Xianwa Niu

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

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. (Hepatology 2015;61:1555–1564)

Quantitative study of starving platelets in a minimal medium: maintenance by acetate or plasma but not by glucose

Summary. The requirement of donor platelets for fuels, plasma and calcium were studied using platelets washed, filtered to remove leucocytes and resuspended in a new glucose‐free minimal platelet storage medium with low citrate (3 mmol/1), low buffer capacity and no calcium. This is the first study of platelets stored without plasma, glucose or calcium and it was shown that platelets continued to aggregate with collagen plus adrenaline for 48 h and showed only a 50% fall in ‘swirl index’, an objective morphology score, after 3 days, showing that by these criteria human platelets do not require glucose. Sodium acetate extended the storage time by between 2 and 4 days, depending on the index parameter. This is the first evidence showing that failure of platelets in these conditions is at least partly due to exhaustion of fuel, and the first evidence that acetate prolongs in vitro survival. As little as 10% low‐glucose plasma extended the storage time, but it was no better than acetate. New observations using this system included a very rapid fall in pH during resuspension of the washed platelet pellet, a rising pH in the absence of added fuel and an increased pH with added acetate.

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

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.

Method for measuring a comprehensive energy budget in a proliferating cell system over multiple cell cycles

Isolated cell systems are now being used very effectively to study a range of important biochemical questions, but their energy metabolism has never been comprehensively investigated. We have developed a system, using J2E cells, which enables us to measure total ATP turnover and the contribution of various fuels and pathways to this total in a dynamic, proliferating preparation. Cells are cultured in 500 ml airtight glass containers which enables (1) the measurement of oxygen consumption, (2) the collection and measurement of 14CO2 production from labelled fuels, and (3) the measurement of metabolite utilization and production. Data on cell numbers are then used to produce a curve of cell number vs. time, the area under which (cell numbers · hour) is used as a base by which all measurements and experiments are compared. To our knowledge this is the first time a comprehensive energy budget has been measured in a proliferating cell system over a period that covers multiple cell cycles. J Cell Physiol 170:1–7, 1997

Carbohydrate metabolism in human platelets in a low glucose medium under aerobic conditions

The metabolism of human platelets has been the subject of investigation for at least three decades, at the level of basic metabolism, and because of the increasing requirement for platelet storage. Platelets are relatively active metabolically and are typical cells in terms of fuels and metabolic pathways. They contain glycogen and utilize glucose and demonstrate aerobic glycolysis and carbohydrate oxidation. Both glycolysis and carbohydrate oxidation contribute significantly to total ATP turnover, so platelets are an ideal system in which to study the partitioning of carbohydrate metabolism between the two available fuels and the two available pathways, in the presence of adequate oxygen. We have designed a system whereby we can study carbohydrate metabolism in relatively pure human platelets, under sterile conditions, over long periods. The system enables us to determine total ATP turnover and, with the aid of a mathematical model, the contribution to this turnover of glycolysis and the oxidation of glucose/glycogen and lactate. When glucose and glycogen are present, most of the glucose and glycogen utilised is converted to lactate, but lactate is being oxidised at this time. When glucose/glycogen stores are exhausted lactate oxidation continues and increases with the result that carbohydrate oxidation accounts for 41% of total ATP turnover over 48 h.

Types and sources of fuels for platelets in a medium containing minimal added fuels and a low carryover plasma

The storage of platelets in synthetic media can result in plasma savings and reduced transfusion reactions. Accordingly, a wide range of storage formulations have been developed with the aim of replacing at least a proportion of the plasma in the storage medium. However, the concentrations and types of fuels in the carryover plasma, and the utilization of these fuels by platelets in storage, has not been investigated. We have developed a system which can measure total ATP turnover, and the contribution to total ATP turnover by the oxidation of various fuels and by lactate production, in a bag of partially purified platelets in a buffered saline with minimal carryover citrate phosphate double dextrose (CP2D) plasma.

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 (R2 = 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. Liver Transpl 20:904–911, 2014.

Iron and oxidative stress in cold-initiated necrotic death of rat hepatocyte.

Iron chelators and antioxidants have been shown to prevent hypothermia-induced apoptosis in hepatocytes. This study examined whether iron chelation and antioxidants could also prevent hypothermia-induced necrosis. Isolated rat hepatocytes were incubated at 4 degrees C for 6 hours and then rewarmed at 37 degrees C for 18 hours with or without the iron chelator deferoxamine and a selection of antioxidants. There was no evidence of increased cell death or adenosine triphosphate depletion during hypothermic incubation. After hypothermia and rewarming, the majority of rat hepatocytes died of necrosis as indicated by the absence of DNA fragmentation, caspase 3 activity, and apoptotic bodies. Cell death was significantly reduced if deferoxamine or a selection of antioxidants were present during hypothermia and rewarming. Deferoxamine was more effective in preventing cell death when added prior to hypothermia, indicating cell death processes were likely initiated during hypothermia.

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.

Evaluation of the trisodium salt of 3-phosphoglycerate as a fuel for red cell storage

During red cell storage the pH of the medium and that of the intracellular fluid decreases. The decrease in pH is due to glycolysis and is a likely contributor to red cell storage lesion. The trisodium salt of 3-phosphoglycerate (PGA) would be an ideal non-acid producing fuel for stored red cells as it would enter the glycolytic pathway below the acid-producing steps (glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase), and in comparison with glucose, would enable the production of equivalent amounts of ATP per carbon of the fuel. A medium containing PGA (PGA) was evaluated for 21 days in comparison with a standard dextrose-phosphate-citrate additive solution (DCP) and a medium containing minimal fuel (NF). Fuel utilisation, lactate and pyruvate production, pH, adenylates, Hb, GSH, Na+ and K+ were measured, and in vitro incubations were used to assess the potential for flux through glycolysis and the pentose pathway. DCP was the superior medium, although this is not evident for the first 7 days. 3-Phosphoglycerate did enter the cells, but was metabolised too slowly to be a useful fuel. The cells in PGA were similar to those in DCP in one respect in that they maintained their pentose pathway potential. Apart from flux through the pentose pathway, PGA and NF were identical in that all tests indicated substantial storage lesion.