Towards collaborative management of non-alcoholic fatty liver disease: a 'real-world' pathway for fibrosis risk assessment in primary care.

BACKGROUND: The optimal strategy to support primary care practitioners (PCP) to assess fibrosis severity in non-alcoholic fatty liver disease (NAFLD) and thereby make appropriate management decisions remains unclear. AIMS: To examine the feasibility of using a two-step pathway that combined simple scores (NAFLD Fibrosis Score and Fibrosis-4 Index) with transient elastography (FibroScan) to streamline NAFLD referrals from a 'routine' primary care population to specialist hepatology management clinics (HMC). METHODS: The two-step 'Towards Collaborative Management of NAFLD' (TCM-NAFLD) fibrosis risk assessment pathway was implemented at two outer metropolitan primary healthcare practices in Brisbane. Patients aged ≥18 years with a new or established PCP-diagnosis of NAFLD were eligible for assessment. The pathway triaged patients at 'high risk' of clinically significant fibrosis to HMC for specialist review, and 'low risk' patients to receive ongoing management and longitudinal follow up in primary care. RESULTS: A total of 162 patient assessments between June 2019 and December 2020 were included. Mean age was 58.7 ± 11.7 years, 30.9% were male, 54.3% had type 2 diabetes or impaired fasting glucose, and mean body mass index was 34.2 ± 6.9 kg/m2 . A total 122 patients was considered 'low risk' for clinically significant fibrosis, two patients had incomplete assessments and 38 (23.5%) were triaged to HMC. Among 31 completed HMC assessments to date, 45.2% were considered to have clinically significant (or more advanced) fibrosis, representing 9.2% of 153 completed assessments. CONCLUSION: Implementation of the two-step TCM-NAFLD pathway streamlined hepatology referrals for NAFLD and may facilitate a more cost-effective and targeted use of specialist hepatology resources.

Iron depletion attenuates steatosis in a mouse model of non-alcoholic fatty liver disease: Role of iron-dependent pathways.

BACKGROUND & AIMS: Iron has been proposed as influencing the progression of liver disease in subjects with non-alcoholic fatty liver disease (NAFLD). We have previously shown that, in the Hfe-/- mouse model of hemochromatosis, feeding of a high-calorie diet (HCD) leads to increased liver injury. In this study we investigated whether the feeding of an iron deficient/HCD to Hfe-/- mice influenced the development of NAFLD. METHODS: Liver histology was assessed in Hfe-/- mice fed a standard iron-containing or iron-deficient diet plus or minus a HCD. Hepatic iron concentration, serum transferrin saturation and free fatty acid were measured. Expression of genes implicated in iron regulation and fatty liver disease was determined by quantitative real-time PCR (qRT-PCR). RESULTS: Standard iron/HCD-fed mice developed severe steatosis whereas NAS score was reduced in mice fed iron-deficient HCD. Mice fed iron-deficient HCD had lower liver weights, lower transferrin saturation and decreased ferroportin and hepcidin gene expression than HCD-fed mice. Serum non-esterified fatty acids were increased in iron-deficient HCD-fed mice compared with standard iron HCD. Expression analysis indicated that genes involved in fatty-acid binding and mTOR pathways were regulated by iron depletion. CONCLUSIONS: Our results indicate that decreasing iron intake attenuates the development of steatosis resulting from a high calorie diet. These results also suggest that human studies of agents that modify iron balance in patients with NAFLD should be revisited.

Liver Function Test Abnormalities in Experimental and Clinical Plasmodium vivax Infection.

Liver transaminase elevations after treatment in malaria volunteer infection studies (VISs) have raised safety concerns. We investigated transaminase elevations from two human Plasmodium vivax VISs where subjects were treated with chloroquine (n = 24) or artefenomel (n = 8) and compared them with studies in Thailand (n = 41) and Malaysia (n = 76). In the VISs, alanine transaminase (ALT) increased to ≥ 2.5 × upper limit of normal (ULN) in 11/32 (34%) volunteers, peaking 5-8 days post-treatment. Transaminase elevations were asymptomatic, were not associated with elevated bilirubin, and resolved by day 42. The risk of an ALT ≥ 2.5 × ULN increased more than 4-fold (odds ratio [OR] 4.28; 95% CI: 1.26-14.59; P = 0.02) for every log10 increase in the parasite clearance burden (PCB), defined as the log-fold reduction in parasitemia 24 hours post-treatment. Although an elevated ALT ≥ 2.5 × ULN was more common after artefenomel than after chloroquine (5/8 [63%] versus 6/24 [25%]; OR 5.0; 95% CI: 0.91-27.47; P = 0.06), this risk disappeared when corrected for PCB. Peak ALT also correlated with peak C-reactive protein (R = 0.44; P = 0.012). Elevations in ALT (≥ 2.5 × ULN) were less common in malaria-endemic settings, occurring in 1/41 (2.5%) Thai patients treated with artefenomel, and in none of 76 Malaysians treated with chloroquine or artemisinin combination therapy. Post-treatment transaminase elevations are common in experimental P. vivax infection but do not appear to impact on participant safety. Although the mechanism of these changes remains uncertain, host inflammatory response to parasite clearance may be contributory.

Assessment and Transplantation of Orphan Donor Livers: A Back-to-Base Approach to Normothermic Machine Perfusion.

Globally, a large proportion of donor livers are discarded due to concerns over inadequate organ quality. Normothermic machine perfusion (NMP) allows for hepatocellular and biliary viability assessment prior to transplantation and might therefore enable the safe use of these orphan donor livers. We describe here the first Australasian experience of NMP-preserved liver transplants using a 'back-to-base' approach, where NMP was commenced at the recipient hospital following initial static cold storage. In the preclinical phase, 10 human donor livers declined for transplantation (7 from donation after circulatory death [DCD] and 3 from donation after brain death [DBD]) were perfused using a custom-made NMP setup. Subsequently, 10 orphan donor livers (5 from DCD and 5 from DBD) underwent NMP and viability assessment on the OrganOx metra device (OrganOx Limited, Oxford, United Kingdom). Both hepatocellular and biliary viability criteria were used. The median donor risk index was 1.53 (1.16-1.71), and the median recipient Model for End-Stage Liver Disease score was 17 (11-21). In the preclinical phase, 'back-to-base' NMP was deemed suitable and feasible. In the clinical phase, each graft met predefined criteria for implantation during NMP and was subsequently transplanted. Five (50%) recipients developed early allograft dysfunction based on peak aspartate aminotransferase. To date, all grafts function satisfactorily, and none of the 5 recipients who received a DCD liver have developed cholangiopathy. The OrganOx metra using a back-to-base approach has enabled the safe use of 10 high-risk orphan donor livers with 100% 6-month patient and graft survival. NMP improved surgeon confidence to use orphan donor livers and has enabled a safe expansion of the donor pool.

Nonalcoholic Fatty Liver Disease: Interface Between Primary Care and Hepatology Clinics.

Primary care physicians (PCPs) have the primary role in the diagnosis and management of nonalcoholic fatty liver disease (NAFLD), and in selecting patients for referral to a hepatologist for further evaluation. This study aimed to characterize PCP referrals for patients diagnosed with NAFLD at a major referral hospital, and to determine the severity of liver disease and patient pathway following evaluation in secondary care. New patients seen in the hepatology outpatient clinic (HOC) with a secondary care diagnosis of NAFLD were identified from the HOC scheduling database. PCP referrals for these patients were retrieved from the electronic medical records and reviewed by study clinicians, along with the hepatologists' clinic notes and letters. Over a 14-month period, 234 new PCP referrals received a diagnosis of NAFLD, accounting for 20.4% of the total number of new cases (n = 1,147) seen in the HOC. The 234 referrals were received from 170 individual PCPs at 135 practices. Most patients with NAFLD (88.5%) were referred for investigation of abnormal liver enzymes or other clinical concerns, including abnormal iron studies, hepatomegaly, and abdominal pain. Only 27 (11.5%) referrals included an assessment of liver disease severity. Following evaluation in the liver clinic, 175 patients (74.8%) were found to have a low risk of advanced fibrosis, and most (n = 159; 90.9%) were discharged back to their PCP for ongoing follow-up in primary care. Conclusion: In addition to better access to noninvasive fibrosis tests, educational strategies to enhance awareness and recognition of NAFLD as a cause for many of the initial concerns prompting patient referral might improve risk stratification and increase the appropriateness of PCP referrals.

Hepatic iron concentration correlates with insulin sensitivity in nonalcoholic fatty liver disease.

Rodent and cell-culture models support a role for iron-related adipokine dysregulation and insulin resistance in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, substantial human data are lacking. We examined the relationship between measures of iron status, adipokines, and insulin resistance in patients with NAFLD in the presence and absence of venesection. This study forms part of the Impact of Iron on Insulin Resistance and Liver Histology in Nonalcoholic Steatohepatitis (IIRON2) study, a prospective randomized controlled trial of venesection for adults with NAFLD. Paired serum samples at baseline and 6 months (end of treatment) in controls (n = 28) and patients who had venesection (n = 23) were assayed for adiponectin, leptin, resistin, retinol binding protein-4, tumor necrosis factor α, and interleukin-6, using a Quantibody, customized, multiplexed enzyme-linked immunosorbent assay array. Hepatic iron concentration (HIC) was determined using MR FerriScan. Unexpectedly, analysis revealed a significant positive correlation between baseline serum adiponectin concentration and HIC, which strengthened after correction for age, sex, and body mass index (rho = 0.36; P = 0.007). In addition, there were significant inverse correlations between HIC and measures of insulin resistance (adipose tissue insulin resistance (Adipo-IR), serum insulin, serum glucose, homeostasis model assessment of insulin resistance, hemoglobin A1c, and hepatic steatosis), whereas a positive correlation was noted with the insulin sensitivity index. Changes in serum adipokines over 6 months did not differ between the control and venesection groups. Conclusion: HIC positively correlates with serum adiponectin and insulin sensitivity in patients with NAFLD. Further study is required to establish causality and mechanistic explanations for these associations and their relevance in the pathogenesis of insulin resistance and NAFLD. (Hepatology Communications 2018;2:644-653).

Ferroportin Expression in Adipocytes Does Not Contribute to Iron Homeostasis or Metabolic Responses to a High Calorie Diet.

BACKGROUND & AIMS: Iron has an increasingly recognized role in the regulation of adipose tissue function, including the expression of adipokines involved in the pathogenesis of nonalcoholic fatty liver disease. The cellular iron exporter, ferroportin, has been proposed as being a key determinant of adipocyte iron homeostasis. METHODS: We studied an adipocyte-specific ferroportin (Fpn1) knockout mouse model, using an Adipoq-Cre recombinase driven Fpn1 deletion and fed mice according to the fast food diet model of nonalcoholic steatohepatitis. RESULTS: We showed successful selective deletion of Fpn1 in adipocytes, but found that this did not lead to increased adipocyte iron stores as measured by atomic absorption spectroscopy or histologically quantified iron granules after staining with 3,3'-diaminobenzidine-enhanced Perls' stain. Mice with adipocyte-specific Fpn1 deletion did not show dysregulation of adiponectin, leptin, resistin, or retinol-binding protein-4 expression. Similarly, adipocyte-specific Fpn1 deletion did not affect insulin sensitivity during hyperinsulinemic-euglycemic clamp studies or lead to histologic evidence of increased liver injury. We have shown, however, that the fast food diet model of nonalcoholic steatohepatitis generates an increase in adipose tissue macrophage infiltration with crown-like structures, as seen in human beings, further validating the utility of this model. CONCLUSIONS: Ferroportin may not be a key determinant of adipocyte iron homeostasis in this knockout model. Further studies are needed to determine the mechanisms of iron metabolism in adipocytes and adipose tissue.

Combination curcumin and vitamin E treatment attenuates diet-induced steatosis in Hfe-/- mice.

AIM: To investigate the synergistic hepato-protective properties of curcumin and vitamin E in an Hfe-/- high calorie diet model of steatohepatitis. METHODS: Hfe-/- C57BL/6J mice were fed either a high calorie diet or a high calorie diet with 1 mg/g curcumin; 1.5 mg/g vitamin E; or combination of 1 mg/g curcumin + 1.5 mg/g vitamin E for 20 wk. Serum and liver tissue were collected at the completion of the experiment. Liver histology was graded by a pathologist for steatosis, inflammation and fibrosis. RNA and protein was extracted from liver tissue to examine gene and protein expression associated with fatty acid oxidation, mitochondrial biogenesis and oxidative stress pathways. RESULTS: Hfe-/- mice fed the high calorie diet developed steatohepatitis and pericentral fibrosis. Combination treatment with curcumin and vitamin E resulted in a greater reduction of percent steatosis than either vitamin E or curcumin therapy alone. Serum alanine aminotransferase and non-alcoholic fatty liver disease (NAFLD) activity score were decreased following combination therapy with curcumin and vitamin E compared with high calorie diet alone. No changes were observed in inflammatory or fibrosis markers following treatment. Epididymal fat pad weights were significantly reduced following combination therapy, however total body weight and liver weight were unchanged. Combination therapy increased the mRNA expression of AdipoR2, Ppar-α, Cpt1a, Nrf-1 and Tfb2m suggesting enhanced fatty acid oxidation and mitochondrial biogenesis. In addition, combination treatment resulted in increased catalase activity in Hfe-/- mice. CONCLUSION: Combination curcumin and vitamin E treatment decreases liver injury in this steatohepatitis model, indicating that combination therapy may be of value in NAFLD.

The Implications of the Shift Toward Donation After Circulatory Death in Australia.

BACKGROUND: In recent years, an increasing number of donor livers are being declined for transplantation in Australia. The aim of this study was to evaluate the impact of donation after cardiac death and other factors associated with organ quality on liver utilization rates in Australia. METHODS: Data on organ donors who donated at least 1 organ between 2005 and 2014 were obtained from the Australia and New Zealand organ donation registry. Temporal changes in donor characteristics were assessed and a logistical regression analysis was performed to evaluate their association with liver nonuse. RESULTS: The number of organ donors increased from 175 in 2005 to 344 in 2014, with overall 19% being donation after cardiac death donors (P < 0.001). The percentage of livers deemed unsuitable for transplantation increased from 24% in 2005 to 41% in 2014 (P < 0.001). Donation after cardiac death was identified as the most important risk factor for nonuse with an odds ratio of 25.88 (95% confidence interval, 18.84-35.56), P < 0.001) followed by donor age, obesity, and diabetes. DISCUSSION: This study shows that livers donated after circulatory death are an underused resource in Australia. Better use of these currently available organs would be a highly cost-effective way of reducing waiting list mortality in liver transplantation.

Low-Dose Lipopolysaccharide Causes Biliary Injury by Blood Biliary Barrier Impairment in a Rat Hepatic Ischemia/Reperfusion Model.

This study explored whether bacterial endotoxins, in the form of lipopolysaccharides (LPS), could have an injurious effect on the biliary tract in conjunction with ischemia. A total of 64 rats were randomly assigned to 4 groups: sham operation (sham group), 1 mg/kg LPS intraperitoneal (LPS group), hepatic ischemia/reperfusion (IR; IR group), and IR combined with LPS (IR+LPS group). Following 1 or 6 hours of reperfusion, serum liver tests, bile duct histology, immunofluorescence microscopy (zonula occludens-1 [ZO-1]), bile composition (bile salts, phospholipids, lactate dehydrogenase), hepatic gene expression (bile salt transporters and inflammatory mediators), as well as serum and biliary cytokine concentrations were quantified and compared between the study groups. In addition, the integrity of the blood biliary barrier (BBB) was assayed in vivo using horseradish peroxidase (HRP). LPS administration induced severe small bile duct injury following 6 hours of reperfusion. Furthermore, total bile salts and bilirubin concentrations in serum were increased in the LPS groups compared with sham controls (LPS, + 3.3-fold and +1.9-fold; IR+LPS, + 3.8-fold and +1.7-fold, respectively). The BBB was impaired in the LPS groups as evidenced by elevated levels of HRP in bile (+4.9-fold), and decreased expression of claudin 1 (-6.7-fold) and claudin 3 (-3.6-fold). LPS was found to be a potent inducer of small bile duct injury following hepatic ischemia and 6 hours of reperfusion. This injury was associated with increased permeability of the BBB and impaired hepatic bile salt clearance. Liver Transplantation 23 194-206 2017 AASLD.

Iron and non-alcoholic fatty liver disease.

The mechanisms that promote liver injury in non-alcoholic fatty liver disease (NAFLD) are yet to be thoroughly elucidated. As such, effective treatment strategies are lacking and novel therapeutic targets are required. Iron has been widely implicated in the pathogenesis of NAFLD and represents a potential target for treatment. Relationships between serum ferritin concentration and NAFLD are noted in a majority of studies, although serum ferritin is an imprecise measure of iron loading. Numerous mechanisms for a pathogenic role of hepatic iron in NAFLD have been demonstrated in animal and cell culture models. However, the human data linking hepatic iron to liver injury in NAFLD is less clear, with seemingly conflicting evidence, supporting either an effect of iron in hepatocytes or within reticulo-endothelial cells. Adipose tissue has emerged as a key site at which iron may have a pathogenic role in NAFLD. Evidence for this comes indirectly from studies that have evaluated the role of adipose tissue iron with respect to insulin resistance. Adding further complexity, multiple strands of evidence support an effect of NAFLD itself on iron metabolism. In this review, we summarise the human and basic science data that has evaluated the role of iron in NAFLD pathogenesis.

Heterozygous Hfe gene deletion leads to impaired glucose homeostasis, but not liver injury in mice fed a high-calorie diet.

Heterozygous mutations of the Hfe gene have been proposed as cofactors in the development and progression of nonalcoholic fatty liver disease (NAFLD). Homozygous Hfe deletion previously has been shown to lead to dysregulated hepatic lipid metabolism and accentuated liver injury in a dietary mouse model of NAFLD We sought to establish whether heterozygous deletion of Hfe is sufficient to promote liver injury when mice are exposed to a high-calorie diet (HCD). Eight-week-old wild-type and Hfe(+/-) mice received 8 weeks of a control diet or HCD Liver histology and pathways of lipid and iron metabolism were analyzed. Liver histology demonstrated that mice fed a HCD had increased NAFLD activity score (NAS), steatosis, and hepatocyte ballooning. However, liver injury was unaffected by Hfe genotype. Hepatic iron concentration (HIC) was increased in Hfe(+/-) mice of both dietary groups. HCD resulted in a hepcidin-independent reduction in HIC Hfe(+/-) mice demonstrated raised fasting serum glucose concentrations and HOMA-IR score, despite unaltered serum adiponectin concentrations. Downstream regulators of hepatic de novo lipogenesis (pAKT, SREBP-1, Fas, Scd1) and fatty acid oxidation (AdipoR2, Pparα, Cpt1) were largely unaffected by genotype. In summary, heterozygous Hfe gene deletion is associated with impaired iron and glucose metabolism. However, unlike homozygous Hfe deletion, heterozygous gene deletion did not affect lipid metabolism pathways or liver injury in this model.

The diagnosis and management of hereditary haemochromatosis.

Hereditary haemochromatosis (HH) is a common genetic disorder of iron metabolism in individuals of Northern European ancestry which leads to inappropriate iron absorption from the intestine and iron overload in susceptible individuals. Iron overload is suggested by elevations in serum ferritin and transferrin saturation. The majority of patients with clinically significant iron overload are homozygous for the C282Y mutation of the HFE gene, however only a minority of C282Y homozygotes fully express the disease clinically. Those with a high serum ferritin (>1000 microg/L) and additional hepatic insults from cofactors are more likely to develop cirrhosis and its complications. The mainstay of treatment is venesection. Those without cirrhosis who undergo appropriate venesection have a normal life expectancy. Family screening is recommended for all first degree relatives of an individual with the disease.