De Novo Non-Alcoholic Fatty Liver Disease Following Orthotopic Liver Transplantation
*Corresponding author: Fred Poordad, firstname.lastname@example.org
Non-alcoholic fatty liver disease (NAFLD) is an increasingly recognized clinico-pathologic entity typically associated with obesity, type II diabetes and hyperlipidemia. It has been noted to recur after orthotopic liver transplantation (OLT). We report four patients who developed de novo NAFLD within 3 months of OLT without the typical predisposing factors of diabetes mellitus or obesity. Three of the four patients underwent OLT for hepatitis C-related cirrhosis, and the other for alcoholic cirrhosis. Examination of the liver explants revealed no evidence of steatosis. No surreptitious alcohol use or a drug-induced process could be identified in these patients. Treatment of recurrent hepatitis C infection in one patient with interferon and ribavirin led to sustained suppression of the viral RNA to undetectable levels, but no improvement in histology or liver enzymes. All four patients had histologic evidence of preservation injury on the initial post-OLT biopsies, but the significance of this finding in relationship to the development of NAFLD is unknown. NAFLD can develop without any of the known predisposing conditions after transplantation, and this raises further questions about the pathogenesis of this condition.
Non-alcoholic fatty liver disease (NAFLD) includes a spectrum of histologic lesions ranging from steatosis to nonalcoholic steatohepatitis (NASH), the latter being increasingly recognized as a significant cause of cirrhosis (1–3). The prevalence of NAFLD in European epidemiologic studies of chronic liver disease is roughly 20% (4), and may be as high as 24% in the United States (5). The two most common predisposing risk factors are insulin resistance and obesity (6–10). Many of the patients with these risk factors also have hyperlipidemia, and this may also be a contributing factor. Other causes include rapid weight loss (11), protein-calorie malnutrition (12,13), total parenteral nutrition (14,15), drugs including amiodarone (16,17), long-term glucocorticoids (18), synthetic estrogens (19), perhexilline maleate (20) and several rare metabolic disorders (21,22). In some cases, predisposing factors are not identified, as demonstrated by a study of 33 patients with NASH, 42% of whom did not have the usual risk factors (23). Many patients with cryptogenic cirrhosis are now thought to have NAFLD (24) and the recurrence of NAFLD post orthotopic liver transplantation (OLT) is well described (25–28). At least 50% of patients experience weight gain in the first year post-OLT. The use of corticosteroids and other immunosuppressive drugs may further contribute to weight gain, diabetes and hyperlipidemia (29), all of which are predisposing factors for NAFLD. However, rapid onset of NAFLD after liver transplantation in those transplanted for other indications and without obvious risk factors, has not been recognized.
We report four patients who developed severe, de novo NAFLD within 12 weeks of OLT without significant risk factors. The disease persisted in three patients and resolved in one during follow-up.
Materials and Methods
Clinical data were obtained from chart review of 88 adult patients who underwent 89 liver transplants between January 1, 1997 and January 1, 1999 at California Pacific Medical Center, San Francisco. The 1-year actuarial patient survival for this group of patients was 94.7%. Four patients transplanted during this period developed NAFLD of unknown cause after OLT (Table 1). Two other patients with NAFLD before and after OLT, presumably due to severe obesity, were not included in this study. All post-OLT patients had laboratory testing including fasting glucose and lipid panel twice weekly for the first month after OLT, then weekly for 1 month, and then at monthly intervals, which sometimes varied as determined by the physician and clinical status of the patient. Patients were weighed at each clinic visit. The body mass index (BMI) of the four patients was calculated. Demographic information collected included age, race, gender, medications, alcohol use, and diagnoses.
Table 1. General features of cases 1–4
|1||Alcohol||55/F||58/23||189/138||Yes||No|| 9 weeks|| 7.5|
|2||HCV||64/M||60/19||171/112||Yes||No|| 4 weeks||15|
|3||HCV||44/M||65/24||102/165||Yes||No|| 8 weeks||10|
The typical immunosuppressive regimen consisted of tacrolimus (FK506, Prograf®, Fujisawa), mycophenolate mofetil (Cellcept®, Roche) and prednisone. Tacrolimus was started after OLT as soon as patients were able to tolerate oral intake and had stable renal function. The dose was adjusted to maintain trough levels of 8–12 ng/mL (by IMX MEIA whole blood method) during months 1–2, 6–10 ng/mL during months 2–6, and 5–8 ng/mL after 6 months. Mycophenolate mofetil was initially given at a dose of 1500 mg twice daily and then tapered to discontinuation by 3 months after OLT. Patients received methylprednisolone 1000 mg intravenously in the operating room and 1000 mg on day 1, followed by a rapid taper from 200 mg to 20 mg daily at day 7 after OLT. Prednisone was substituted for methylprednisolone when tolerated, and the dose was tapered gradually to 5 mg by 42 days after OLT, and then tapered to discontinuation after 180 days.
Prophylaxis against cytomegalovirus (CMV) consisted of intravenous ganciclovir at 10 mg/kg daily for 14 days, followed by oral acyclovir 200 mg twice daily until discontinuation at 3 months. Nystatin 5 mL 4 times daily for 2 months was used for fungal prophylaxis. All patients without allergy to sulfa drugs received trimethoprim-sulfamethoxazole single-strength tablets twice weekly indefinitely for prophylaxis against Pneumocystis carini pneumonia. Omeprazole 20 mg twice daily was given as long as patients were on mycophenolate mofetil, or as clinically indicated.
Biopsies were performed for elevated liver chemistries. All liver biopsy specimens and the explanted liver of the four patients with hepatic steatosis were reviewed by a single liver pathologist. Grading of fatty liver was as follows: mild (1 +), moderate (2 +) or severe (3 +) according to the criteria of Diehl et al. (8). Mild was defined as less than 20% fat, moderate 20–50%, and severe greater than 50%. NASH was defined by histologic criteria of (macrovesicular) steatosis with parenchymal inflammation, with or without fibrosis and Mallory bodies as originally described (6,7).
A 55-year-old Caucasian woman underwent OLT for alcoholic cirrhosis. She was discharged from the hospital 8 days after surgery. Liver biopsy for persistently elevated aminotransferases on day 7 revealed residual preservation injury. Persistent mild elevations in her liver chemistries 2 months after OLT [total bilirubin (TB) of 1.2 mg/dL, alkaline phosphatase (AP) 115 U/L, ALT 72 U/L, AST 48 U/L, GGT 211 U/L] led to another liver biopsy (#2), which revealed moderate macrovesicular steatosis. Her medical history was negative for diabetes or hyperlipidemia. She had maintained a stable weight of 58 kg with body mass index (BMI) of 23. She denied any alcohol consumption. Evaluations included negative blood and urine alcohol, negative carbohydrate-deficient transferrin by immunoassay at <2.5% [10–20], and normal carnitine at 41 nmol/mL [23–56]. Progressive worsening of her liver enzymes was seen over the ensuing 2 months with peak TB of 3.7 mg/dL. Repeat liver biopsies (#3 and 4) at 3 and 4 months after OLT showed severe steatosis involving >75% of the liver. Rapid tapering of immunosuppression led to an episode of histologically confirmed rejection at 5 months after OLT, necessitating high-dose steroids and reinstitution of mycophenolate mofetil. Two additional liver biopsies at 8 and 12 months after OLT, respectively, showed persistence of severe steatosis without evidence of rejection. At follow-up 17 months after OLT, laboratory tests revealed TBil of 5.4 mg/dL, AP 567 U/L, ALT 109 U/L, AST 376 U/L, and GGT of 2275 U/L. Both MRCP and ERCP failed to show mechanical obstruction or significant biliary tract disease.
A 64-year-old Caucasian man with hepatitis C virus (HCV) cirrhosis underwent OLT, complicated by primary graft nonfunction, necessitating re-transplantation on postoperative day 3. He was discharged from the hospital 8 days later. One month after OLT, elevations in liver enzymes were noted with TB of 1.3 mg/dL, AP of 128 U/L, ALT of 124 U/L, and AST of 83 U/L. Liver biopsy (#1) revealed residual preservation injury without rejection or evidence of recurrent hepatitis. Two months after OLT, further elevations in liver enzymes were seen with TB of 1.6 mg/dL, AP 123 U/L, ALT 165 U/L, AST of 110 U/L. A repeat liver biopsy (#2) revealed severe macrovesicular steatosis. Blood screens for drugs and alcohol were negative. His medical history was negative for previous alcohol dependence, diabetes or hyperlipidemia. His weight was 60 kg with BMI of 19. He had no recent history of weight gain or rapid weight loss. Tapering of immunosuppression led to further elevation in liver enzyme with TB of 10.9 mg/dL, AP 196 U/L, ALT 394 U/L, AST 400 U/L, GGT 556 U/L. Repeat liver biopsy (#3) 4 months after OLT revealed perivenular hepatocellular dropout and necrosis with mild cellular rejection. Prominent steatosis was still present on the liver biopsy. The patient was treated with high-dose steroids, and mycophenolate mofetil was restarted. His liver enzymes gradually stabilized but remained mildly elevated. At follow-up 14 months after OLT, laboratory data revealed TB of 1.6 mg/dL, AP 288 U/L, ALT 58 U/L, AST 75 U/L, and GGT of 159 U/L.
A 44-year-old Hispanic man underwent OLT for cirrhosis secondary to HCV and alcohol. He was discharged from the hospital on postop day 9. Liver biopsy on day 7 showed mild preservation injury. One month after OLT, he was admitted to the hospital with fever and peritonitis. Peritoneal fluid culture grew methicillin-resistant Staphylococcus aureus, most likely from a wound infection. He was treated with intravenous vancomycin as well as surgical exploration and drainage of intra-abdominal fluid. Elevated liver enzymes were also noted during the hospitalization with TB of 0.8 mg/dL, AP 1580 U/L, GGT 703 U/L, AST of 20 U/L. Liver biopsy (#2) revealed moderate to severe macrovesicular steatosis with mild fibrosis. This patient had no history of diabetes or hyperlipidemia. Alcohol and drug screens were repeatedly negative. He weighed 65 kg with a BMI of 24. Endoscopic retrograde cholangio-pancreatography revealed multiple donor common bile duct strictures. He underwent balloon dilatations, sphincterotomy, and placement of a 10 French, 5-cm biliary stent. Hepatic angiogram revealed no hepatic artery thrombosis. The patient then developed significant bleeding at the sphincterotomy site, culminating in exploratory laparotomy with sphincteroplasty. Repeat liver biopsy (#3) 3 months after OLT again showed moderate to severe macrovesicular steatosis and pseudoductular proliferation in some portal tracts consistent with biliary obstruction. Marked elevation of AP and GGT persisted. Follow-up liver biopsy (#4) at 4 months post-OLT showed persistent macrovesicular steatosis. He was started on ursodeoxycholic acid (UDCA) and required multiple additional ERCP procedures for extraction of biliary calculi and sludge. His repeat liver biopsy (#5) at 10 months after OLT showed bridging fibrosis with pseudoductular proliferation but resolution of steatosis. He underwent biliary revision surgery with Roux-en-Y anastomosis at 14 months after OLT. Liver biopsy at 38 months after OLT revealed bridging fibrosis presumably secondary to his biliary complications, without evidence of steatosis.
A 47-year-old Hispanic man underwent liver re-transplantation as a result of recurrent HCV and chronic rejection. There was no evidence of steatosis after his first OLT. He received total parenteral nutrition (TPN) for 2 weeks prior to re-transplant. Immunosuppression consisted of tacrolimus and prednisone without mycophenolate mofetil. Daclizumab (Zenapax®, Roche), a monoclonal antibody against interleukin-2 was given at a dose of 1 mg/kg intraoperatively, then every 2 weeks for 5 doses. His postoperative course was complicated by hemorrhagic pancreatitis and retroperitoneal hematoma that required two subsequent exploratory laparotomies. He was also treated with bowel rest and TPN for 10 days. Liver biopsy on day 8 showed only preservation injury. He underwent physical therapy and rehabilitation before being discharged to home 50 days after OLT.
Three months after OLT, increased liver enzymes were noted with TB of 1.4 mg/dL, AP 140 U/L, ALT 494 U/L, AST 155 U/L, GGT of 564 U/L. Liver biopsy (#2) showed severe macrovesicular steatosis and mild inflammatory activities in the portal tracts suggestive of recurrent hepatitis C infection. HCV genotype was 3A, and quantitative HCV RNA titer was 100 000 copies/mL. Interferon and ribavirin were subsequently started with prompt virologic response. His HCV RNA by PCR became undetectable within 1 month of starting therapy and remained persistently undetectable thereafter. However, his liver enzymes remained elevated. He had no other known risk factors for fatty liver disease, including diabetes, hyperlipidemia or alcohol consumption prior to OLT. He did develop hypertriglyceridemia, ranging from 300 to 600 within 30 days of surgery. He lost 8 kg in weight over the first 6 weeks after OLT but returned to his premorbid weight of 80 kg (BMI 25) by postop month 3. Liver biopsy (#3) 8 months after OLT revealed severe steatohepatitis. His liver profile 13 months after OLT revealed TB of 1.5 mg/dL, AP 119 U/L, ALT 485 U/L, AST 185 U/L, and GGT of 93 U/L.
Non-alcoholic steatohepatitis is a pathological entity which shares many of the histologic features of alcohol liver disease (6,8). The histology may show focal or diffuse steatosis, usually macrovesicular, associated with a mixed lobular inflammatory cellular infiltrate, Mallory's hyaline, and perivenular fibrosis. The true incidence of histologic progression from steatosis to steatohepatitis and then to cirrhosis is not known, but may occur in up to 30% of patients (7).
We describe four patients who developed severe NAFLD within 12 weeks of OLT. Their pertinent clinical and laboratory data are summarized in Table 1. Steatosis developed rapidly after transplantation, and none of the patients had NAFLD prior to OLT. The explanted livers did not reveal features of NAFLD, nor did the initial post-OLT biopsies. None of the patients were obese (BMI > 30) or had significant weight change during the early postoperative period. They did not have diabetes mellitus or hyperlipidemia prior to OLT, and only one developed post-OLT hypertriglyceridemia, which was controlled with medications over several months. Careful history and alcohol screening did not reveal surreptitious alcohol consumption in any patient. The immunosuppression regimen consisted of tacrolimus, mycophenolate mofetil and prednisone. This combination has been previously studied in OLT with favorable graft and patient survival without predisposition to the development of NAFLD (30–33). Rapid taper of immunosuppression resulted in mild rejection in two patients, without significant improvement in hepatic steatosis. In all four patients, steatosis persisted after discontinuation of prednisone. One of the four patients received TPN for 4 weeks peri-operatively but resumed oral intake and maintained adequate nutritional status. Severe hepatic steatosis and subsequent progression to steatohepatitis in this patient was unlikely to be the result of TPN, since hepatic dysfunction is almost always self-limited and resolves following discontinuation of TPN (15).
Three of the four patients had underlying hepatitis C, which is frequently associated with steatosis, but is typically mild and focal (34). It is unlikely that steatosis would develop so quickly after transplantation solely as a result of recurrent hepatitis C, since histologic changes associated with hepatitis C post transplantation occur on average 3–4 months after surgery (35). Furthermore, genotype 3a HCV is most commonly associated with steatosis, and only one patient had this, while the other two were both genotype 1a. One patient had had documented alcohol-induced cirrhosis and developed steatosis within 2 months of transplant, but denied alcohol use. She was seen twice weekly in clinic, had a supportive family, and was screened for alcohol use, so the likelihood of surreptitious alcohol abuse post transplant was felt to be very low.
NAFLD resolved in one patient (patient #3) 9 months after the initial diagnosis although the histologic picture progressed to bridging fibrosis. This patient was placed on UDCA for cholestasis, and a preliminary study of UDCA in 24 patients has shown modest improvements in the grade of steatosis and liver chemistries (36). It is not clear if it affected the steatosis in this case.
Two of the cases discussed had bacterial infections secondary to intra-abdominal processes and required antibiotics. The link between bacterial infections and liver injury in NAFLD is not clear, but increased sensitivity to endotoxin and tumor necrosis factor (TNF) may play a role (37). Further support for this hypothesis comes from reports of NASH being associated with small bowel intestinal overgrowth, and prevention or regression of steatosis in jejuno-ileal bypass patients with antibiotic therapy aimed at intestinal flora (38,39). It is unlikely that these infections played a significant role in these patients.
Interestingly, all four patients received organs that had moderately severe preservation injury (PI). Mild PI is not uncommon post OLT and is not thought to be associated with NASH. In our experience, severe PI resulting in prolonged liver enzyme or histologic abnormalities occurs in fewer than 10% of cases. The mechanism of injury in PI does, however, share some features with presumptive mechanisms of injury in NASH. After cold ischemia, mitochondrial function deteriorates (40) and oxidative phosphorylation abnormalities are known to result in reactive oxygen species (ROS), which are implicated in lipid peroxidation (41). Lipid peroxidation results in cell death, but also releases malondialdehyde and 4-hydroxynonenal, which induce collagen synthesis by stellate cells, may be involved in Mallory body formation, and act as chemoattractants; all of these findings are observed in steatohepatitis (42). Malnutrition has also been implicated in the pathogenesis of NASH and may have played a role in this group as well (43–45). Poor nutritional status is common in transplant patients both pre- and peri-operatively.
In summary, the four patients presented had de novo NAFLD within 12 weeks of OLT. It is clear that NASH can occur in the post-transplant setting in patients with other underlying liver disease, and raises the question of whether the etiology is host dependent, or perhaps donor liver dependent, or an interplay between the two.