Liver-protecting effects of omega-3 fish oil lipid emulsion in liver transplantation

Abstract

AIM: To investigate the liver-protecting effect of parenteral nutrition (PN) support with omega-3 fatty acids in a randomized controlled clinical trial.

METHODS: Sixty-six patients with the diagnosis of end-stage liver disease or hepatic cellular carcinoma were admitted to the Affiliated Drum Tower Hospital, Nanjing University, China for orthotopic liver transplantation. The patients were randomly divided into two groups: PN group (n = 33) and polyunsaturated fatty acid (PUFA) group (n = 33). All patients received isocaloric and isonitrogenous PN for seven days after surgery, and in PUFA group omega-3 fish oil lipid emulsion replaced part of the standard lipid emulsion. Liver function was tested on days 2 and 9 after surgery. Pathological examination was performed after reperfusion of the donor liver and on day 9. Clinical outcome was assessed based on the post-transplant investigations, including: (1) post-transplant mechanical ventilation; (2) total hospital stay; (3) infectious morbidities; (4) acute and chronic rejection; and (5) mortality (intensive care unit mortality, hospital mortality, 28-d mortality, and survival at a one-year post-transplant surveillance period).

RESULTS: On days 2 and 9 after operation, a significant decrease of alanine aminotransferase (299.16 U/L ± 189.17 U/L vs 246.16 U/L ± 175.21 U/L, P = 0.024) and prothrombin time (5.64 s ± 2.06 s vs 2.54 s ± 1.15 s, P = 0.035) was seen in PUFA group compared with PN group. The pathological results showed that omega-3 fatty acid supplement improved the injury of hepatic cells. Compared with PN group, there was a significant decrease of post-transplant hospital stay in PUFA group (18.7 d ± 4.0 d vs 20.6 d ± 4.6 d, P = 0.041). Complications of infection occurred in 6 cases of PN group (2 cases of pneumonia, 3 cases of intra-abdominal abscess and 1 case of urinary tract infection), and in 3 cases of PUFA group (2 cases of pneumonia and 1 case of intra-abdominal abscess). No acute or chronic rejection and hospital mortality were found in both groups. The one-year mortality in PN group was 9.1% (3/33), one died of pulmonary infection, one died of severe intra-hepatic cholangitis and hepatic dysfunction and the other died of hepatic cell carcinoma recurrence. Only one patient in PUFA group (1/33, 3.1%) died of biliary complication and hepatic dysfunction during follow-up.

CONCLUSION: Post-transplant parenteral nutritional support combined with omega-3 fatty acids can significantly improve the liver injury, reduce the infectious morbidities, and shorten the post-transplant hospital stay.

INTRODUCTION

Liver transplantation has dramatically improved the prognosis of end-stage liver disease. The progress made in the immunosuppressive regimens and surgical techniques has yielded a better outcome of the patients, and the 5-year survival after liver transplantation is 70%-80%[1]. The liver recipients with liver insufficiency are in fact known to experience a higher incidence of severe protein/calorie malnutrition, which is associated with a greater risk of postoperative complications and mortality in patients undergoing liver transplantation[2,3]. And ischemia/reperfusion (I/R) injury associated with liver transplantation often leads to hepatic dysfunction despite the improvement in surgical techniques and perioperative medication. I/R injury of the liver is an event involving multiple factors, such as hypoxia during inflow occlusion of the liver and inflammatory reactions after reperfusion[4,5], and the mechanisms of the reperfusion injury, including the release of inflammatory cytokines, the generation of oxygen free radicals, Kupffer cell activation and leukocyte-endothelial cell interaction[6,7]. Based on the pathophysiology of hepatic I/R injury, the current study particularly focused on various perioperative approaches to protect the liver from these inflammatory reactions and microcirculatory disturbances.

Omega-3 (N-3) fatty acids, which are derived from fish oil, are essential polyunsaturated fatty acids (PUFAs) for humans. Omega-3 fatty acids exert anti-inflammatory and immunomodulatory properties through their ability to modulate the synthesis of different eicosanoids[8,9]. Perioperative administration of omega-3 fatty acids reduces plasma and tissue levels of the eicosanoids, specific leukotrienes, thromboxanes, and prostaglandins, all of which have pro-inflammatory effects[10,11]. Recent studies described that supplementation with omega-3 fatty acids decreases the rate of inflammatory complications, the length of hospital stay, and the mortality after major abdominal surgeries[12-14]. Their protective effects on hepatic I/R injury and inflammatory responses have been increasingly investigated.

In this study, we investigated the liver-protecting effects of parenteral nutrition (PN) supplemented with omega-3 fish oil lipid emulsion in patients undergoing liver transplantation.

MATERIALS AND METHODS

Ethics

This study was carried out in the Department of Hepatobiliary Surgery of the Affiliated Drum Tower Hospital, Medical School of Nanjing University, China according to the principles and guidelines of the Helsinki Declaration of 1975 revised in 2000. The protocol was approved by the Ethics Committee of the Affiliated Drum Tower Hospital. All patients fully understood the objective and adverse reactions of the study, and signed the written informed consent voluntarily prior to study enrollment.

Patients and randomization

From January 2006 to July 2010, we prospectively investigated 66 patients (45 men and 21 women; mean age, 51.6 years; range, 34-64 years) who underwent orthotopic liver transplantation in the Department of Hepatobiliary Surgery at the Affiliated Drum Tower Hospital of Medical School of Nanjing University. Recipients with manifest metabolic diseases (e.g., diabetes mellitus and hyperthyroidism) or severe renal abnormality were excluded. No acute rejection, primary transplanted liver dysfunction or second operation, which may affect the evaluation of liver function, were seen during the first 9 d after transplantation. The selection criteria for donors in this study included: (1) age < 50 years, matched ABO blood group and no history of chronic liver disease; (2) no evidence of malignant tumor, viral hepatitis or other viral infections; (3) no cirrhosis, mass or severe fatty degeneration of the donor liver seen during organ harvesting; and (4) liver biopsies of each donor liver taken before transplantation were reviewed by two pathologists. Donor livers with normal pathology or mild fatty change (10%-30%) were included in this study (Figure 1).

Figure 1.

Pathology of liver biopsy of donor liver taken before transplantation. Mild steatosis was observed in donor liver. (Hematoxylin and eosin staining; paraffin-embedded 5 μm thick sections; ×200).

After transplantation, these patients were randomized into two groups based on the randomization chart generated by the Statistical Analysis System (SAS): (1) PN group (33 patients), without supplementation of omega-3 fatty acids in addition to routine treatment; and (2) PUFA group (33 patients), with PN supplemented with omega-3 fatty acids in addition to routine treatment.

This is a randomized controlled clinical study carried out in the Department of Hepatobiliary Surgery of our hospital. Nutrition Risk Screening 2002 (NRS 2002) scoring system was used, and the post-operative NRS 2002 score was ≥ 3 in all the patients, which meant that all the patients need nutritional support.

Treatment

The PN was given around the clock for seven days from the second day after operation. The two nutritional support groups were isonitrogenous and isocaloric. Nitrogen intake was 0.16 g/kg body weight per day, caloric intake was 104.5 kJ/kg per day, and lipid intake was 1.0 g/kg per day. The nonprotein calories were provided with dextrose (4.0 g/kg per day) and fat emulsion in a ratio of 2:1. The only source of lipids in PN group was the standard lipid emulsion (20% emulsion, with a ratio of long-chain triglycerides to medium-chain triglycerides of 1:1, Huarui Pharmaceuticals, Jiangsu, China), and in PUFA group omega-3 fish oil lipid emulsion (Omegaven, 10%, 2 mL/kg per day, Fresenius Kabi Co., Austria) replaced part of the standard lipid emulsion. Both groups received 1.0 g amino acid/kg per day, and they were administered a commercially available branched-chain amino acid solution (Branched-chain amino acid solution 20%, Huarui Pharmaceuticals, Jiangsu, China). The ratio of nonprotein calories to nitrogen in both nutritional support groups was 653 kJ:1 g. The omega-3 fish oil lipid emulsion-containing solutions were prepared by the clinical pharmacist under aseptic condition and adjusted according to the weight of each individual patient. The amino acids, fat emulsion and dextrose mixture with electrolytes, vitamins, and trace elements were administered through a central venous catheter. As soon as the bowel function returned on days 3 or 4 after transplantation, all patients in the two groups were given liquid carbohydrate and cow’s milk protein.

The surgical treatment was standardized, and modified piggyback orthotopic liver transplantation was performed by three groups of surgeons using the same approach. After operation, all the patients in the two groups were treated with the same antibiotics and antivirutics, and 20 g of albumin was administered intravenously daily for five days to prevent any complications caused by hypoalbuminemia.

Assessment

Venous heparin blood samples were obtained on days 1 (the day before transplantation), 2 and 9 after surgery and liver function assessment was made. Serum total bilirubin (TB), direct bilirubin (DB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and prothrombin time (PT) were measured by an automatic biochemical analyzer (HITACHI 7600, Japan).

Liver biopsy with fine needle was conducted after reperfusion of donor liver and on day 9 after surgery, respectively. Hepatic specimens for light microscopy were fixed with formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin for histological examination. Portal inflammation in the liver biopsy specimens was semiquantified by calculating inflammatory cells in portal tracts based on the Knodell histology activity index (HAI)[15]. Portal inflammation was scored as 0, no portal inflammation; 1, mild (sprinkling of inflammatory cells in < 1/3 of portal tracts); 3, moderate (increased inflammatory cells in 1/3-2/3 of portal tracts); and 4, marked (dense packing of inflammatory cells in > 2/3 of portal tracts).

The assessment of clinical outcome was based on post-transplant investigations as shown by: (1) post-transplant mechanical ventilation; (2) total hospital stay; (3) infectious morbidities (pneumonia, intra-abdominal abscess, central line sepsis, wound infection, and urinary tract infection); (4) acute and chronic rejection; (5) mortality (intensive care unit mortality, hospital mortality, 28-d mortality, and survival at one year post-transplant surveillance period).

These post-transplant parameters were investigated and documented daily during the patients’ post-transplant hospital stay and the period of one-year postoperative follow-up.

Statistical analysis

The results were expressed as mean ± SD. Data were analyzed using the SAS. Differences between means were evaluated using Student t test when normal distribution was confirmed by Shapiro-Wilks test. When the hypothesis of normal distribution was rejected, differences between groups were tested by nonparametric statistics using Mann-Whitney test for unpaired samples and Wilcoxon criteria for paired samples. Fisher’s exact test was used for analysis of categorical values when appropriate. A P value of < 0.05 was considered significant.

RESULTS

A total of 66 patients were enrolled in this study, including 33 patients in PN group and 33 patients in PUFA group. The mean age of the subjects was 51.6 years (range, 34-64 years). The clinical diagnosis of these patients included: hepatic cell carcinoma (27 cases), post-hepatitis B liver cirrhosis (35 cases), alcoholic liver cirrhosis (1 case), primary biliary liver cirrhosis (2 cases) and congenital polycystic liver (1 case). Demographic and clinical data (including age, sex, clinical diagnosis, Child-Pugh classification of hepatic function, warm ischemic time, cold ischemic time, operative time, anhepatic phase and post-operative immunosuppression) are summarized in Table 1. With respect to warm ischemic time, cold ischemic time, operative time, anhepatic phase, ratio of Child-Pugh classification, immunosuppression and clinical diagnosis, there were no significant differences between the two groups in any of these above parameters (P > 0.05).

Table 1.

Clinical data of the enrolled patients

	PN group	PUFA group
Sex (M/F)	23/10	22/11
Age, yr	48.62 ± 14.61	51.52 ± 12.41
Clinical diagnosis
Hepatic cell carcinoma	13	14
Post- hepatitis B liver cirrhosis	17	18
Alcoholic liver cirrhosis	1	0
Primary biliary liver cirrhosis	1	1
Congenital polycystic liver	1	0
Child-Pugh classification (A/B/C)	14/10/9	13/10/10
Warm ischemic time (min)	3.91 ± 1.16	4.15 ± 1.32
Cold ischemic time (min)	524.28 ± 132.83	506.56 ± 151.26
Operation period (min)	651.27 ± 181.42	626.39 ± 192.86
Anhepatic phase (min)	119.81 ± 82.35	142.15 ± 58.75
Immunosuppressive therapy	22/11/0	21/11/1
(FK506 + P/CSA + P/CSA + P + MMF)

PN: Parenteral nutrition; PUFA: Polyunsaturated fatty acid; P: Prednisone; CSA: Ciclosporin A; MMF: Mycophenolate mofetil; FK506: Tacrolimus.

Liver function assessment

No significant difference of pre-operative liver function was observed between the two groups. On days 2 and 9 after operation, a significant decrease of ALT (299.16 U/L ± 189.17 U/L vs 246.16 U/L ± 175.21 U/L, P = 0.024) and PT (5.64 s ± 2.06 s vs 2.54 s ± 1.15 s, P = 0.035) was seen in PUFA group compared with PN group. And there was no significant decrease of the following parameters tested on days 2 and 9: AST (116.31 U/L ± 42.19 U/L vs 121.09 U/L ± 53.14 U/L, P=0.682), TB (93.93 μmol/L ± 45.49 μmol/L vs 87.20 μmol/L ± 61.12 μmol/L, P = 1.439), DB (42.74 μmol/L ± 17.36 μmol/L vs 36.22 μmol/L ± 21.63 μmol/L, P = 0.815) and LDH (156.12 U/L ± 89.20 U/L vs 119.10 U/L ± 69.72 U/L, P = 1.112) in PUFA group compared with PN group (Table 2).

Table 2.

Effect of parenteral nutritional support with Omega-3 fatty acids on liver function

	Normal value	Group	Day 1	Day 2	Day 9	Decrease (Day 2-Day 9)
ALT (U/L)	5-40	PN group	198.16 ± 117.13	401.32 ± 215.35	155.16 ± 108.41b	246.16 ± 175.21
		PUFA group	227.16 ± 121.17	410.98 ± 201.64	101.82 ± 71.24b	299.16 ± 189.17c
AST (U/L)	8-40	PN group	95.12 ± 61.79	203.25 ± 73.49	82.16 ± 46.16b	121.09 ± 53.14
		PUFA group	115.62 ± 81.27	185.12 ± 42.16	68.81 ± 24.32b	116.31 ± 42.19
TB (μmol/L)	5-20.5	PN group	92.16 ± 42.15	158.32 ± 65.41	71.12 ± 55.12a	87.20 ± 61.12
		PUFA group	116.82 ± 61.65	160.34 ± 68.24	66.41 ± 61.52b	93.93 ± 45.49
DB (μmol/L)	1.7-6.8	PN group	52.15 ± 32.95	76.46 ± 31.28	40.24 ± 26.69b	36.22 ± 21.63
		PUFA group	47.39 ± 27.19	81.25 ± 26.32	38.51 ± 19.87b	42.74 ± 17.36
LDH (U/L)	109-245	PN group	266.25 ± 132.42	476.25 ± 98.15	357.15 ± 192.52a	119.10 ± 69.72
		PUFA group	226.45 ± 172.24	416.38 ± 151.14	260.26 ± 111.32b	156.12 ± 89.20
PT (s)	10-15	PN group	18.76 ± 3.21	17.16 ± 4.05	14.62 ± 3.87a	2.54 ± 1.15
		PUFA group	19.12 ± 4.16	17.81 ± 3.82	12.17 ± 3.69b	5.64 ± 2.06c

PN: Parenteral nutrition; PUFA: Polyunsaturated fatty acid; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TB: Total bilirubin; DB: Direct bilirubin; LDH: Lactate dehydrogenase; PT: Prothrombin time.

^aP < 0.05,

^bP < 0.01 vs day 2;

^cP < 0.05 vs PN group.

Light microscopy

The histological examination after reperfusion revealed some swelling hepatocytes and inflammatory cell infiltration in the portal areas, and no significant difference of numerical score of portal inflammation was observed between the two groups.

Histological examination on day 9 in PN group revealed more inflammatory cells aggregating in hepatic sinusoid lumen, extensive swelling and some balloon-like degeneration of hepatocytes, extensive congestion, and bilirubin deposit in the hepatic plasma (Figure 2A). These were ameliorated markedly by parenteral nutritional support with omega-3 fatty acids (Figure 2B), and the numerical score of portal inflammation was significantly lowered in PUFA group (Table 3). There was no sign of acute rejection in both groups.

Figure 2.

Histological appearance of the transplanted liver on day 9 after surgery. A: Extensive swelling, balloon-like degeneration and necrosis of hepatocytes, extensive congestion and bilirubin deposit were observed in the hepatic plasma in parenteral nutrition group; B: The results of histological examination were ameliorated markedly in the polyunsaturated fatty acid group. (Hematoxylin and eosin staining; paraffin-embedded 5 μm thick sections; ×200).

Table 3.

Effect of parenteral nutritional support with Omega-3 fatty acids on numerical score of portal inflammation

Group	Day 0	Day 9
PN group	3.3 ± 0.5	2.7 ± 0.9
PUFA group	3.6 ± 0.4	1.8 ± 0.6a

PN: Parenteral nutrition; PUFA: Polyunsaturated fatty acid.

^aP < 0.05 vs PN group.

Clinical outcome

There was no significant difference of post-transplant mechanical ventilation between the two groups (P > 0.05). Compared with PN group, the post-transplant hospital stay was significantly shortened in PUFA group (P < 0.05). Infectious complications occurred in 6 cases of PN group (2 cases of pneumonia, 3 cases of intra-abdominal abscess and 1 case of urinary tract infection), and in 3 cases of PUFA group (2 cases of pneumonia, 1 case of intra-abdominal abscess). No acute or chronic rejection and hospital mortality were found in the two groups. All patients were followed up, and the one-year mortality in PN group was 9.1% (3/33), one died of pulmonary infection, one died of severe intra-hepatic cholangitis and hepatic dysfunction and the other of hepatic cell carcinoma recurrence. Only one patient in PUFA group (1/33, 3.1%) died of biliary complication and hepatic dysfunction during follow-up (Table 4).

Table 4.

Effect of parenteral nutritional support with Omega-3 fatty acids on clinical outcome

	PN group	PUFA group
Post-transplant mechanical ventilation (h)	12.1 ± 5.1	10.8 ± 5.4
Post-transplant hospital stay (d)	20.6 ± 4.6	18.7 ± 4.0a
Infectious morbidities	6/33	3/33a
Acute/chronic rejection	0/33	0/33
ICU mortality	0/33	0/33
Hospital mortality	0/33	0/33
28-d mortality	0/33	0/33
One-year mortality	3/33	1/33

PN: Parenteral nutrition; PUFA: Polyunsaturated fatty acid; ICU: Intensive care unit.

^aP < 0.05 vs PN group.

DISCUSSION

An impairment of nutritional status is a frequent finding in patients with end-stage liver disease. Malnutrition adversely affects the prognosis of these patients and is associated with the morbidity and mortality after liver transplantation[16]. Malnutrition has been shown to be the only independent risk factor for the length of stay in the intensive care and the total number of days spent in the hospital, and the liver recipient’s nutritional status also influences the incidence of post-transplant complications and may therefore increase the costs of liver transplant[17,18]. After liver transplantation, surgical stress, postoperative fasting, and the possible occurrence of complications suggest the need for nutritional support. The primary goal of the nutrition support in the immediate post-transplant period is to provide adequate nutrition to promote recovery and replenishment of the depleted nutrient stores. Although most transplant centers use the similar post-transplant nutritional support as for other major abdominal operations, few studies have elucidated the role of postoperative nutritional support in the liver recipients.

Enteral nutrition is safer and less expensive than PN, and enteral nutrition has the potential advantage of maintaining intestinal trophism more effectively[19]. This effect may help prevent bacterial translocation and enteric-origin infections in patients treated with transplantation[20,21]. All patients in this study resumed their daily oral diet postoperatively as soon as bowel function returned to maintain intestinal trophism, but the recipients could not endure a large amount of liquid diet even with nasoenteric tube at the early phase after transplantation because of obvious abdominal pain, distention or diarrhea in our previous experience. The bowel function in all the patients in this study returned on day 3 or 4 after transplantation, and PN support discontinued on day 8 after surgery when the patients were able to maintain an adequate oral intake.

Omega-3 fatty acids mainly act as eicosapentaenoic acids (EPA) and docosahexaenoic acids (DHA), both had anti-inflammatory effects. EPA and DHA reduce the release of arachidonic acid-derived pro-inflammatory eicosanoids, and generate a group of lipid mediators called resolvins (E- and D-series) and protectins with potent anti-inflammatory and inflammation resolution properties[22,23]. Studies with experimental models of liver reperfusion injury have reported the beneficial actions of n-3 PUFA-derived resolvins and protectins in preventing liver DNA damage and oxidative stress, thus significantly ameliorating the necroinflammatory liver injury and hepatic steatosis[24-26]. The liver-protecting effects of postoperative PN support supplemented with omega-3 fatty acids were evaluated in this study. Liver enzyme of ALT released after I/R was significantly suppressed by the supplements of omega-3 fatty acids. PT, as an important parameter in evaluating the synthesis function of liver, was significantly decreased in PUFA group. And the results of histological examination on day 9 revealed that the hepatocyte injury and inflammatory cell aggregation were ameliorated markedly in PUFA group. PUFA therapy could also decrease the infectious morbidities, and shorten the post-transplant hospital stay significantly. The possible mechanisms of omega-3 fatty acids include down-regulation of the inflammatory responses to surgery and immune modulation rather than a sole nutritional effect.

Some of the patients exhibited an obvious nitrogen accumulation disorder reflected by either encephalopathy or an excessive rise in blood urea nitrogen in the immediate postoperative period. Branched-chain amino acids were chosen for this study because it can promote protein synthesis in patients with chronic liver diseases and avoid the additional metabolic load of transplanted liver[27]. Medium-chain triglycerides were included in the regimen to avert glucose intolerance and deposits in the transplanted liver. Based on the results of this study, we think that post-transplant nutritional support in the form of a solution enriched with branched-chain amino acids, dextrose, medium-chain triglycerides and omega-3 fatty acids might offer a benefit in terms of preserved liver function and better clinical outcome, including the decreased infectious morbidities and post-transplant hospital stay.

In conclusion, we have shown that omega-3 fatty acids-supplemented PN significantly improves the injury of transplanted liver, decreases the infectious morbidities, and shortens the post-transplant hospital stay.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Chen Jun for his contribution to the pathological analysis.

COMMENTS

Background

The liver recipients with liver insufficiency are known to experience a higher incidence of severe protein/calorie malnutrition, and malnutrition is associated with a greater risk of postoperative complications and mortality in patients undergoing liver transplantation. And ischemia/reperfusion injury associated with liver transplantation often leads to hepatic dysfunction despite the improvements in surgical techniques and perioperative medication.

Research frontiers

Omega-3 fatty acids exert anti-inflammatory and immunomodulatory properties through their ability to modulate the synthesis of different eicosanoids. Recent studies have described that supplementation with omega-3 fatty acids decreases the rate of inflammatory complication, the length of hospital stay, and the mortality after major abdominal surgeries.

Innovations and breakthroughs

Although most transplant centers use the similar post-transplant nutritional support as for other major abdominal operations, few studies have elucidated the role of postoperative nutritional support in the organ recipient. Based on the results of this study, the authors have shown that post-transplant nutritional support in the form of a solution enriched with branched-chain amino acids, dextrose, medium-chain triglycerides and omega-3 fatty acids might offer a benefit in terms of preserved liver function and better clinical outcome, including the decreased infectious morbidities and post-transplant hospital stay.

Applications

This study has shown that omega-3 fatty acids-supplemented parenteral nutrition (PN) significantly improves the injury of transplanted liver, decreases the infectious morbidities, and shortens the post-transplant hospital stay. The nutritional support strategy is recommended in patients undergoing liver transplantation.

Peer review

The manuscript evaluates the potential for supplementation with polyunsaturated fatty acid to ameliorate hepatic injury associated with reperfusion and PN. It provides evidences about an efficient nutritional support strategy for liver transplanted patients.