C. Berardo

Fatty liver oxidative events monitored by autofluorescence optical diagnosis: Comparison between subnormothermic machine perfusion and conventional cold storage preservation

Livers with moderate steatosis are currently recruited as marginal organs to face donor shortage in transplantation, even though lipid excess and oxidative stress increase preservation injury risk. Sensitive, real‐time detection of liver metabolism engagement could help donor selection and preservation procedures, ameliorating the graft outcome. Hence, we investigated endogenous biomolecules with autofluorescence (AF) properties as biomarkers supporting the detection of liver oxidative events and the assessment of metabolic responses to external stimuli.

Changes in ADMA/DDAH Pathway after Hepatic Ischemia/Reperfusion Injury in Rats: The Role of Bile

We investigated the effects of hepatic ischemia/reperfusion (I/R) injury on asymmetric dimethylarginine (ADMA, a nitric oxide synthase inhibitor), protein methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) (involved, resp., in ADMA synthesis and degradation), and the cationic transporter (CAT). Male Wistar rats were subjected to 30 or 60 min hepatic ischemia followed by 60 min reperfusion. ADMA levels in serum and bile were determined. Tissue ADMA, DDAH activity, DDAH-1 and CAT-2 protein, DDAH-1 and PRMT-1 mRNA expression, GSH/GSSG, ROS production, and lipid peroxidation were detected. ADMA was found in bile. I/R increased serum and bile ADMA levels while an intracellular decrease was detected after 60 min ischemia. Decreased DDAH activity, mRNA, and protein expression were observed at the end of reperfusion. No significant difference was observed in GSH/GSSG, ROS, lipid peroxidation, and CAT-2; a decrease in PRMT-1 mRNA expression was found after I/R. Liver is responsible for the biliary excretion of ADMA, as documented here for the first time, and I/R injury is associated with an oxidative stress-independent alteration in DDAH activity. These data are a step forward in the understanding of the pathways that regulate serum, tissue, and biliary levels of ADMA in which DDAH enzyme plays a crucial role.

Liver plays a central role in asymmetric dimethylarginine- mediated organ injury

Asymmetric-dimethylarginine (ADMA) competes with L-arginine for each of the three isoforms of nitric oxide synthase: endothelial; neuronal; inducible. ADMA is synthesized by protein methyltransferases followed by proteolytic degradation. ADMA is metabolized to citrulline and dimethylamine, by dimethylarginine dimethylaminohydrolase (DDAH) and enters cells through cationic amino-acid transporters extensively expressed in the liver. The liver plays a crucial role in ADMA metabolism by DDAH-1 and, as has been recently demonstrated, it is also responsible for ADMA biliary excretion. A correlation has been demonstrated between plasma ADMA levels and the degree of hepatic dysfunction in patients suffering from liver diseases with varying aetiologies: plasma ADMA levels are increased in patients with liver cirrhosis, alcoholic hepatitis and acute liver failure. The mechanism by which liver dysfunction results in raised ADMA concentrations is probably due to impaired activity of DDAH due to severe inflammation, oxidative stress, and direct damage to DDAH. High plasma ADMA levels are also relevant as they are associated with the onset of multi-organ failure (MOF). Increased plasma concentration of ADMA was identified as an independent risk factor for MOF in critically-ill patients causing enhanced Intensive Care Unit mortality: a significant reduction in nitric oxide synthesis, leading to malperfusion in various organs, eventually culminating in multi organs dysfunction.

Oxygen tension-independent protection against hypoxic cell killing in rat liver by low sodium

The role of Na+ in hypoxic injury was evaluated by a time-course analysis of damage in isolated livers perfused with N2-saturated buffer containing standard (143 mM) or low (25 mM) Na+ levels. Trypan blue uptake was used to detect non-viable cells. Under hypoxia with standard-Na+, trypan blue uptake began at the border between pericentral areas and periportal regions and increased in the latter zone; using a low-Na+ buffer, no trypan blue zonation occurred but a homogenous distribution of dye was found associated with sinusoidal endothelial cell (SEC) staining. A decrease in hyaluronic acid (HA) uptake, index of SEC damage, was observed using a low-Na+ buffer. A time dependent injury was confirmed by an increase in LDH and TBARS levels with standard-Na+ buffer. Using low-Na+ buffer, SEC susceptibility appears elevated under hypoxia and hepatocytes was protected, in an oxygen independent manner.

Autofluorescence discrimination of metabolic fingerprint in nutritional and genetic fatty liver models

Liver tissue autofluorescence (AF) has been characterized in two models with a different potential to undergo disease progression to steatohepatitis: Wistar rats, administered with a methionine, choline deficient diet (MCD), and Zucker (fa/fa) rats, homozygous for a spontaneous mutation of leptin receptor. AF spectra were recorded from liver tissue cryostatic sections by microspectrofluorometry, under 366nm excitation. Curve fitting analysis was used to estimate the contribution of different endogenous fluorophores (EFs) to the overall AF emission: i) fluorescing fatty acids, a fraction of liver lipids up to now poorly considered and complicated to detect by conventional procedures; ii) lipofuscin-like lipopigments, biomarkers of oxidizing events; iii) NAD(P)H and flavins, biomarkers of energy metabolism and tissue redox state. AF data and biochemical correlates of hepatocellular injury resulted to depend more on rat strain than on intratissue bulk lipid or ROS levels, reflecting a different metabolic ability of the two models to counteract potentially harmful agents. AF analysis can thus be proposed for extensive applications ranging from experimental hepatology to the clinics. AF based diagnostic procedures are expected to help both the prediction of the risk of fatty liver disease progression and the prescreening of marginal organs to be recruited as donors for transplantation. A support is also foreseen in the advancement and personalization of strategies to ameliorate the donor organ preservation outcome and the follow up of therapeutic interventions.

Changes in Biliary Levels of Arginine and its Methylated Derivatives after Hepatic Ischaemia/Reperfusion

Arginine (Arg) can be methylated to form symmetrical dimethylarginine (SDMA) and asymmetrical dimethylarginine (ADMA), the latter an endogenous inhibitor of nitric oxide synthase (NOS). SDMA is excreted in the urine, while ADMA is mainly subjected to degradation in the liver. Arg competes with ADMA and SDMA for cellular transport across cationic amino‐acid transporters (CATs). We evaluated the changes in serum, tissue and biliary levels of Arg, citrulline (Cit), ADMA and SDMA and the modifications in CATs after ischaemia‐reperfusion (I/R). Male Wistar rats were subjected to 30‐min. partial‐hepatic ischaemia or sham‐operated. After 60‐min. reperfusion, the concentrations of ADMA, SDMA, Arg and Cit in serum, tissue and bile were measured. Serum levels of AST, ALT and alkaline phosphatase (AP) levels were determined. mRNA of cationic transporter 2A (CAT‐2A) and 2B (CAT‐2B) were also quantified. An increase in ADMA and a decrease in SDMA were observed in bile at the end of reperfusion. On the contrary, lower tissue ADMA levels and higher SDMA levels were quantified. No serum changes in ADMA and SDMA were found. A decrease in Arg and an increase of Cit were detected in serum, bile and tissue after I/R. A marked increase in AST, ALT and AP levels in serum confirmed I/R injury. A decrease in mRNA transporter CAT‐2A but not in CAT‐2B was detected. This study supported a biliary CAT‐2B–dependent transport of ADMA and demonstrated, for the first time, that the liver is also responsible for the biliary excretion of SDMA into the bile.

Fluorescing fatty acids in rat fatty liver models

The autofluorescence (AF) of NAD(P)H and flavins has been at the basis of many in-situ studies of liver energy metabolism and functionality. Conversely, few data have been so far reported on fluorescing lipids. In this work we investigated the AF of liver lipid extracts from two fatty liver models, Wistar rats fed with MCD diet for 12 days (Wi-MCD), and obese (fa/fa) Zucker rats. Among the most abundant fatty acids in the lipid extracts, indicated by mass spectrometry, arachidonic acid (AA) exhibited higher quantum yield than the other fluorescing fatty acids (FLFA), and red shifted AF spectrum. This allowed to estimate the AA contribution to the overall emission of lipid extracts by curve fitting analysis. AA prevailed in obese Zucker livers, accounting for the different AF spectral profiles between the two models. AF and mass spectrometry indicated also a different balance between the fluorescing fraction and the overall amount of AA in the two models. The ability of AF to detect directly AA and FLFA was demonstrated, suggesting its supportive role as tool in wide-ranging applications, from the control of animal origin food, to experimental investigations on liver fat accumulation, lipotoxicity and disease progression, with potential translation to the clinics.

NAD(P)H and Flavin Autofluorescence Correlation with ATP in Rat Livers with Different Metabolic Steady State Conditions

The monitoring of NAD(P)H and flavin autofluorescence (AF) is at the basis of numerous investigations on energy metabolism. Nevertheless, the ability of these AF biomarkers to accurately represent the energy currency, ATP, is poorly explored. Here, we focused on the AF/ATP correlation in lean and fatty livers with different steady‐state metabolic conditions, achieved after organ isolation, preservation and recovery, in a likely dependence on both liver intrinsic metabolic features and externally induced perturbations. Within these eventual, various conditions, a significant correlation was detected between liver NAD(P)H and flavin AF, measured via fiber‐optic probe, and biochemical ATP data, strengthening AF as biomarker of energy metabolism in steady‐state conditions for wide‐ranging experimental and diagnostic applications.

Liver Graft Susceptibility during Static Cold Storage and Dynamic Machine Perfusion: DCD versus Fatty Livers

We compared static preservation (cold storage, CS, 4 °C) with dynamic preservation (machine perfusion, MP, 20 °C) followed by reperfusion using marginal livers: a model of donation after cardiac death (DCD) livers and two models of fatty livers, the methionine-choline deficient (MCD) diet model, and obese Zucker (fa/fa) rats. CS injury in DCD livers was reversed by an oxygenated washout (OW): hepatic damage, bile flow, and the ATP/ADP ratio in the OW + CS group was comparable with the ratio obtained with MP. Using fatty livers, CS preservation induced a marked release in hepatic and biliary enzymes in obese Zucker rats when compared with the MCD group. The same trend occurred for bile flow. No difference was found when comparing MP in MCD and obese Zucker rats. Fatty acid analysis demonstrated that the total saturated (SFA)/polyunsaturated fatty acid (PUFA) ratio was, respectively, 1.5 and 0.71 in obese Zucker and MCD rats. While preservation damage in DCD livers is associated with the ATP/ADP recovered with OW, injury in fatty livers is linked to fatty acid constituents: livers from obese. Zucker rats, with greater content in saturated FA, might be more prone to CS injury. On the contrary, MCD livers with elevated PUFA content might be less susceptible to hypothermia.

Machine perfusion at 20°C prevents ischemic injury and reduces hypoxia-inducible factor-1α expression during rat liver preservation

BACKGROUND Ischemic cholangitis is the main cause of liver failure after transplantation and subnormothermic machine perfusion may represent a better strategy than conventional cold storage, minimizing preservation injury. We compared livers preserved by machine perfusion at 20°C (MP20) or by cold storage at 4°C (CS4) with regard to hypoxia-inducible factor (HIF)-1α mRNA expression and protein stabilization in hypoxic conditions. MATERIAL AND METHODS Livers from male Wistar rats were stored on ice at 4°C in UW solution (CS4) or perfused with oxygenated Krebs-Henseleit buffer at 20°C (MP20) for six hours. After preservation, the livers were reperfused for two hours with oxygenated Krebs-Henseleit buffer at 37°C to simulate reimplantation. We collected bile, perfusate, and tissue samples. Transaminases, lactate dehydrogenase, bilirubin, and lactic acid were assayed in the perfusate and bile. ATP/ADP, glycogen, HIF-1α mRNA, and protein expression were measured in the tissue homogenates. RESULTS At the end of preservation, as well as after reperfusion, HIF-1α mRNA expression was significantly higher in the ischemic CS4 livers. Although the hypoxic conditions found in CS4 preservation stabilized HIF-1α protein was significantly higher in the CS4 livers at the end of preservation, no difference was observed after reperfusion, likely because of the oxygen in the reperfusion medium. After reperfusion, the MP20 livers released less transaminases and LDH. The MP20 livers had higher ATP/ADP, glycogen, and biliary bilirubin after both preservation and reperfusion when compared with the CS4 livers. CONCLUSIONS The data demonstrated that MP20 was associated with a lower HIF-1α expression and organ injury with respect to CS4, suggesting that oxygen provided by this preservation setting might approximate the organ request, thus avoiding the ischemic injury usually observed during organ preservation by cold storage.