- Top of page
- Supporting Information
Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in the western world and its incidence, as well as that of its complications continues to rise. The impact of NAFLD on health resources is significant, with costs and utilisation reported to be 26% higher than healthy individuals. As such, its treatment has become an area of much interest. Nonpharmacological lifestyle interventions positively impact NAFLD and the metabolic syndrome and are recommended first-line therapies.[2, 3] Unfortunately, these changes are difficult for patients to sustain long term and so other treatment options continue to be investigated. To date, however, there remains a lack of convincing data supporting the widespread use of pharmacological agents or bariatric surgery.
Hyperferritinemia is common in the metabolic syndrome and NAFLD. Evidence is mounting that elevated iron is linked to increased risk of complications including cardiovascular outcomes, diabetes severity and progression of hepatic disease.[7, 8] Recently, one of the largest studies evaluating iron and liver histology in NAFLD demonstrated that elevated serum ferritin (SF) identified NAFLD patients with more advanced disease. Previous studies have suggested a benefit of iron reduction therapy on NAFLD severity and insulin sensitivity.[10-12] To date, however, the impact of this therapy on liver histology, considered to be the gold standard for evaluating efficacy of liver disease treatment, remains uncertain.
The purpose of a Phase II trial is to obtain some evidence of treatment response and safety in small groups of patients. While Phase II results are not definitive; they are helpful for justifying and designing larger Phase III trials.
The primary purpose of this study was to conduct a Phase II trial of the effects of phlebotomy in 31 individuals with biopsy proven NAFLD. A secondary aim was to use the estimated effect size to calculate the sample size needed for a definitive Phase III trial.
- Top of page
- Supporting Information
Phase II studies are appropriate for preliminary evaluations of treatments, particularly in conditions that lack a widely accepted efficacious, safe and efficient treatment. This small Phase II study showed statistically significant reductions in the NAS score, ALT, AST, SF and hepatic iron concentration. Although not statistically significant, improvements in steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis were all in the predicted direction. The proportion of NASH patients who improved was 26% (95% CI: 11.3–46.6). No important adverse events were noted. Taken together, these results suggest that phlebotomy to near-iron depletion could improve liver histology and function in patients with NAFLD in a safe and well-tolerated fashion.
In this study, each patient served as his or her own control, and the measures used were objective. However, the absence of an untreated control group means that the findings must be viewed with caution. While not as strong as a concurrent control group, changes in the same outcome in untreated NAFLD patients from RCTs of other treatments can put the present findings in perspective. Two RCTs, with placebo groups of 83 and 29, reported mean changes in NAS scores of −0.5 and −0.1, respectively, for a weighted average of −0.4. When subtracted from the mean change in NAS, we observed of −0.74, the difference is −0.34 or 0.19 of one standard deviation. As a treatment effect size, this is considered small. To have 80% power to detect this difference or larger at the 5% level of significance would require two groups of about 450 patients.
Aside from recommendations regarding appropriate caloric intake and lifestyle changes, no other specific interventions were instituted and no reduction in weight or BMI was seen over the study period, which suggests that the effects noted on liver histology are due to the effect of phlebotomy. Although the majority of outcome measures changed in the predicted direction with treatment, the lack of improvement in weight and BMI reflects the difficulty this group of patients has in achieving these changes over the period of follow-up, despite being part of a clinical trial with regular medical follow-up. While the effect of phlebotomy on insulin resistance was not statistically significant, a reduction in HOMA-IR scores of approximately one quarter of a standard deviation was seen. It is noteworthy that the estimated effect size of 0.26 was consistent with that observed in the treatment group of trials of pentoxifylline (ES = 0.32) and pioglitazone (ES = 0.18).[25, 26] As shown above, a larger sample size and concurrent random controls are needed to better estimate the effect of phlebotomy on IR in these patients.
The results of this study are consistent with previously published work demonstrating that subjects with elevated baseline SF are likely to receive the greatest benefit from iron reduction. Also in keeping with previous studies,[27-31] an important observation in this trial was that despite the majority of subjects having elevated SF, only two had hepatic iron overload (HIC > 36 μmol/g) at baseline. There was, however, a significant reduction in HIC in the overall study population, indicating that phlebotomy successfully removes tissue iron. As most subjects had normal HIC at baseline, we feel it is unlikely that the improvement in liver histology is solely due to reduction in liver iron. It is quite possible that iron reduction has anti-inflammatory effects independent from decreasing tissue iron.[32, 33] Iron is a known potent catalyst of oxidative stress[34, 35] and may play a role in inducing fibrogenesis.
There is evidence to support haemoglobin as a risk factor for NAFLD, particularly in individuals without the metabolic syndrome.[37-39] Potential mechanisms of liver injury have been suggested to relate to higher iron accumulation as well as increased blood viscosity raising peripheral resistance and reducing hepatic blood flow.[37, 40] Although the majority of patients in this study met diagnostic criteria for the metabolic syndrome, it is interesting to note that subjects with higher baseline haemoglobin had more severe histological disease. As well, iron reduction therapy resulted in an improvement in the NAS. These findings support this relationship and warrant further evaluation of the role haemoglobin level may play in NAFLD pathogenesis.
The impact of HFE gene status on iron accumulation in NAFLD has been evaluated. A recent large multicentre study concluded that although hepatocyte iron accumulation was associated with a higher risk of moderate/severe fibrosis, no significant association between the presence of HFE mutations and liver disease severity was found. It was concluded that HFE genetic testing has limited value in predicting those patients with more severe disease. Similar results were seen in our study where of the six subjects carrying HFE mutations, only one demonstrated hepatic iron accumulation. This is in keeping with a relatively weak association between HFE mutations and hepatic iron deposition, which has been suggested to be due to the low penetrance of HFE mutations.
We recognise that the results of the present trial are limited by the lack of concurrent random controls.[25, 26] Additional limitations include the relatively mild overall degree of iron overload and liver disease at baseline (mean NAS = 3.8), combined with variability in the severity of individual NAS parameters and clinical characteristics, including degrees of hepatic iron deposition. To reliably assess changes in liver fibrosis, studies with a 12-month treatment duration are recommended. In the case of phlebotomy for iron reduction, treatment duration cannot be standardised, as time to iron depletion varies in each individual. An attempt was made to control for this by obtaining the final liver biopsy 6 months following the end of treatment, thus demonstrating the longer term histological effect of this intervention. As the sample size was relatively small, it was not possible to perform multivariable analysis to control confounding and examine interactions.
NAFLD patients have been shown to have increased overall mortality compared with matched control populations.[42-44] NASH is presently the third most common reason for liver transplantation in the United States and is projected to become the most common indication in coming years. The identification of treatment modalities for NAFLD is therefore an important, albeit challenging area of research. Despite a significant amount of work to date, we still have little to offer these patients. It has been stated that the ideal treatment ‘should be one that decreases overall mortality, including liver-related and cardiovascular deaths, while remaining safe, widely available, and relatively inexpensive.’ Phlebotomy is appealing as it easily fulfils the final three criteria, something that cannot be said for some other therapies that have been evaluated for this condition. Although our results suggest that phlebotomy has a positive impact on liver disease in NAFLD as assessed by the NAS, no statistically significant improvement in fibrosis or steatohepatitis was seen. An additional important consideration is whether the estimated treatment effect is clinically significant enough to warrant a randomised trial with an untreated control group, particularly given the ethical and feasibility challenges posed by serial liver biopsies in several hundred patients.
This Phase II trial helps to address the important clinical question of whether iron depletion could have a beneficial effect on liver histology in NAFLD and was undertaken as a hypothesis-generating study to determine whether a larger, Phase III randomised trial would be prudent. On the basis of our results, we suggest that future studies of iron reduction therapy be focused on those patients with baseline SF of at least 300 μg/L and steatohepatitis on liver biopsy to more definitively ascertain the magnitude of benefit in this condition. Clinicaltrials.gov registration number; NCT 00641524.