likely reasons for children being more susceptible than adults to plasma fibronectin depletion after cardiovascular operations. (J THORAC CARDIOVASC SURG 1993;105:31-6)

Richard Hackbarth, MD, Ashok P. Sarnaik, MD, Kathleen Meert, MD, Devendra R. Deshmukh, PhD, and Eduardo Arciniegas, MD, Detroit, Mich.

Plasma fibronectin (PFn) is a 450,OOO-dalton protein that plays an important role in maintaining capillary endothelial integrity and host defense mechanisms.l" Depletion of PFn levels has been shown to occur in adults after septic shock.>? major trauma, or burn injury.r!" Several investigators demonstrated PFn depletion in infants and children with bacterial sepsis. 11, 12 Limited studies in animals and adults suggest a correlation between decreased PFn levels and alterations in lung capillary permeability and tissue perfusion. 13-17 Children undergoing cardiac operations for repair of their congenFrom the Children's Hospital of Michigan, Wayne State University School of Medicine, Detroit, Mich. Presented at the Annual Meeting of the Society for Pedia tric Research, New Orleans, La., April 1991. Received for publication Aug. 22, 1991. Accepted for publication Feb. 24,1992. Address for reprints: Ashok P. Sarnaik, MD, Director, Critical Care Medicine, Children's Hospital of Michigan, 3901 Beaubien, Detroit, MI 4820 I.

12/1/38579 0022-5223/93/$1.00/+ 0.10

ital heart defects demonstrate marked changes in capillary permeability.l'' Alterations in PFn levels, if they occur in these patients, could contribute to abnormalities in pulmonary function, tissue perfusion, and host defense mechanisms. Postoperative changes in PFn have not been described in this group of patients.

Materials and methods Children 6 months of age or older who were admitted for electivecardiac operations for congenital heart lesionsduring a I-year study periodwereconsidered for enrollment in this study. The study protocol was approved by the institutional review board. Informed consent was obtained from the parents of the children. Assent was also obtained from the children when appropriate. Children were excluded from the study if they had a coexistent disease known to affect PFn levels. Patients were also excluded if their nutritional status was significantly compromised (weight <3rd percentile for age), because proteincalorie malnutrition also affects PFn. 19 One patient with sickle cell disease and four patients with poor nutritional status were thus excluded. Two additional patients were excluded after institution of extracorporeal membrane oxygenation postoperatively, because systemic heparinization interferes with the accuracy of PFn rneasurements.P In all, 20 patients were

31

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The Journal of Thoracic and Cardiovascular Surgery

Hackbarth et at.

Table I. Patient characteristics Patient No. I

2 3 4 5 6 7 8 9 10 II

12 13 14 15 16 17 18 19 20

Age (yr)

2.2 2.5 1.2

2.5 1.2 0.6 3 6 12 7 5 5 1.5 3.8 3 0.7 4.5 0.6 1.2

0.5

Sex

Lesion

Repair

TlSS score

M F F F M M M F F M M M F M M M F M F M

VSD ASDLVOTO VSD ASDPAPVR TOF AV canal ASD ASD ASD PAPVR LVOTO DORVDTGA TOF ASD Single ventricle ASDVSD Tricuspid atresia DORVVSD AV canal VSD

Closure VSD Repair ASD LVOTO Closure VSD Repair ASD PAPVR TOF repair AV canal repair Closure ASD Closure ASD Closure ASD Repair PAPVR Relief LVOTO Fontan TOF repair Closure ASD Fontan Closure ASD VSD Fontan Conduit VSD to aorta AV canal repair Closure VSD

33 49 40 32 42 45 25 27

27 34 34 43 49 26 50 50 43 48 55 54

ASD, Atrial septal defect; AV, atrioventricular; DORV, double-outlet right ventricle; DTGA. dextrotransposition of great arteries; LVOTO. left ventricular outflow tract obstruction; PAPVR. partial anomalous pulmonary venous return; TOF, tetralogy of Fallot; VSD, ventricular septal defect.

enrolled and studied for the duration of their intensive care unit

ucu: stay (Table I).

Blood samples for PFn determination were obtained from venous catheters before thoracotomy, immediately after surgery, and at 24-hour intervals thereafter while the patients were in the ICU. The samples were collected into calcium ethylenediaminetetraacetic acid containing plastic tubes and centrifuged at 10,700 g for 3 minutes at room temperature. The plasma was then decanted off and mixed with 0.05 ml of bovine lung aprotonin, a trypsin inhibitor (Sigma Chemicals A6279), to impede proteolysis. Samples were then immediately frozen at -70 0 C and assayed by the immunoturbidometric method as described by Saba and coworkers.P All samples were assayed within 7 days of collection. No significant changes in PFn levelsoccurred due to storage less than 10 days. Arterial lactic acid, serum albumin, and venous and arterial blood gases were also measured at the same time intervals. Intrapulmonary shunt fraction, arterial venous oxygen difference, and alveolar-arterial oxygen difference were calculated from blood gas data. The amount of blood and blood products used to prime the cardiopulmonary bypass circuit and to replace the blood loss was noted intraoperatively and on subsequent days. Therapeutic intervention scoring system (TISS) scores" were computed postoperatively for each patient as an objective measure of complexity of the surgical procedure. This scoring system is based on 57 different therapeutic and monitoring techniques employed in critically ill patients. Each of these interventions carry a score from I to 4, depending on the invasiveness of the procedure and the level of intensity of care provided. Higher scores reflect greater degrees of therapeutic intervention. PFn levels at various time intervals after surgery were compared with the preoperative values by one way analysis ofvariance followed by Scheffe's multiple comparison procedure.

Change in PFn at 24 hours from preoperative values was analyzed separately for patients with TISS scores less than 35 and greater than 35 by paired t tests. The magnitude of change in PFn at 24 hours was compared in patients with TISS score less than 35 and greater than 35 by Student's t test. The relationship of cardiopulmonary bypass time to the change in PFn levels between preoperative and postoperative samples was analyzed by Pearson's product moment correlation.

Results The children ranged in age from 6 months to 12 years, with a mean age of 3.2 years. TISS scores averaged 41 ± 10. Twelve patients were male and eight female. Of the total amount of blood and blood products required for these patients, 64% ± 18% was used to prime the cardiopulmonary bypass circuit and to replace the intraoperative blood loss. Mean preoperative PFn levels were 267 ± 11 Jlg/ml (mean ± standard error of the mean), well within the range of normal values previously described for this age group.F There was no significant difference in PFn levelsbetween male and female patients (p > 0.4). PFn levels declined on postoperative days 1, 2, 3, 4, and 5 (p < 0.05) (Fig. I). A nadir was reached on day 3 after operation with tendency toward recovery thereafter (Table 11). However, the PFn levels were still less than preoperative values at the time of dischargefrom the leu (up to 8 postoperative days). There was no significant difference in serum albumin levelsthroughout the study period.

Volume 105 Number 1

Plasma fibronectin changes in children

January 1993

..

300

:i

...E

CIt

3

...J W

> W

33

250

II

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SEM

200

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ow z 0

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II: III

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ii: 2

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8

POST-OPERATIVE DAY

8

10

Fig. 1. Postoperative plasma fibronectin levels. Time zero represents preoperative plasma fibronectin level. Table III. Therapeutic intervention and PFn

Table II. PFn levels during clinical course Time Preoperatively Postoperative day 1

2 3 4

5

6 7 8

PFn (J.Lgfml) 267 ± 223 ± 162 ± 95 ± 84 ± 100 ± 128 ± 124 ± 125 ± 180 ±

11 14 16* 10*

11 *

9* 9* 5

No. ofpatients 20 20 20 13

9 6 4

2 1 1

Values expressed as mean ± standard error of mean. "p < 0.05 by one-way analysis of variance followed by Scheffe's test.

PFn level (J.Lg/ml)

Time Preoperatively Postoperative day 1

2

TISS 267 242 212 210

< 35

± 16 (8) ± 16 (8) ± 20* (8)

± (I)

TISS> 35 267 209 128 86

± 16 (12) ± 21 (12) ± 17t,:!: (12)

± 8 (12)

Valuesexpressed as mean ± standard error of mean; number of patients shown in parentheses. 'Less than preoperative value (p < 0.05). [Less than preoperative value (p < 0.001). :j:Decline from preoperative valuegreater than in patientswith TISS lessthan 35 (p < 0.005).

difference, and arterial lactate levels did not show significance by multiple regression analysis. Patients were divided into two groups by TISS score, those with scores less than 35 and those with greater than 35 to separate patients with less complex repairs from those with more complicated repairs. Serial PFn levels in each patient are shown in Fig. 2. Compared with the preoperative values, PFn levelswere significantly lower at 24 hours in patients with TISS scores less than 35 (p < 0.05) and in those with TISS scores greater than 35 (p < 0.001). However, patients with TISS scores greater than 35 had greater PFn declines than those with TISS scores less than 35 (p < 0.005; Table III). The length of ICU stay was also longer for patients with TISS scores greater than 35 (5.3 ± 2 vs 2.1 ± 0.4 days; p < 0.002 by t test). Cardiopulmonary bypass time did not correlate with postoperative PFn depletion (r = 0.07). Relationships between PFn levels and intrapulmonary shunt fraction, arterial venous oxygen difference, alveolar arterial oxygen

Discussion We have shown that a significant depletion of PFn occurs in children after repair of congenital heart disease. Levels of this protein decreased 40% by the first postoperative day. Depletion continued until the postoperative day 3, when PFn levels were less than one third of the preoperative levels. Until now, only a transient depletion of PFn had been shown in adults after cardiovascular operations.P: 24 In these studies the PFn levels were lowest immediately after completion of the operation with a subsequent rise toward preoperative levelswithin the first 48 hours. Furthermore, the mean PFn levels were observed to be greater than the lower limits of normal during the study period." Our findings differ significantly from these observations regarding the magnitude and duration of PFn depletion. These children continued to show a decline in PFn to subnormal levels, with the max-

34

The Journal of Thoracic and Cardiovascular Surgery

Hackbarth et af.

::I

400

..E. o

2-

...J

W

>

300

---- group A

-group B

W ...J

Z

i=

o

w z oa:

200

lD

u:

-e

100

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~

11.

oL-_...L-_....I...._---l.._--l_ _.l..-_-'-_---'--_--.L_ _' - - _

o

PRE

POST

234

5

6

7

8

DAY

Fig. 2. Serial plasmafibronectin levels in individual patients. PatientswithTISS scores greater than 35 had greater decline in PFn levels on day I compared with patients with TISS scores less than 35 (p < 0.005).

imum depletion occurring on the postoperative day 3. Although there was a subsequent tendency toward recovery, PFn levels continued to be less than postoperative values for up to 8 days after operation. This difference may be accounted for at least in part by the more invasive nature of the surgical repair of congenital heart defects compared with the coronary artery bypass grafting performed in the majority of the patients in the adult studies. Although all patients in our series had a significant depletion of PFn at 24 hours, patients requiring a greater degree of therapeutic intervention (TISS >35) showed a more marked PFn decline. The reasons for PFn depletion after cardiovascular operations are unclear. Possibilities include loss of the protein from the intravascular space caused by changes in vascular permeability, hemodilution, increase in consumption or degradation of the protein, and a decrease in hepatic PFn synthesis. Loss from the vascular compartment is unlikely because other proteins of comparable size such as albumin would also be depleted. Postoperative albumin levelsin the patients in our study did not decrease appreciably from preoperative levels. Degradation of the protein would occur either by mechanical destruction or protease activation by the bypass circuit.!" This also appears unlikely because no correlation between PFn depletion and duration of bypass could be demonstrated. Hemodilution resulting from the administered blood products and colloids could conceivably lower PFn levels. It has been reported that stored homologous blood has normal PFn levels, although PFn may have less biologic activity.P: 26 Administration of blood and blood products may therefore be expected to have little effect on the PFn antigen as measured by immunoturbidometric assay.

Additionally, priming of the cardiopulmonary bypass circuit and replacement of intraoperative blood loss accounted for most of the blood products used. Thus these patients received 64% ± 18% of the total amount of Ifhemodiblood administered before arriving atthe lution was the major reason for low PFn levels in our patients, we would have seen the greatest PFn decline immediately after the operation. Although the immediate postoperative levels were somewhat lower than the preoperative level, this difference was not statistically significant. Hemodilution therefore does not appear to be a major mechanism for depressed PFn levels. The most likely explanation for the depletion of PFn seen in these patients is increased consumption and decreased synthesis. Several mechanisms are likely to be involved. PFn is known to bind to collagen and to other proteins that may be exposed at sites of tissue injury." Consumption of PFn at surgical sites is therefore to be expected. PFn is probably incorporated into the vascular endothelium to preserve endothelial integrity in response to the increased capillary permeability caused by the release of inflammatory mediators associated with surgery and cardiopulmonary bypass.l'' PFn also plays an important role in the stabilization of fibrin clot" and would be expected to be decreased for this reason as well. All these mechanisms may be compounded if production of PFn by the liver is decreased. Recent observations have shown that the hepatic fractional synthetic rate is much slower in infants than in adults. Infants produce 15% of the PFn pool every 24 hours,27 whereas adults have a turnover of 35% during the same period.P These studies suggest that the fibronectin biosynthesis in human beings is a maturational process. Children might therefore be

leu.

Volume 105 Number 1 January 1993

expected to restore the depleted PFn pool at a slower rate than adults. Proteins synthesized by the liver with relatively short half-lives such as PFn 29 would be expected to be affected earlier than proteins with longer half-lives such as albumin. This was indeed the case in our patient population. Abnormalities of fibronectin metabolism could have a great deal of significance in children after cardiovascular operations. Several authors have shown that PFn depletion adversely affects lung capillary permeability in animal models of septic shock and adults with bacterial sepsis. IS, 17,30-32 In an experimental model of postoperative bacteremia Cohler and co-workers-' demonstrated that depletion of PFn was associated with increased pulmonary capillary permeability. Furthermore, treatment with fibronectin was shown to protect the pulmonary capillary integrity. Limited experience in adults has shown improvement in intrapulmonary shunt fraction and dead space ventilation with repletion of the PFn levels in patients with sepsis. We were unable to demonstrate significant correlation between pulmonary shunt fraction and alveolar-arterial oxygen gradient, and PFn. This may be due to the relatively small number of patients remaining in the ICU for longer than 3 days postoperatively. More direct and objective measurements of pulmonary capillary permeability and lung water content, in a larger number of patients, are necessary to determine the pathophysiologic effects of PFn depletion in this clinical setting.

1. 2.

3. 4.

5. 6. 7.

8. 9.

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26. Blumenstock FA, Valeri CR, Saba TM, et al. Progressive loss of fibronectin-mediated opsonic activity in plasma cryoprecipitate with storage. Vox Sang 1988;54:129-37. 27. Polin RA, Yoder MC, DouglasSD, McNelis W, Nissim I, Yudkoff M. Fibronectin turnover in the premature neonate measured with [15N] glycine. Am J Clin Nutr 1989;49: 314-9. 28. Thompson S, Blumenstock FA, Saba TM, et al. Plasma fibronectin synthesis in normal and injured humans as determined by stable isotope incorporation. J Clin Invest 1989;84:1226-35. 29. Deno DC, Lewis EP, Saba TM. Clearance from the vascular compartment of endogenously labelled fibronectin. J Trauma 1984;24:91-8.

The Journal of Thoracic and Cardiovascular Surgery

30. Scovill WA, Annest SJ, Saba TM, et al. Cardiovascular hemodynamics after opsonic alpha-2-surface binding glycoprotein therapy in injured patients. Surgery 1979;86:28493. 31. Saba TM, Blumenstock FA, Shah DM, et al. Reversal of fibronectin and opsonic deficiency in patients: a controlled study. Ann Surg 1984;199:87-96. 32. Saba TM, Jaffe E. Plasma fibronectin (opsonic glycoprotein): its synthesis by vascular endothelial cells and role in cardiopulmonary integrity after trauma as related to reticuloendothelial function. Am J Med 1980;68:577-94. 33. Cohler LF, Saba TM, Lewis E, Vincent PA, Charash WE. Plasma fibronectin therapy and lung protein clearance with bacteremia after surgery. J Appl Physiol 1989;63:623-33.

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