- Email: [email protected]
Necrotizing enterocolitis and total parenteral nutrition-associated cholestasis
APPLIED
NUTRITIONAL
INVESTIGATION
Nutrition Vol. 12, No. 5, 1996
Necrotizing Enterocolitis and Total Parenteral Nutrition-Associated Cholestasis R. LAWRENCE MOSS, MD,* JOHN B. DAS, MB, PHD,t AND JOHN G. RAFFENSPERGER, MDt"
From the *Department of Surgery, University of New Mexico School of Medicine, Albuquerque, New Mexico, and the 7~Department of Surgery, Children's Memorial Hospital, Chicago, Illinois, USA Date accepted: 5 August 1995 ABSTRACT The proportion of patients with total parenteral nutrition (TPN)-associated cholestasis (TPN-AC) who have necrotizing enterocolitis (NEC) has increased markedly in the past ten years. Little is known about how these diseases affect each other. We retrospectively studied 24 patients with NEC and bowel necrosis or perforation who required surgical intervention. Patients were divided into two groups: those who had received TPN (NEC + TPN, n = 17) and those who had not (NEC, n = 7). As cholestasis was present clinically, or prolonged TPN was anticipated, liver biopsy was done. Bile acid levels were measured in both serum and bile in 13 patients. Six patients, who underwent bowel resection and enterostomy, had a second liver biopsy and measurement of bile acid levels at stoma closure. Our results showed that in 13 patients for whom bile acid levels were measured (NEC + TPN, n = 6) (NEC, n = 7), serum bile acid level was significantly elevated in both groups over normal for age. Biliary bile acid levels were correspondingly depressed in both groups suggesting a failure of bile acid transport. All patients had abnormal liver histology, but the pattern of injury differed between the two groups. Those in the NEC group had biliary stasis and mild hepatocyte degeneration. In contrast, 15 of 17 in the NEC + • TPN group had advanced injury specific for TPN-AC. All six patients managed on TPN and partial enteral feeding before a second biopsy had no change in bile acid levels and progression of histologic injury. We conclude that NEC alone can cause functional cholestasis and histologic liver injury but does not cause the specific progressive damage caused by TPN. NEC may make the liver more susceptible to the effects of TPN. Partial enteral feeding does not halt or reverse this injury. Nutrition 1996; 12:340-343 Key words: hyperalimentation, necrotizing enterocolitis, cholestasis, liver disease bile acids
INTRODUCTION In the early 1970s, Peden et al. 1 and Touloukian et al. 2 first noted an association between total parenterai nutrition (TPN) and jaundice. Since then, many retrospective studies have attempted to identify those babies at greatest risk of developing the syndrome of TPN-associated cholestasis ( T P N - A C ) . Periera et al. 3 showed a higher incidence of sepsis in babies with TPN-AC compared with other neonates on TPN. Recent surveys have shown a strong propensity for infants with necrotizing enterocolitis (NEC) to develop TPN cholestasis. In the first reported series of patients with T P N - A C in the 1970s, less than 25% of the patients had NEC. 2"4 By the 1990s, over 75% of patients with T P N - A C had NEC. 5"6 Seventy-eight percent of patients with T P N - A C at Children's Memorial Hospital over the past five years were neonates with NEC. 6 GinnPease et al. 7 found that patients with NEC were more likely to develop jaundice than other patients on TPN. Despite the
association between NEC and TPN-AC, little is known about how these diseases affect each other. W e reviewed our experience with NEC as it relates to liver function and TPN. METHODS W e retrospectively studied 24 patients treated at Children's Memorial Hospital with NEC who required surgical exploration for intestinal perforation or clinical signs of sepsis suggesting intestinal necrosis. As cholestasis was present clinically or prolonged TPN was anticipated, liver biopsy was done in all patients. A sample of gall bladder bile was obtained via aspiration of the fundus with a 22-gauge needle through a small pursestring suture. Bile acid level was measured in both serum and bile in 13 patients. Six patients who underwent bowel resection and enterostomy had a second liver biopsy and measurement of bile acid levels at a subsequent operation for stoma closure. In an earlier report, we detailed the unique sequential histologic
Correspondence to: R. Lawrence Moss, MD, University of New Mexico, School of Medicine, Department of Surgery, 2211 Lomas Blvd. NE, Albuquerque, NM 87131-5341, USA.
Nutrition 12:340-343, 1996 ©Elsevier Science Inc. 1996 Printed in the USA. All rights reserved.
ELSEVIER
0899-9007/96/$15.00 PII S0899-9007(96)00062-2
N E C R O T I Z I N G E N T E R O C O L I T I S A N D TPN C H O L E S T A S I S
34l
TABLE 1. CLINICAL DATA NEC Number Gestational age (weeks) Birthweight (g) Direct bilirubin mg/dL Hyaline Membrane Disease
7 30 (3.6) 1186 (105) 3.8 (1.2) 4
NEC + TPN 17 29 (4.1) 1104 (165) 4.1 (1.5) 10
Student's t test; p not significant for all groups. Numbers in parentheses are standard deviations.
changes seen in TPN-AC. 6 Some of the histologic data used in that study are reanalyzed here with specific reference to NEC. TPN was provided for full caloric support ( 1 2 0 - 1 5 0 kcal. k g - ' . d - l ) . Infants received 2 . 5 - 3 . 0 g. kg -~. d -J of protein, 1 2 - 1 8 g . k g - ~ . d -1 of glucose, and 2 - 3 g - k g - l - d ~ of lipids (Intralipid, KabiVitrum Inc., Alameda, C A ) . The protein source was Aminosyn (Abbott, Chicago, IL) before 1987 and Trophamine (Kendall McGaw, Irvine, C A ) after 1987. Multivitamins and trace minerals were added to the daily infusate. Enteral feeding was begun as early as possible to maintain luminal nutrition in all infants even if continued TPN was necessary for full caloric support (mean duration of TPN to start of enteral feeding 14 days). W e divided these patients into two groups: those who had not received TPN prior to the development of NEC (NEC, n = 7) and those who had ( N E C + TPN, n = 17). The clinical characteristics of the two groups were similar (Table I). All patients in both groups had necrotic or perforated bowel. Patients on TPN prior to the biopsy had received TPN for 1 2 - 8 2 d (mean 31 d). RESULTS In 13 patients for w h o m serum and biliary bile acid levels were available, 6 had received TPN prior to the measurement and 7 had not. Serum total bile acid level was significantly elevated in both groups over normals for age (Table II). There was no significant difference between the two groups. The presence of hepatic secretory failure was confirmed by measurement of biliary bile acid levels. The level of total bile acids in gall bladder bile was markedly depressed in both groups. Liver histology was available in all 24 patients. Six of the
FIG. 1. Liver from a 32-week gestational-age infant with necrotizing enterocolitis who had not received TPN. Hepatocytes are swollen and bile is plugging a canaliculus. seven patients who did not receive TPN ( N E C ) had abnormal liver histology. However, the hepatic injury was mild and nonspecific. Three patients had evidence of liver congestion while three exhibited histologic changes of biliary stasis. Figure 1 shows ballooning and degeneration of hepatocytes and stasis of bile in a canaliculus in a premature infant with NEC who never received TPN. Liver histology in infants with NEC who had received TPN ( N E C + T P N ) differed markedly from the group discussed above. The injury was more severe in the NEC + TPN group compared to the group with NEC alone. Fifteen of 17 liver specimens from this group were abnormal. Five patients had biliary stasis, suggesting relatively mild injury (Figure 2). Ten patients, however, had evidence of advanced structural injury in a pattern specific for T P N - A C (Figure 3). Eight patients had progressed to portal inflammation, two had bile duct proliferation, and two had portal fibrosis. During the midportion of this study, the amino acid source used for premature neonates was changed from Aminosyn to Trophamine. Fourteen patients studied received Aminosyn and ten received Trophamine. This study did not specifically ad-
TABLE II. BILE ACID LEVELS IN INFANTS WITH NEC WITH OR WITHOUT TPN
Group
Total Serum Bile Acids (mM/L)
NEC (n = 7) NEC + TPN (n = 17) Normal
17.65 (4.43) 20.9 (5.21) 11.80"
Biliary Bile Acids (Gall Bladder) (mM/L) 2.0 (0.98) 2.8 (1.1) 7.25**
Mean (_+_SE) * Nijima: Ped Res, 1985 (26) ** Bongiovanni: J Clin Endocrin Metab, 1965 (27) Student's t test: p < 0.05 for all comparisons stated in text.
FIG. 2. Liver from a 32-week gestational-age infant with NEC who had received TPN for 13 days. Bile plugs are seen in canaliculi and hepatocytes appear similar to Figure 1. There is a mild eosinophilic infiltrate.
342
NECROTIZING ENTEROCOLITIS AND TPN CHOLESTASIS
FIG. 3. Liver from an infant with NEC who had been on TPN for 6 weeks. There is an inflammatory portal infiltrate, proliferation of bile ducts, and fibrosis around portal triads.
dress the effect of amino acid composition on cholestasis. However, the incidence of NEC did not change appreciably during the study period and the authors did not observe a difference in the incidence or severity of cholestasis after the implementation of Trophamine. Six patients had two subsequent bile acid measurements and liver biopsies with a period of partial enteral nutrition between the two evaluations. These patients received continuous drip feeding with a predigested formula (Pregestemil, Mead Johnson Nutritional, Evansville, IN). The six infants received 10-25% of their dally caloric requirements enterally. The remaining calories were provided by TPN. This regimen was continued for a mean of 47 days before the second biopsy. Antibiotics were continued for a mean of 12 postoperative days. No patients developed clinical signs of sepsis, recurrent NEC, or positive blood cultures during this period. Serum and biliary bile acid levels were unchanged. They remained abnormal on this regimen of partial enteral feeding. The established hepatic secretory failure did not reverse when nutrition was given by the gut. Examination of liver histology in these six patients was similarly revealing. Liver disease had progressed markedly in all six patients by the time of the second biopsy. Five of the six patients developed fibrosis while being fed. The one patient who did not have fibrosis on the second biopsy had a marked inflammatory infiltrate of the portal tracts, which was not present on the initial biopsy. DISCUSSION The trend toward earlier enteral feeding in otherwise healthy premature infants in neonatal intensive care units has reduced the incidence of TPN-AC. 6 The syndrome has been almost eliminated as a clinically significant problem in babies without major gastrointestinal disease. Even when these infants do develop early hepatic dysfunction on TPN, the cholestasis resolves as TPN is stopped and oral nutrition is instituted. Infants with severe gastrointestinal disease such as NEC still require prolonged intravenous nutritional support, These are the patients who develop the progressive liver damage caused by TPN. Furthermore, with improvements in neonatal intensive care, more premature infants with NEC are surviving the initial insult and developing TPN-induced liver disease later. The data in this report suggest that NEC, by itself, may cause some degree of hepatic injury and cholestasis. Furthermore, NEC
may make the liver more susceptible to the toxic effects of TPN. Cholestasis may result from injury to the hepatocyte and its secretory apparatus; bile flow is reduced and the ability to secrete bile acids and bilirubin is impaired. This is functional cholestasis. The most sensitive indicator of functional cholestasis is a rise in the serum level of bile acids. At this point, liver histology is usually normal. As the process continues, hepatic architecture is disturbed and bile stasis can be seen histologically. This is structural cholestasis. At the stage of functional cholestasis, the hepatic effects due to TPN are indistinguishable from those due to other insults. The bile acid profiles of the infants with NEC, in this study, were abnormal whether or not the infants had been on TPN. Thus NEC alone appears to alter hepatobiliary function. At the structural level, however, the hepatic effects of NEC and TPN are quite different. NEC was associated with biliary and canalicular plugging without other histologic changes. TPN, on the other hand, produced a progressive sequence of histologic changes leading to fibrosis and cirrhosis. In functional cholestasis, the secretory apparatus of the hepatocyte fails to move bile acids against a concentration gradient from the serum into bile. The secretion of bile acids is one of the principal driving forces for bile formation and flow. The hepatic secretory apparatus in the neonate, especially the premature neonate, is immature. Bile flow rates in infants are lower than in adults. 8'9 Bile acid uptake is decreased at the sinusoidal membrane, interaction of bile acids with cytoplasmic binding proteins is altered, and rates of intracellular bile acid conjugation are decreased. 1° For these reasons, Balistreri et al.H concluded that immature hepatic secretory function predisposes the infant to cholestasis. Insults such as NEC and TPN may upset an already precarious balance. As hepatic secretory function fails, bile acids are not effectively transported from serum to bile and therefore found in high concentration in serum and low concentration in bile. Farrell et al.t2 found that a rise in serum bile acids is the earliest and most sensitive indicator of cholestasis, occurring 7 - 2 9 days before histologic changes of cholestasis are seen by light microscopy. On a molecular level, Popper and Schaffner ~3 isolated the conjugation of bile acids to the smooth endoplasmic reticulum. Electron microscopy from infants with early TPNAC showed swelling and abnormal morphology of the smooth endoplasmic reticulum.14 These early changes will not be apparent on light microscopy, thus, histology would appear normal. We found an elevation in serum bile acids and a decrease in biliary bile acid levels in patients with NEC whether or not they had received TPN. Thus it appears that NEC itself may alter hepatocyte function independent of TPN. The mechanism for this is not clear. Nakai and Landing 15 were the first to identify bacterial sepsis as a possible cause of cholestasis. Rooney et al.~6 showed that even clinically occult bacteremia may cause jaundice in neonates. Widespread damage to mucosal integrity with NEC likely leads to an influx of bacteria into the portal venous system. Endotoxin inhibits NA-K ATPase activity and decreases bile acid independent bile flow. ~7'18Sepsis and endotoxemia, common in NEC, also mediate the production of cytokines and leukotrienes in the intestinal mucosa. 19 These may damage the hepatocyte when they enter the portal circuit causing cholestasis. Leukotrienes in particular promote cellular infiltration and edema around bile ducts. 2° Despite this evidence that bacteria may play a role in cholestasis, a causal link has not been established. All patients with NEC in this study received broad-spectrum antibiotics with gram negative and anaerobic coverage. Some authors have speculated that antibiotics that decrease intestinal flora may
N E C R O T I Z I N G E N T E R O C O L I T I S A N D TPN C H O L E S T A S I S have a role in limiting cholestasis. Capron et al. 21 gave metronidazole to adult patients on TPN. They found that transaminase levels were lower in the metronidazole group. Spurr and associates 5 examined bilirubin levels in two groups of premature infants on TPN. One group received oral gentamicin for prophylaxis against NEC. Bilirubin levels were slightly lower in this group. However, no patient in this short-term study developed clinical cholestasis comparable to the patients in our series. These studies suggest a potential role for antibiotic use in this setting, but further study is needed. While the NEC and NEC + TPN groups shared similar functional disturbances, the liver histology varied markedly between the two groups. The syndrome of T P N - A C is characterized by a specific orderly progression of histologic changes. We reported these changes in 34 patients at Children's Memorial Hospital. The sequence of changes observed was: 1) biliary stasis; 2) portal tract inflammation; 3) bile duct proliferation; and 4) portal fibrosis. 6 This type of histologic damage was not observed in any of the patients in the NEC group. It was seen, however, in 88% of the NEC + TPN group. This is far greater than the expected incidence of cholestasis in other groups of infants on TPN. 22 While NEC alone does not cause the same progressive liver damage as TPN, it appears to make the liver more susceptible to the toxic effects of TPN. In patients with severe gastrointestinal diseases, such as
343 NEC, partial enteral feeding has been suggested as a means to prevent progressive liver damage. 23 Proponents of this theorize that even a small amount of feeding by gut will preserve the intestinal mucosa and also promote bile flow. This hypothesis remains unproven. The six patients in our series who underwent liver biopsies before and after a period of TPN and partial enteral feeding allowed us to examine this question. The continued presence of functional cholestasis and marked progression of histologic injury seen in these patients suggest that partial enteral feeding does not prevent cholestasis. In a rat model of TPN administration, Belli et al. 2~ found that TPN decreased bile flow even when rat chow was given concurrently with TPN. In our laboratory, we found that TPN solution caused cholestatic injury whether it was given intravenously or by gut. 25 The liver injury seen in the patients in this series appears to be due to the synergistic hepatotoxic effect of NEC and TPN rather than the absence of enteral feeding. Since the elimination of NEC is not on the horizon, only improvements in TPN solution that result in less hepatic toxicity offer hope in the prevention of cholestasis in these babies. ACKNOWLEDGEMENT The authors would like to thank Dr. Frank Gonzalez-Crussi for assistance with interpretation of the histologic data.
REFERENCES 1. Peden VH, Witzleben CL, Skelton MA. Total parenteral nutrition. Pediatrics 1971;78:180 2. Touloukian RJ, Seashore JH. Hepatic secretory obstruction with total parenteral nutrition in the infant. J Pediatr Surg 1975; 10:353 3. Periera GR, Sherman MS, DiGiacomo J, et al. Hyperalimentation induced cholestasis: increased incidence and severity in premature infants. Am J Dis Child 1981; 135:842 4. Postuma R, Trevenen CL. Liver disease in infants receiving total parenteral nutrition. Pediatrics 1979;63:110 5. Spurr SG, Grylack LJ, Mehta NR. Hyperalimentation associated neonatal cholestasis: effect of oral Gentamicin. JPEN 1989; 13:633 6. Moss RL, Das JB, Raffensperger JG. Total parenteral nutrition associated cholestasis: clinical and histopathologic correlation. J Pediatr Surg 1993;28:1270 7. Ginn-Pease ME, Pantalos D, King DR. TPN associated hyperbilirubinemia: a common problem in surgical neonates. J Pediatr Surg 1985;20:436 8. Shaffer EA, Zahavi I, Gall DG. Postnatal development of hepatic bile formation in the rabbit. Dig Dis Sci 1985;30:558 9. Suita S, Ikeda K, Naito K, et al. Cholelithiasis in infants: association with parenteral nutrition. JPEN 1984;8:568 10. Suchy FJ, Balistreri WF. Uptake of taurocholate by hepatocytes isolated from developing rats. Pediatr Res 1982; 16:282 11. Balistreri WF, Bove KE. Hepatobiliary consequences of parenteral alimentation. Prog Liver Dis 1990;9:567 12. Farrell MK, Gilster S, Balistreri WF. Serum bile acids: an early indicator of parenteral nutrition associated liver disease. Gastroenterology 1984;86:1074 13. Popper H, Schaffner F. Pathophysiology of cholestasis. Hum Path 1970;1:1
14. Dahms BB, Halpin TC. Serial biopsies in parenteral nutrition-associated cholestasis of early infancy. Gastroenterology 1981; 81:136 15. Nakai H, Landing BH. Factors in the genesis of bile stasis in infancy. Pediatrics 1961;27:300 16. Rooney JC, Hill DJ, Danks DM. Jaundice associated with bacterial infection in the newborn. Amer J Dis Child 1971; 122:39 17. Nolan JP. The role of endotoxin in liver injury. Gastroenterology 1975;69:1346 18. Utili R, Abemathy CO, Zimmerman HJ. Cholestatic effects of Escherichia coli endotoxin on the isolated perfused rat liver. Gastroenterology 1976;70:248 19. Cheromcha DP, Hyman PE. Dig Dis Sci 1988;33 (suppl 3):78s 20. Keppler D, Hagmann W, Rapp S, et al. The relation of leukotrienes to liver injury. Hepatol0gY 1985;5:883 21. Capron JP, Herve MA, Gineston JL, et al. Metronidazole in prevention of cholestasis associated with total parenteral nutrition. Lancet 1983; 1:446 22. Bell RL, Ferry GD, Smith EO, et al. Total parenteral nutritionrelated cholestasis in infants. JPEN 1986; 10:356 23. Farrell MK, Balistreri WF. Parenteral nutrition and hepatobiliary dysfunction. Clinics Perinatol 1986; 13:197 24. Belli DC, Lyse-Andree F, Lepage G, et al. Total parenteral nutrition-associated cholestasis in rats: comparison of different amino acid mixtures. JPEN 1987; 11:67 25. Moss RL, Das JB, Ansari G, et al. Total parenteral nutrition associated cholestasis is caused by the infusate, not the route of administration. J Pediatr Surg 1993;28:391 26. Nijima SI. Studies in the conjugating activity of bile acids in children. Ped Res 1985; 19:302 27. Bongiovanni AM. J Clin Endocrin Metab 1965;25:678
NUTRITIONAL
INVESTIGATION
Nutrition Vol. 12, No. 5, 1996
Necrotizing Enterocolitis and Total Parenteral Nutrition-Associated Cholestasis R. LAWRENCE MOSS, MD,* JOHN B. DAS, MB, PHD,t AND JOHN G. RAFFENSPERGER, MDt"
From the *Department of Surgery, University of New Mexico School of Medicine, Albuquerque, New Mexico, and the 7~Department of Surgery, Children's Memorial Hospital, Chicago, Illinois, USA Date accepted: 5 August 1995 ABSTRACT The proportion of patients with total parenteral nutrition (TPN)-associated cholestasis (TPN-AC) who have necrotizing enterocolitis (NEC) has increased markedly in the past ten years. Little is known about how these diseases affect each other. We retrospectively studied 24 patients with NEC and bowel necrosis or perforation who required surgical intervention. Patients were divided into two groups: those who had received TPN (NEC + TPN, n = 17) and those who had not (NEC, n = 7). As cholestasis was present clinically, or prolonged TPN was anticipated, liver biopsy was done. Bile acid levels were measured in both serum and bile in 13 patients. Six patients, who underwent bowel resection and enterostomy, had a second liver biopsy and measurement of bile acid levels at stoma closure. Our results showed that in 13 patients for whom bile acid levels were measured (NEC + TPN, n = 6) (NEC, n = 7), serum bile acid level was significantly elevated in both groups over normal for age. Biliary bile acid levels were correspondingly depressed in both groups suggesting a failure of bile acid transport. All patients had abnormal liver histology, but the pattern of injury differed between the two groups. Those in the NEC group had biliary stasis and mild hepatocyte degeneration. In contrast, 15 of 17 in the NEC + • TPN group had advanced injury specific for TPN-AC. All six patients managed on TPN and partial enteral feeding before a second biopsy had no change in bile acid levels and progression of histologic injury. We conclude that NEC alone can cause functional cholestasis and histologic liver injury but does not cause the specific progressive damage caused by TPN. NEC may make the liver more susceptible to the effects of TPN. Partial enteral feeding does not halt or reverse this injury. Nutrition 1996; 12:340-343 Key words: hyperalimentation, necrotizing enterocolitis, cholestasis, liver disease bile acids
INTRODUCTION In the early 1970s, Peden et al. 1 and Touloukian et al. 2 first noted an association between total parenterai nutrition (TPN) and jaundice. Since then, many retrospective studies have attempted to identify those babies at greatest risk of developing the syndrome of TPN-associated cholestasis ( T P N - A C ) . Periera et al. 3 showed a higher incidence of sepsis in babies with TPN-AC compared with other neonates on TPN. Recent surveys have shown a strong propensity for infants with necrotizing enterocolitis (NEC) to develop TPN cholestasis. In the first reported series of patients with T P N - A C in the 1970s, less than 25% of the patients had NEC. 2"4 By the 1990s, over 75% of patients with T P N - A C had NEC. 5"6 Seventy-eight percent of patients with T P N - A C at Children's Memorial Hospital over the past five years were neonates with NEC. 6 GinnPease et al. 7 found that patients with NEC were more likely to develop jaundice than other patients on TPN. Despite the
association between NEC and TPN-AC, little is known about how these diseases affect each other. W e reviewed our experience with NEC as it relates to liver function and TPN. METHODS W e retrospectively studied 24 patients treated at Children's Memorial Hospital with NEC who required surgical exploration for intestinal perforation or clinical signs of sepsis suggesting intestinal necrosis. As cholestasis was present clinically or prolonged TPN was anticipated, liver biopsy was done in all patients. A sample of gall bladder bile was obtained via aspiration of the fundus with a 22-gauge needle through a small pursestring suture. Bile acid level was measured in both serum and bile in 13 patients. Six patients who underwent bowel resection and enterostomy had a second liver biopsy and measurement of bile acid levels at a subsequent operation for stoma closure. In an earlier report, we detailed the unique sequential histologic
Correspondence to: R. Lawrence Moss, MD, University of New Mexico, School of Medicine, Department of Surgery, 2211 Lomas Blvd. NE, Albuquerque, NM 87131-5341, USA.
Nutrition 12:340-343, 1996 ©Elsevier Science Inc. 1996 Printed in the USA. All rights reserved.
ELSEVIER
0899-9007/96/$15.00 PII S0899-9007(96)00062-2
N E C R O T I Z I N G E N T E R O C O L I T I S A N D TPN C H O L E S T A S I S
34l
TABLE 1. CLINICAL DATA NEC Number Gestational age (weeks) Birthweight (g) Direct bilirubin mg/dL Hyaline Membrane Disease
7 30 (3.6) 1186 (105) 3.8 (1.2) 4
NEC + TPN 17 29 (4.1) 1104 (165) 4.1 (1.5) 10
Student's t test; p not significant for all groups. Numbers in parentheses are standard deviations.
changes seen in TPN-AC. 6 Some of the histologic data used in that study are reanalyzed here with specific reference to NEC. TPN was provided for full caloric support ( 1 2 0 - 1 5 0 kcal. k g - ' . d - l ) . Infants received 2 . 5 - 3 . 0 g. kg -~. d -J of protein, 1 2 - 1 8 g . k g - ~ . d -1 of glucose, and 2 - 3 g - k g - l - d ~ of lipids (Intralipid, KabiVitrum Inc., Alameda, C A ) . The protein source was Aminosyn (Abbott, Chicago, IL) before 1987 and Trophamine (Kendall McGaw, Irvine, C A ) after 1987. Multivitamins and trace minerals were added to the daily infusate. Enteral feeding was begun as early as possible to maintain luminal nutrition in all infants even if continued TPN was necessary for full caloric support (mean duration of TPN to start of enteral feeding 14 days). W e divided these patients into two groups: those who had not received TPN prior to the development of NEC (NEC, n = 7) and those who had ( N E C + TPN, n = 17). The clinical characteristics of the two groups were similar (Table I). All patients in both groups had necrotic or perforated bowel. Patients on TPN prior to the biopsy had received TPN for 1 2 - 8 2 d (mean 31 d). RESULTS In 13 patients for w h o m serum and biliary bile acid levels were available, 6 had received TPN prior to the measurement and 7 had not. Serum total bile acid level was significantly elevated in both groups over normals for age (Table II). There was no significant difference between the two groups. The presence of hepatic secretory failure was confirmed by measurement of biliary bile acid levels. The level of total bile acids in gall bladder bile was markedly depressed in both groups. Liver histology was available in all 24 patients. Six of the
FIG. 1. Liver from a 32-week gestational-age infant with necrotizing enterocolitis who had not received TPN. Hepatocytes are swollen and bile is plugging a canaliculus. seven patients who did not receive TPN ( N E C ) had abnormal liver histology. However, the hepatic injury was mild and nonspecific. Three patients had evidence of liver congestion while three exhibited histologic changes of biliary stasis. Figure 1 shows ballooning and degeneration of hepatocytes and stasis of bile in a canaliculus in a premature infant with NEC who never received TPN. Liver histology in infants with NEC who had received TPN ( N E C + T P N ) differed markedly from the group discussed above. The injury was more severe in the NEC + TPN group compared to the group with NEC alone. Fifteen of 17 liver specimens from this group were abnormal. Five patients had biliary stasis, suggesting relatively mild injury (Figure 2). Ten patients, however, had evidence of advanced structural injury in a pattern specific for T P N - A C (Figure 3). Eight patients had progressed to portal inflammation, two had bile duct proliferation, and two had portal fibrosis. During the midportion of this study, the amino acid source used for premature neonates was changed from Aminosyn to Trophamine. Fourteen patients studied received Aminosyn and ten received Trophamine. This study did not specifically ad-
TABLE II. BILE ACID LEVELS IN INFANTS WITH NEC WITH OR WITHOUT TPN
Group
Total Serum Bile Acids (mM/L)
NEC (n = 7) NEC + TPN (n = 17) Normal
17.65 (4.43) 20.9 (5.21) 11.80"
Biliary Bile Acids (Gall Bladder) (mM/L) 2.0 (0.98) 2.8 (1.1) 7.25**
Mean (_+_SE) * Nijima: Ped Res, 1985 (26) ** Bongiovanni: J Clin Endocrin Metab, 1965 (27) Student's t test: p < 0.05 for all comparisons stated in text.
FIG. 2. Liver from a 32-week gestational-age infant with NEC who had received TPN for 13 days. Bile plugs are seen in canaliculi and hepatocytes appear similar to Figure 1. There is a mild eosinophilic infiltrate.
342
NECROTIZING ENTEROCOLITIS AND TPN CHOLESTASIS
FIG. 3. Liver from an infant with NEC who had been on TPN for 6 weeks. There is an inflammatory portal infiltrate, proliferation of bile ducts, and fibrosis around portal triads.
dress the effect of amino acid composition on cholestasis. However, the incidence of NEC did not change appreciably during the study period and the authors did not observe a difference in the incidence or severity of cholestasis after the implementation of Trophamine. Six patients had two subsequent bile acid measurements and liver biopsies with a period of partial enteral nutrition between the two evaluations. These patients received continuous drip feeding with a predigested formula (Pregestemil, Mead Johnson Nutritional, Evansville, IN). The six infants received 10-25% of their dally caloric requirements enterally. The remaining calories were provided by TPN. This regimen was continued for a mean of 47 days before the second biopsy. Antibiotics were continued for a mean of 12 postoperative days. No patients developed clinical signs of sepsis, recurrent NEC, or positive blood cultures during this period. Serum and biliary bile acid levels were unchanged. They remained abnormal on this regimen of partial enteral feeding. The established hepatic secretory failure did not reverse when nutrition was given by the gut. Examination of liver histology in these six patients was similarly revealing. Liver disease had progressed markedly in all six patients by the time of the second biopsy. Five of the six patients developed fibrosis while being fed. The one patient who did not have fibrosis on the second biopsy had a marked inflammatory infiltrate of the portal tracts, which was not present on the initial biopsy. DISCUSSION The trend toward earlier enteral feeding in otherwise healthy premature infants in neonatal intensive care units has reduced the incidence of TPN-AC. 6 The syndrome has been almost eliminated as a clinically significant problem in babies without major gastrointestinal disease. Even when these infants do develop early hepatic dysfunction on TPN, the cholestasis resolves as TPN is stopped and oral nutrition is instituted. Infants with severe gastrointestinal disease such as NEC still require prolonged intravenous nutritional support, These are the patients who develop the progressive liver damage caused by TPN. Furthermore, with improvements in neonatal intensive care, more premature infants with NEC are surviving the initial insult and developing TPN-induced liver disease later. The data in this report suggest that NEC, by itself, may cause some degree of hepatic injury and cholestasis. Furthermore, NEC
may make the liver more susceptible to the toxic effects of TPN. Cholestasis may result from injury to the hepatocyte and its secretory apparatus; bile flow is reduced and the ability to secrete bile acids and bilirubin is impaired. This is functional cholestasis. The most sensitive indicator of functional cholestasis is a rise in the serum level of bile acids. At this point, liver histology is usually normal. As the process continues, hepatic architecture is disturbed and bile stasis can be seen histologically. This is structural cholestasis. At the stage of functional cholestasis, the hepatic effects due to TPN are indistinguishable from those due to other insults. The bile acid profiles of the infants with NEC, in this study, were abnormal whether or not the infants had been on TPN. Thus NEC alone appears to alter hepatobiliary function. At the structural level, however, the hepatic effects of NEC and TPN are quite different. NEC was associated with biliary and canalicular plugging without other histologic changes. TPN, on the other hand, produced a progressive sequence of histologic changes leading to fibrosis and cirrhosis. In functional cholestasis, the secretory apparatus of the hepatocyte fails to move bile acids against a concentration gradient from the serum into bile. The secretion of bile acids is one of the principal driving forces for bile formation and flow. The hepatic secretory apparatus in the neonate, especially the premature neonate, is immature. Bile flow rates in infants are lower than in adults. 8'9 Bile acid uptake is decreased at the sinusoidal membrane, interaction of bile acids with cytoplasmic binding proteins is altered, and rates of intracellular bile acid conjugation are decreased. 1° For these reasons, Balistreri et al.H concluded that immature hepatic secretory function predisposes the infant to cholestasis. Insults such as NEC and TPN may upset an already precarious balance. As hepatic secretory function fails, bile acids are not effectively transported from serum to bile and therefore found in high concentration in serum and low concentration in bile. Farrell et al.t2 found that a rise in serum bile acids is the earliest and most sensitive indicator of cholestasis, occurring 7 - 2 9 days before histologic changes of cholestasis are seen by light microscopy. On a molecular level, Popper and Schaffner ~3 isolated the conjugation of bile acids to the smooth endoplasmic reticulum. Electron microscopy from infants with early TPNAC showed swelling and abnormal morphology of the smooth endoplasmic reticulum.14 These early changes will not be apparent on light microscopy, thus, histology would appear normal. We found an elevation in serum bile acids and a decrease in biliary bile acid levels in patients with NEC whether or not they had received TPN. Thus it appears that NEC itself may alter hepatocyte function independent of TPN. The mechanism for this is not clear. Nakai and Landing 15 were the first to identify bacterial sepsis as a possible cause of cholestasis. Rooney et al.~6 showed that even clinically occult bacteremia may cause jaundice in neonates. Widespread damage to mucosal integrity with NEC likely leads to an influx of bacteria into the portal venous system. Endotoxin inhibits NA-K ATPase activity and decreases bile acid independent bile flow. ~7'18Sepsis and endotoxemia, common in NEC, also mediate the production of cytokines and leukotrienes in the intestinal mucosa. 19 These may damage the hepatocyte when they enter the portal circuit causing cholestasis. Leukotrienes in particular promote cellular infiltration and edema around bile ducts. 2° Despite this evidence that bacteria may play a role in cholestasis, a causal link has not been established. All patients with NEC in this study received broad-spectrum antibiotics with gram negative and anaerobic coverage. Some authors have speculated that antibiotics that decrease intestinal flora may
N E C R O T I Z I N G E N T E R O C O L I T I S A N D TPN C H O L E S T A S I S have a role in limiting cholestasis. Capron et al. 21 gave metronidazole to adult patients on TPN. They found that transaminase levels were lower in the metronidazole group. Spurr and associates 5 examined bilirubin levels in two groups of premature infants on TPN. One group received oral gentamicin for prophylaxis against NEC. Bilirubin levels were slightly lower in this group. However, no patient in this short-term study developed clinical cholestasis comparable to the patients in our series. These studies suggest a potential role for antibiotic use in this setting, but further study is needed. While the NEC and NEC + TPN groups shared similar functional disturbances, the liver histology varied markedly between the two groups. The syndrome of T P N - A C is characterized by a specific orderly progression of histologic changes. We reported these changes in 34 patients at Children's Memorial Hospital. The sequence of changes observed was: 1) biliary stasis; 2) portal tract inflammation; 3) bile duct proliferation; and 4) portal fibrosis. 6 This type of histologic damage was not observed in any of the patients in the NEC group. It was seen, however, in 88% of the NEC + TPN group. This is far greater than the expected incidence of cholestasis in other groups of infants on TPN. 22 While NEC alone does not cause the same progressive liver damage as TPN, it appears to make the liver more susceptible to the toxic effects of TPN. In patients with severe gastrointestinal diseases, such as
343 NEC, partial enteral feeding has been suggested as a means to prevent progressive liver damage. 23 Proponents of this theorize that even a small amount of feeding by gut will preserve the intestinal mucosa and also promote bile flow. This hypothesis remains unproven. The six patients in our series who underwent liver biopsies before and after a period of TPN and partial enteral feeding allowed us to examine this question. The continued presence of functional cholestasis and marked progression of histologic injury seen in these patients suggest that partial enteral feeding does not prevent cholestasis. In a rat model of TPN administration, Belli et al. 2~ found that TPN decreased bile flow even when rat chow was given concurrently with TPN. In our laboratory, we found that TPN solution caused cholestatic injury whether it was given intravenously or by gut. 25 The liver injury seen in the patients in this series appears to be due to the synergistic hepatotoxic effect of NEC and TPN rather than the absence of enteral feeding. Since the elimination of NEC is not on the horizon, only improvements in TPN solution that result in less hepatic toxicity offer hope in the prevention of cholestasis in these babies. ACKNOWLEDGEMENT The authors would like to thank Dr. Frank Gonzalez-Crussi for assistance with interpretation of the histologic data.
REFERENCES 1. Peden VH, Witzleben CL, Skelton MA. Total parenteral nutrition. Pediatrics 1971;78:180 2. Touloukian RJ, Seashore JH. Hepatic secretory obstruction with total parenteral nutrition in the infant. J Pediatr Surg 1975; 10:353 3. Periera GR, Sherman MS, DiGiacomo J, et al. Hyperalimentation induced cholestasis: increased incidence and severity in premature infants. Am J Dis Child 1981; 135:842 4. Postuma R, Trevenen CL. Liver disease in infants receiving total parenteral nutrition. Pediatrics 1979;63:110 5. Spurr SG, Grylack LJ, Mehta NR. Hyperalimentation associated neonatal cholestasis: effect of oral Gentamicin. JPEN 1989; 13:633 6. Moss RL, Das JB, Raffensperger JG. Total parenteral nutrition associated cholestasis: clinical and histopathologic correlation. J Pediatr Surg 1993;28:1270 7. Ginn-Pease ME, Pantalos D, King DR. TPN associated hyperbilirubinemia: a common problem in surgical neonates. J Pediatr Surg 1985;20:436 8. Shaffer EA, Zahavi I, Gall DG. Postnatal development of hepatic bile formation in the rabbit. Dig Dis Sci 1985;30:558 9. Suita S, Ikeda K, Naito K, et al. Cholelithiasis in infants: association with parenteral nutrition. JPEN 1984;8:568 10. Suchy FJ, Balistreri WF. Uptake of taurocholate by hepatocytes isolated from developing rats. Pediatr Res 1982; 16:282 11. Balistreri WF, Bove KE. Hepatobiliary consequences of parenteral alimentation. Prog Liver Dis 1990;9:567 12. Farrell MK, Gilster S, Balistreri WF. Serum bile acids: an early indicator of parenteral nutrition associated liver disease. Gastroenterology 1984;86:1074 13. Popper H, Schaffner F. Pathophysiology of cholestasis. Hum Path 1970;1:1
14. Dahms BB, Halpin TC. Serial biopsies in parenteral nutrition-associated cholestasis of early infancy. Gastroenterology 1981; 81:136 15. Nakai H, Landing BH. Factors in the genesis of bile stasis in infancy. Pediatrics 1961;27:300 16. Rooney JC, Hill DJ, Danks DM. Jaundice associated with bacterial infection in the newborn. Amer J Dis Child 1971; 122:39 17. Nolan JP. The role of endotoxin in liver injury. Gastroenterology 1975;69:1346 18. Utili R, Abemathy CO, Zimmerman HJ. Cholestatic effects of Escherichia coli endotoxin on the isolated perfused rat liver. Gastroenterology 1976;70:248 19. Cheromcha DP, Hyman PE. Dig Dis Sci 1988;33 (suppl 3):78s 20. Keppler D, Hagmann W, Rapp S, et al. The relation of leukotrienes to liver injury. Hepatol0gY 1985;5:883 21. Capron JP, Herve MA, Gineston JL, et al. Metronidazole in prevention of cholestasis associated with total parenteral nutrition. Lancet 1983; 1:446 22. Bell RL, Ferry GD, Smith EO, et al. Total parenteral nutritionrelated cholestasis in infants. JPEN 1986; 10:356 23. Farrell MK, Balistreri WF. Parenteral nutrition and hepatobiliary dysfunction. Clinics Perinatol 1986; 13:197 24. Belli DC, Lyse-Andree F, Lepage G, et al. Total parenteral nutrition-associated cholestasis in rats: comparison of different amino acid mixtures. JPEN 1987; 11:67 25. Moss RL, Das JB, Ansari G, et al. Total parenteral nutrition associated cholestasis is caused by the infusate, not the route of administration. J Pediatr Surg 1993;28:391 26. Nijima SI. Studies in the conjugating activity of bile acids in children. Ped Res 1985; 19:302 27. Bongiovanni AM. J Clin Endocrin Metab 1965;25:678