Development and validation of the AMMON-OHE model to predict risk of overt hepatic encephalopathy occurrence in outpatients with cirrhosis.

BACKGROUND & AIMS: Neuropsychological and psychophysical tests are recommended to assess the risk of overt hepatic encephalopathy (OHE), but their accuracy is limited. Hyperammonaemia is central in the pathogenesis of OHE, but its predictive utility is unknown. In this study, we aimed to determine the role of neuropsychological or psychophysical tests and ammonia, and to develop a model (AMMON-OHE) to stratify the risk of subsequent OHE development in outpatients with cirrhosis. METHODS: This observational, prospective study included 426 outpatients without previous OHE from three liver units followed for a median of 2.5 years. Psychometric hepatic encephalopathy score (PHES) <-4 or critical flicker frequency (CFF) <39 was considered abnormal. Ammonia was normalized to upper limit of normal (AMM-ULN) at the respective reference laboratory. Multivariable frailty competing risk and random survival forest analyses were performed to predict future OHE and to develop the AMMON-OHE model. External validation was carried out using 267 and 381 patients from two independent units. RESULTS: Significant differences were found in time-to-OHE (log-rank p <0.001) according to PHES or CFF and ammonia, with the highest risk in patients with abnormal PHES plus high AMM-ULN (hazard ratio 4.4; 95% CI 2.4-8.1; p <0.001 compared with normal PHES and AMM-ULN). On multivariable analysis, AMM-ULN but not PHES or CFF was an independent predictor of the development of OHE (hazard ratio 1.4; 95% CI 1.1-1.9; p = 0.015). The AMMON-OHE model (sex, diabetes, albumin, creatinine and AMM-ULN) showed a C-index of 0.844 and 0.728 for the prediction of a first episode of OHE in two external validation cohorts. CONCLUSIONS: In this study, we developed and validated the AMMON-OHE model, comprising readily available clinical and biochemical variables that can be used to identify outpatients at the highest risk of developing a first episode of OHE. IMPACT AND IMPLICATIONS: In this study, we aimed to develop a model to predict which patients with cirrhosis are at risk of developing overt hepatic encephalopathy (OHE). Using data from three units and including 426 outpatients with cirrhosis, we developed the AMMON-OHE model - comprising sex, diabetes, albumin, creatinine and ammonia levels - which demonstrated good predictive ability. The AMMON-OHE model performs better than PHES and CFF to predict the first episode of OHE in outpatients with cirrhosis. This model was validated in 267 and 381 patients from two independent liver units. The AMMON-OHE model is available online for clinical use.

Plasma ammonia levels predict hospitalisation with liver-related complications and mortality in clinically stable outpatients with cirrhosis.

BACKGROUND & AIMS: Hyperammonaemia is central in the pathogenesis of hepatic encephalopathy. It also has pleiotropic deleterious effects on several organ systems, such as immune function, sarcopenia, energy metabolism and portal hypertension. This study was performed to test the hypothesis that severity of hyperammonaemia is a risk factor for liver-related complications in clinically stable outpatients with cirrhosis. METHODS: We studied 754 clinically stable outpatients with cirrhosis from 3 independent liver units. Baseline ammonia levels were corrected to the upper limit of normal (AMM-ULN) for the reference laboratory. The primary endpoint was hospitalisation with liver-related complications (a composite endpoint of bacterial infection, variceal bleeding, overt hepatic encephalopathy, or new onset or worsening of ascites). Multivariable competing risk frailty analyses using fast unified random forests were performed to predict complications and mortality. External validation was carried out using prospective data from 130 patients with cirrhosis in an independent tertiary liver centre. RESULTS: Overall, 260 (35%) patients were hospitalised with liver-related complications. On multivariable analysis, AMM-ULN was an independent predictor of both liver-related complications (hazard ratio 2.13; 95% CI 1.89-2.40; p <0.001) and mortality (hazard ratio 1.45; 95% CI 1.20-1.76; p <0.001). The AUROC of AMM-ULN was 77.9% for 1-year liver-related complications, which is higher than traditional severity scores. Statistical differences in survival were found between high and low levels of AMM-ULN both for complications and mortality (p <0.001) using 1.4 as the optimal cut-off from the training set. AMM-ULN remained a key variable for the prediction of complications within the random forests model in the derivation cohort and upon external validation. CONCLUSION: Ammonia is an independent predictor of hospitalisation with liver-related complications and mortality in clinically stable outpatients with cirrhosis and performs better than traditional prognostic scores in predicting complications. LAY SUMMARY: We conducted a prospective cohort study evaluating the association of blood ammonia levels with the risk of adverse outcomes in 754 patients with stable cirrhosis across 3 independent liver units. We found that ammonia is a key determinant that helps to predict which patients will be hospitalised, develop liver-related complications and die; this was confirmed in an independent cohort of patients.

The Macrophage Activation Marker Soluble CD163 is Associated With Early Allograft Dysfunction After Liver Transplantation.

BACKGROUND/OBJECTIVES: Soluble CD163 (sCD163), a macrophage activation marker, is upregulated in conditions of macrophage proliferation and activation. Elevated sCD163 levels have been associated with liver disease severity and progression. During liver transplantation, the implanted liver is exposed to ischaemia and reperfusion injury, resulting in an acute inflammatory response and macrophage activation. The relationship between sCD163 levels during liver transplantation and the development of early allograft dysfunction (EAD) has not been investigated. METHODS: We included 27 cirrhosis patients (age 55 [range 32-72] years, 23 men) on the waiting list for liver transplantation. Alcohol consumption and viral hepatitis were the most frequent causes for cirrhosis. Patients were characterised by standard biochemical analysis and based on clinical disease severity scores. Information about donor, graft and course of the liver transplantation was recorded. sCD163 levels were measured at the time of liver transplantation before surgery, 2 h after reperfusion, and then at 24 h after transplantation. RESULTS: We observed above-normal sCD163 levels at baseline (5.9 mg/L [4.7-8.8]). Two hours after reperfusion, sCD163 levels increased significantly from baseline (8.4 mg/L [7.4-10.9]; P < 0.01). Twenty-four hours after transplantation, sCD163 levels were significantly reduced compared with baseline (3.7 mg/L [2.9-5.5]; P < 0.01). However, in patients with EAD (n = 16), sCD163 levels were increased compared with patients without EAD (4.1 [3.2-7.4] vs. 3.1 [2.8-3.8] mg/L; P = 0.03). CONCLUSIONS: We observed elevated sCD163 levels in patients with EAD after liver transplantation, confirming macrophage activation to play a role in EAD. Thus, sCD163 may be used as an early marker for EAD after liver transplantation, but larger studies are warranted to validate these findings.

The IGF system in patients with inflammatory bowel disease treated with prednisolone or infliximab: potential role of the stanniocalcin-2 / PAPP-A / IGFBP-4 axis.

BACKGROUND: Patients with inflammatory bowel disease (IBD) present with reduced serum insulin-like growth factor I (IGF-I). Anti-inflammatory treatment with prednisolone or infliximab ameliorates symptoms and increases circulating IGF-I, but prednisolone induces catabolism, whereas infliximab may promote protein synthesis. Recently, stanniocalcin-2 (STC2) was discovered as a novel inhibitor of the enzyme pregnancy-associated plasma protein-A (PAPP-A), which modulates IGF-I activity. PAPP-A can cleave IGF binding protein-4 (IGFBP-4), upon which IGF-I is liberated. We hypothesized that prednisolone and infliximab exert different effects on levels of STC2, PAPP-A, and IGFBP-4, thereby explaining the distinct metabolic effects of prednisolone and infliximab. METHODS: Thirty-eight patients with active IBD treated with either prednisolone (n = 17) or infliximab (n = 21) were examined before and after 7 days of treatment. Circulating levels of IGF-I, IGF-II, IGFBP-3, PAPP-A, and STC2 were measured by immunoassays. Intact IGFBP-4 and two IGFBP-4 fragments were determined by a novel immunoassay. Bioactive IGF was assessed by cell-based IGF receptor activation assay. Concentrations of IGFBP-4, PAPP-A, and STC2 on day 0 and 7 were compared to healthy control subjects. RESULTS: Following seven days of prednisolone treatment, total and bioactive IGF-I were increased (p < 0.001 and p < 0.05, respectively). Upon infliximab treatment, total IGF-I levels were augmented (p < 0.05), yet IGF bioactivity remained unaltered. Intact IGFBP-4 and the two IGFBP-4 fragments generated upon cleavage by PAPP-A were all decreased following treatment with either prednisolone or infliximab (all p < 0.05). PAPP-A levels were only increased by infliximab (p = 0.005), whereas the inhibitor STC2 did not respond to any of the treatments. CONCLUSION: IGF-I and IGFBP-4 concentrations were markedly altered in patients with IBD and near-normalized with disease remission following treatment with prednisolone or infliximab. Thus, IGFBP-4 may modulate IGF bioavailability in IBD. The effect of immunosuppression did not appear to extend beyond the regulation of IGF and IGFBP-4, as neither PAPP-A nor STC2 were discernibly affected. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00955123 . Date of registration: August 7, 2009 (retrospectively registered).

Bariatric surgery in patients with non-alcoholic fatty liver disease - from pathophysiology to clinical effects.

Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized as a significant liver disease, and it covers the disease spectrum from simple steatosis with a risk of development of non-alcoholic steatohepatitis (NASH) to fibrosis, subsequent cirrhosis, end-stage liver failure, and liver cancer with a potential need for liver transplantation. NAFLD and NASH are closely related to obesity, metabolic syndrome, and type 2 diabetes (T2D). The role of gut hormones, especially glucagon-like peptide 1 (GLP-1), is important in NAFLD. Bariatric surgery has the potential for inducing great weight loss and may improve the symptoms of metabolic syndrome and T2D. Recent data demonstrated significant effects of bariatric surgery on GLP-1 and other gut hormones and important lipid metabolic and inflammatory abnormalities in the pathophysiology of NAFLD. Therefore, bariatric surgery may reverse the pathological liver changes in NAFLD and NASH patients. In the present review, we describe NAFLD and NASH pathophysiology and the primary effects of bariatric surgery on metabolic pathways. We performed a systematic review of the beneficial and harmful effects and focused on changes in liver disease severity in NAFLD and NASH patients. The specific focus was liver histopathology as assessed by the invasive liver biopsy. Additionally, we reviewed several non-invasive methods used for the assessment of liver disease severity following bariatric surgery.

Hepatic transcriptome signatures in patients with varying degrees of nonalcoholic fatty liver disease compared with healthy normal-weight individuals.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of conditions ranging from simple steatosis (NAFL), over nonalcoholic steatohepatitis (NASH) with or without fibrosis, to cirrhosis with end-stage disease. The hepatic molecular events underlying the development of NAFLD and transition to NASH are poorly understood. The present study aimed to determine hepatic transcriptome dynamics in patients with NAFL or NASH compared with healthy normal-weight and obese individuals. RNA sequencing and quantitative histomorphometry of liver fat, inflammation and fibrosis were performed on liver biopsies obtained from healthy normal-weight ( n = 14) and obese ( n = 12) individuals, NAFL ( n = 15) and NASH ( n = 16) patients. Normal-weight and obese subjects showed normal liver histology and comparable gene expression profiles. Liver transcriptome signatures were largely overlapping in NAFL and NASH patients, however, clearly separated from healthy normal-weight and obese controls. Most marked pathway perturbations identified in both NAFL and NASH were associated with markers of lipid metabolism, immunomodulation, extracellular matrix remodeling, and cell cycle control. Interestingly, NASH patients with positive Sonic hedgehog hepatocyte staining showed distinct transcriptome and histomorphometric changes compared with NAFL. In conclusion, application of immunohistochemical markers of hepatocyte injury may serve as a more objective tool for distinguishing NASH from NAFL, facilitating improved resolution of hepatic molecular changes associated with progression of NAFLD. NEW & NOTEWORTHY Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in Western countries. NAFLD is associated with the metabolic syndrome and can progress to the more serious form, nonalcoholic steatohepatitis (NASH), and ultimately lead to irreversible liver damage. Using gold standard molecular and histological techniques, this study demonstrates that the currently used diagnostic tools are problematic for differentiating mild NAFLD from NASH and emphasizes the marked need for developing improved histological markers of NAFLD progression.

Non-alcoholic steatohepatitis weakens the acute phase response to endotoxin in rats.

BACKGROUND & AIMS: Patients with non-alcoholic steatohepatitis (NASH) have increased mortality, including from infections. We, therefore, tested in a rodent model of steatohepatitis whether the hepatic acute phase response is intact. METHODS: Steatohepatitis was induced in rats by feeding a high-fat, high-cholesterol diet for 4 (early) and 16 weeks (advanced NASH). 2 h after low-dose LPS (0.5 mg/kg i.p.), we measured the serum concentrations of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). We also measured liver mRNA's and the serum concentrations of acute phase proteins 24 h after LPS. RESULTS: Non-alcoholic steatohepatitis in itself increased the liver mRNA levels of TNF-α and IL-6 and also the liver mRNA and serum levels of the acute phase proteins. The exposure to LPS increased serum TNF-α in both early and advanced NASH and more so than in the control rats. However, the increases in acute phase protein genes in liver tissue and proteins in the blood were lower than in the control rats. CONCLUSION: In rats with early or advanced experimental NASH, LPS despite an increased interleukin release resulted in a blunted acute phase protein response. This tachyphylaxis may be part of the mechanism for the increased infection susceptibility of patients with NASH. We speculate that the steatosis-related interleukin release desensitises the signalling pathway leading to acute phase protein synthesis.

Prednisolone but not infliximab aggravates the upregulated hepatic nitrogen elimination in patients with active inflammatory bowel disease.

BACKGROUND: Catabolism and weight loss are serious problems in patients with active inflammatory bowel disease (IBD). The body nitrogen (N) depletion is partly related to increased hepatic capacity for the elimination of N through urea synthesis. This is probably caused by the inflammation per se, and the treatment with prednisolone may aggravate the problem, whereas the effect of biological therapy is unknown. Therefore, we examined the effects of prednisolone or infliximab on the regulation of urea synthesis in patients with active IBD. METHODS: Urea synthesis was quantified by the functional hepatic nitrogen clearance (FHNC), i.e., the slope of the linear relationship between the urea nitrogen synthesis rate and the blood α-amino nitrogen concentration during alanine infusion. Thirty-seven patients with active IBD treated with either prednisolone or infliximab were examined before and after 7 days of treatment. RESULTS: At baseline, the FHNC was similar in the 2 treatment groups (36 L/h). After 7 days, prednisolone increased the FHNC by 40% (55 L/h) (P = 0.03), whereas infliximab tended to reduce the FHNC by 15% (30 L/h) (P = 0.09). The changes in the FHNC differed significantly between the 2 treatment groups (P < 0.01). CONCLUSIONS: Prednisolone treatment further upregulated urea synthesis, which increases the hepatic loss of nitrogen and promotes body catabolism. In contrast, infliximab treatment caused no such aggravation and likely reduced the N loss. These results may argue in favor of infliximab therapy for IBD and add to the pathophysiological understanding of the interplay between inflammation, catabolism, and anti-inflammatory treatment.

IL-6 has no acute effect on the regulation of urea synthesis in vivo in rats.

BACKGROUND: Clinical or experimentally induced, active inflammation up-regulates the in vivo capacity of urea synthesis (CUNS), which promotes nitrogen removal from the body and metabolic catabolism. We have shown that tumor necrosis factor α (TNF-α) up-regulates CUNS and increases interleukin 6 expression (IL-6) within hours of administration. The described effect of TNF-α on nitrogen homeostasis may, therefore, depend on IL-6. METHODS: Three hours after the i.v. injection of 125 µg.kg⁻¹ of IL-6 or placebo, we evaluated the CUNS, hepatocyte urea cycle enzyme protein levels and the mRNA levels of the urea cycle enzyme genes in rats. The prevailing rat serum acute phase proteins and their liver mRNA levels were also measured. RESULTS: IL-6 did not change CUNS or hepatocyte urea cycle enzyme protein levels, whereas urea cycle enzyme mRNA levels, except for ornithine transcarbamylase (OTC), decreased by approximately 20%. The liver mRNA levels of α2MG, haptoglobin and α1AGP all increased by 1.5- to 2-fold (p < 0.001). In serum, only the α2MG concentration slightly increased (p < 0.001), whereas the levels of the other circulating acute phase proteins remained unchanged. CONCLUSION: IL-6 is not the mediator of the in vivo CUNS up-regulation observed 3 h after TNF-α administration, but it may be involved in the down-regulation of urea cycle genes. IL-6 may also mediate TNF-α effects on acute phase protein gene expression. Thus, IL-6 did not contribute to the in vivo hepatic component of inflammation-associated catabolism.

Unchanged capacity of urea synthesis during acute phase response in rats.

BACKGROUND: The acute phase response presents a catabolic event related to increased waste of amino-N via hepatic urea synthesis despite an increased need for amino-N incorporation into acute phase proteins. In our previous studies, tumour necrosis factor-α (TNF-α) acutely up-regulated the in vivo capacity of urea-nitrogen synthesis (CUNS) in rats before the hepatic acute phase response was established. To extend these observations, this study aimed to clarify the regulation of N elimination via urea during the later stages of the acute phase response. METHODS: Twenty-four hours after i.v. injection of 25 µg kg(-1) TNF-α or placebo, we determined the in vivo CUNS, hepatocyte urea cycle enzyme protein levels and mRNA levels of the urea cycle enzyme genes in pair-fed rats. In addition, serum acute phase proteins and their liver mRNA levels were measured. RESULTS: After TNF-α, CUNS and hepatocyte urea cycle enzyme protein expressions were unchanged while urea cycle enzyme mRNA levels decreased. Liver mRNA levels of α2MG, haptoglobin and α1AGP rose and their serum levels increased equally. CONCLUSION: Despite a fully established 24-h acute phase response, there was no change in the in vivo capacity for disposal of amino-N by urea synthesis or in the urea cycle enzyme proteins, although the expression of the urea cycle enzyme genes was decreased. Thus, in vivo urea synthesis was not orchestrated together with acute phase protein synthesis so as to limit N waste despite genetic regulation to this effect. This may contribute towards catabolism of inflammation.

Effects of insulin-like growth factor-I administration on in vivo regulation of urea synthesis in normal subjects and patients with cirrhosis.

BACKGROUND: The anabolic effects of insulin-like growth factor-I (IGF-I) may involve a decrease of hepatic nitrogen (N) clearance, but this has never been studied in humans. Patients with cirrhosis have low levels of IGF-I and might benefit from IGF-I therapy. Conversely, a possible decrease in hepatic N clearance by IGF-I could increase the risk of hepatic encephalopathy. AIMS: To examine the effects of 1-week IGF-I administration on the functional hepatic N clearance (FHNC), viz. the linear slope of the relationship between blood-α-amino-N concentration and urea-N synthesis rate as controlled by an infusion of alanine. METHODS: A randomized sequence-crossover placebo-controlled study. Eight healthy volunteers and eight patients with alcoholic cirrhosis received injections of saline or IGF-I twice daily (50 µg/kg) for 7 days. RESULTS: IGF-I levels at baseline were lower in the patients than those in the controls. The IGF-I treatment normalized patient levels and caused an increase in the controls to supra-physiological levels. FHNC was lower in patients compared with healthy subjects (23.0 vs 36.5 L/h, P=0.03). IGF-I treatment reduced FHNC by 30% in healthy subjects (from 36.5 to 25.7 L/h, P = 0.02), whereas no effect was found in the patients. CONCLUSION: IGF-I downregulates urea synthesis in normal subjects. This may be part of the explanation behind the anabolic effects of IGF-I. The normalization of IGF-I in cirrhosis patients without an effect on urea synthesis implies that the patients were resistant to IGF-I with regard to reduction of hepatic amino-N elimination. IGF-I treatment of cirrhosis patients evidently carries no risk of N accumulation.

Tumor necrosis factor-alpha acutely up-regulates urea synthesis in vivo in rats--a hepatic component of inflammatory catabolism?

BACKGROUND: Catabolism is a serious problem in patients with active inflammation. The tissue nitrogen (N) depletion is related to increased hepatic capacity for elimination of N via conversion of amino-N into urea-N. This is caused by the inflammatory process, but the mediators responsible are unknown. Tumor necrosis factor-alpha (TNF-alpha) plays a key role in inflammation, and we hypothesized that TNF-alpha up-regulates urea synthesis. METHODS: We examined the in vivo capacity of urea-N synthesis (CUNS) and mRNA levels of urea cycle enzyme genes 3 h after TNF-alpha injection in rats. Circulating concentrations of glucagon, corticosterone, insulin, glucose, cytokines and acute phase proteins and their liver tissue gene expressions were measured. RESULTS: TNF-alpha increased CUNS by 40% (p=0.03) despite decreased urea-cycle enzyme gene expression. TNF-alpha increased interleukin 6 (IL-6) (p < 0.001); circulating acute phase proteins were unchanged. CONCLUSION: TNF-alpha in rats caused an acute up-regulation of the in vivo capacity of urea synthesis which may promote loss of nitrogen from the body and catabolism. The results indicate that TNF-alpha has a post-transcriptional effect on regulation of urea synthesis that is independent of the acute phase protein synthesis. Effects of IL-6 may be involved.