The impact of phlebotomy in nonalcoholic fatty liver disease: A prospective, randomized, controlled trial.

UNLABELLED: Iron is implicated in the pathogenesis of liver injury and insulin resistance (IR) and thus phlebotomy has been proposed as a treatment for nonalcoholic fatty liver disease (NAFLD). We performed a prospective 6-month randomized, controlled trial examining the impact of phlebotomy on the background of lifestyle advice in patients with NAFLD. Primary endpoints were hepatic steatosis (HS; quantified by magnetic resonance imaging) and liver injury (determined by alanine aminotransaminase [ALT] and cytokeratin-18 [CK-18]). Secondary endpoints included insulin resistance measured by the insulin sensitivity index (ISI) and homeostasis model of assessment (HOMA), and systemic lipid peroxidation determined by plasma F2-isoprostane levels. A total of 74 subjects were randomized (33 phlebotomy and 41 control). The phlebotomy group underwent a median (range) of 7 (1-19) venesection sessions and had a significantly greater reduction in ferritin levels over 6 months, compared to controls (-148 ± 114 vs. -38 ± 89 ng/mL; P < 0.001). At 6 months, there was no difference between phlebotomy and control groups in HS (17.7% vs. 15.5%; P = 0.4), serum ALT (36 vs. 46 IU/L; P = 0.4), or CK-18 levels (175 vs. 196 U/L; P = 0.9). Similarly, there was no difference in end-of-study ISI (2.5 vs. 2.7; P = 0.9), HOMA (3.2 vs. 3.2; P = 0.6), or F2-isoprostane levels (1,332 vs. 1,190 pmmol/L; P = 0.6) between phlebotomy and control groups. No differences in any endpoint were noted in patients with hyperferritinemia at baseline. Among patients undergoing phlebotomy, there was no correlation between number of phlebotomy sessions and change in HS, liver injury, or IR from baseline to end of study. CONCLUSION: Reduction in ferritin by phlebotomy does not improve liver enzymes, hepatic fat, or IR in subjects with NAFLD.

Iron-induced oxidative rat liver injury after non-heart-beating warm ischemia is mediated by tumor necrosis factor α and prevented by deferoxamine.

This study investigated iron-induced injury after warm ischemia in a non-heart-beating (NHB) rat liver model and the effects of deferoxamine (DFO). Livers from heart-beating (HB) rats or rats that were NHB for 60 minutes were stored in University of Wisconsin solution for 5 hours at 4°C [cold storage (CS)] and then were subjected to 2 hours of machine reperfusion (MRP) at 37°C. Three NHB groups were compared: (1) no DFO, (2) DFO 30 minutes before cardiac arrest and during CS and MRP, and (3) DFO during CS and MRP. Aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels in the NHB perfusate were significantly elevated (P < 0.01) in comparison with levels in HB controls after CS and MRP. After CS, the levels of iron and tumor necrosis factor α (TNF-α) were 0.077 ± 0.007 µmol/g and 151 ± 26 pg/g, respectively, in the NHB group and 0.022 ± 0.004 µmol/g and 17 ± 7 pg/g, respectively, in the HB group (P < 0.01). After MRP, LDH significantly correlated with iron (R(2) = 0.81, P < 0.01). The DFO pretreatment of NHB donors decreased AST (7.3 ± 0.8 versus 4.0 ± 0.5 U/g of liver, P < 0.05) and LDH (42.5 ± 4.1 versus 20.4 ± 2.5 U/g of liver, P < 0.05) with 2 hours of MRP and increased bile flow during MRP (142 ± 34 versus 240 ± 18 µL/g, P < 0.05). It also reduced the levels of iron (0.077 ± 0.007 versus 0.050 ± 0.008 µmol/g, P < 0.05) and TNF-α (151 ± 26 versus 51 ± 13 pg/g, P < 0.05) after CS and the levels of lipid peroxidation products F2-isoprostane (149 ± 11 versus 99 ± 10 ng/g, P < 0.05) and malondialdehyde (1.58 ± 0.1 versus 1.14 ± 0.08 µmol/g, P < 0.05) after MRP. In conclusion, iron-initiated oxidative stress is likely involved in NHB donor liver injury, and importantly, DFO pretreatment reduces liver damage.

Oxidative stress causes a decline in lysosomal integrity during hypothermic incubation of rat hepatocytes.

Oxidative stress during cold preservation has been identified as a significant cause of cell injury but the process by which injury occurs is poorly understood. We examined loss of lysosomal integrity as a possible cause of cell injury during extended cold storage of isolated rat hepatocytes. After 21 h of hypothermia, there was a marked decline in lysosomal integrity, which was correlated with an increase in lipid peroxidation. When lipid peroxidation was prevented with the antioxidant Trolox (a vitamin E analog) or the iron chelator desferrioxamine, lysosomal integrity was preserved. In contrast, increasing lysosomal iron with ferric chloride caused an increase in lipid peroxidation and decreased lysosomal integrity. Loss of lysosomal integrity during cold preservation in this experimental model was consistent with iron-initiated oxidative stress. The progressive loss of lysosomal integrity during hypothermic incubation has the potential to affect liver function after transplantation.

Desferrioxamine in warm reperfusion media decreases liver injury aggravated by cold storage.

AIM: To evaluate whether desferrioxamine decreases ischemia and perfusion injury aggravated by cold storage (CS) in a rat liver perfusion model. METHODS: Isolated rat livers were kept in CS in University of Wisconsin Solution for 20 h at 4 °C, then exposed to 25 min of warm ischemia (WI) at 37 °C followed by 2 h of warm perfusion (WP) at 37 °C with oxygenated (95% oxygen and 5% carbon dioxide) Krebs-Henseleit buffer. Desferrioxamine (DFO), an iron chelator, was added at different stages of storage, ischemia and perfusion: in CS only, in WI only, in WP only, in WI and perfusion, or in all stages. Effluent samples were collected after CS and after WI. Perfusate samples and bile were collected every 30 min (0, 0.5, 1, 1.5 and 2 h) during liver perfusion. Cellular injury was assessed by the determination of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in the effluent and perfusate samples. Total iron was analysed in the perfusate samples. After WP, the liver was collected for the determination of liver swelling (wet to dry ratio) and liver morphological examination (hematoxylin and eosin staining). RESULTS: Increased CS time caused increased liver dysfunction during WP. After 2 h of WP, liver injury was indicated by increased release of AST (0.5 h CS: 9.4 ± 2.2 U/g liver vs 20 h CS: 45.9 ± 10.8 U/g liver, P < 0.05) and LDH (0.5 h CS: 59 ± 14 U/g liver vs 20 h CS: 297 ± 71 U/g liver, P < 0.05). There was an associated increase in iron release into the perfusate (0.5 h CS: 0.11 ± 0.03 µmoL/g liver vs 20 h CS: 0.58 ± 0.10 µmoL/g liver, P < 0.05) and reduction in bile flow (0.5 h CS: 194 ± 12 µL/g vs 20 h CS: 71 ± 8 µL/g liver, P < 0.05). When DFO was added during WI and WP following 20 h of CS, release of iron into the perfusate was decreased (DFO absent 0.58 ± 0.10 µmoL/g liver vs DFO present 0.31 ± 0.06 µmoL/g liver, P < 0.05), and liver function substantially improved with decreased release of AST (DFO absent 45.9 ± 10.8 U/g liver vs DFO present 8.1 ± 0.9 U/g liver, P < 0.05) and LDH (DFO absent 297 ± 71 U/g liver vs DFO present 56 ± 7 U/g liver, P < 0.05), and increased bile flow (DFO absent 71 ± 8 µL/g liver vs DFO present 237 ± 36 µL/g liver, P < 0.05). DFO was also shown to improve liver morphology after WP. Cellular injury (the release of LDH and AST) was significantly reduced with the addition of DFO in CS medium but to a lesser extent compared to the addition of DFO in WP or WI and perfusion. There was no effect on liver swelling or bile flow when DFO was only added to the CS medium. CONCLUSION: DFO added during WI and perfusion decreased liver perfusion injury aggravated by extended CS.

Does sequencing the PKRBD of hepatitis C virus NS5A predict therapeutic response to combination therapy in an Australian population?

UNLABELLED: Abstract Background and Aim: The presence of four or more amino acid substitutions within the interferon sensitivity determining region (ISDR) of the hepatitis C virus (HCV) genotype 1b NS5A gene determines sensitivity to interferon (IFN) monotherapy in Japanese patients. Resistance of HCV genotype 1 to IFN-alpha has been attributed to the functional inhibition of a RNA dependent protein kinase (PKR) by the HCV NS5A PKR binding domain (PKRBD), which includes the ISDR. The ability of the ISDR and PKRBD sequence to predict a response to IFN-alpha and ribavirin combination therapy was investigated in an Australian population. METHODS: The sequence of the PKRBD of NS5A, including the ISDR, for the dominant quasi-species of HCV was determined in 37 genotype 1 (genotype 1a: n = 26, genotype 1b: n = 11) and 13 genotype 3a infected patients. RESULTS: The number of PKRBD amino acid substitutions in HCV genotype 1 infected patients with a sustained virological response was significantly higher than that in patients with a non-response to treatment (P = 0.047). It was found that only 2/37 HCV genotype 1 infected patients had four or more amino acid substitutions relative to the prototype ISDR sequence (HCV-J). Importantly, a sustained virological response was not found in any of the HCV infected patients who had a prototype ISDR genotype 1 sequence (n = 5). CONCLUSIONS: There are relatively few amino acid mutations within the ISDR of this Western Australian patient population. Patients infected with a HCV genotype 1 prototype sequence should be counseled before receiving combination IFN-alpha and ribavirin therapy as they have a poor response to treatment.

Disease stage-dependent accumulation of lipid and protein oxidation products in human atherosclerosis.

Oxidative modification of low-density lipoprotein is thought to promote arterial lipid accumulation and atherogenesis. Previous studies reported on the presence of certain lipid or protein oxidation products in lesions, although a systematic investigation measuring several oxidation parameters and the accumulation of nonoxidized lipids and antioxidants at various stages of atherosclerosis has not been performed in the same tissue. Using the intimal lipoprotein-containing fraction of human aortic lesions, we demonstrate here that cholesterol accumulated with lesion development and that this increase was already significant at the fatty streak stage. By comparison, cholesterylesters increased significantly only in fibro-fatty and more complex lesions that also contained significantly increased amounts of cholesterylester hydro(pero)xides and 27-hydroxycholesterol. Cholesterylester hydroxides were the major lipid oxidation product detected. Despite accumulation of oxidized lipid, alpha-tocopherol was also present and maintained at a comparable level over the disease process. Of the oxidized protein moieties measured only o,o-dityrosine increased with disease, although chlorotyrosines were present at relatively high levels in all lesions compared to healthy vessels. Our data show that accumulation of nonoxidized lipid precedes that of oxidized lipid in human aortic lesions.