Purpose/BackgroundMethylphenidate (MPH) is widely used to reduce symptoms of attention-deficit/hyperactivity disorder. Methylphenidate is metabolized by the carboxylesterase 1 (CES1) enzyme. Some patients need a very high dose of MPH to reach desired clinical effects, without having adverse effects. This may be due to differences in MPH pharmacokinetics (PK), potentially caused by DNA variants in CES1, the gene encoding the enzyme that metabolizes MPH. Here we describe 3 patients requiring high-dose MPH and investigated the CES1 gene.Methods/ProceduresThe 3 patients were using short-acting MPH in a dose of 180 to 640 mg instead of the maximum advised dose of around 100 mg MPH in the Netherlands. Plasma concentrations of MPH were determined at scheduled time points (day-curve). Methylphenidate plasma concentrations were used for PK analysis using an earlier published 2-compartment PK population model of MPH. Individual data of the 3 patients were compared with simulated population data, when equivalent doses were used. In addition, CES1 was genotyped (number of gene copies and single nucleotide polymorphisms) using real-time polymerase chain reaction.Findings/ResultsPharmacokinetic analysis in all 3 patients showed lower plasma concentrations of MPH in comparison with the population data. The mean absorption time and volume of distribution of the central compartment were equal, but the elimination clearance was higher. However, CES1 genotyping revealed no variations that could explain a higher metabolism of MPH.Implications/ConclusionsIn these 3 cases, we could not demonstrate a correlation between MPH clearance and known genetic variants of the CES1 gene.

Metformin may act renoprotective prior to kidney transplantation by reducing ischemia‐reperfusion injury (IRI). This study examined whether metformin preconditioning and postconditioning during ex vivo normothermic machine perfusion (NMP) of rat and porcine kidneys affect IRI. In the rat study, saline or 300 mg/kg metformin was administered orally twice on the day before nephrectomy. After 15 minutes of warm ischemia, kidneys were preserved with static cold storage for 24 hours. Thereafter, 90 minutes of NMP was performed with the addition of saline or metformin (30 or 300 mg/L). In the porcine study, after 30 minutes of warm ischemia, kidneys were preserved for 3 hours with oxygenated hypothermic machine perfusion. Subsequently, increasing doses of metformin were added during 4 hours of NMP. Metformin preconditioning of rat kidneys led to decreased injury perfusate biomarkers and reduced proteinuria. Postconditioning of rat kidneys resulted, dose‐dependently, in less tubular cell necrosis and vacuolation. Heat shock protein 70 expression was increased in metformin‐treated porcine kidneys. In all studies, creatinine clearance was not affected. In conclusion, both metformin preconditioning and postconditioning can be done safely and improved rat and porcine kidney quality. Because the effects are minor, it is unknown which strategy might result in improved organ quality after transplantation.

IntroductionMetformin can accumulate and cause lactic acidosis in patients with renal insufficiency. Metformin is known to inhibit mitochondria, while renal secretion of the drug by proximal tubules indirectly requires energy. We investigated whether addition of metformin before or during ex vivo isolated normothermic machine perfusion (NMP) of porcine and rat kidneys affects its elimination.Research design and methodsFirst, Lewis rats were pretreated with metformin or saline the day before nephrectomy. Subsequently, NMP of the kidney was performed for 90 min. Metformin was added to the perfusion fluid in one of three different concentrations (none, 30 mg/L or 300 mg/L). Second, metformin was added in increasing doses to the perfusion fluid during 4 hours of NMP of porcine kidneys. Metformin concentration was determined in the perfusion fluid and urine by liquid chromatography-tandem mass spectrometry.ResultsMetformin clearance was approximately 4–5 times higher than creatinine clearance in both models, underscoring secretion of the drug. Metformin clearance at the end of NMP in rat kidneys perfused with 30 mg/L was lower than in metformin pretreated rats without the addition of metformin during perfusion (both p≤0.05), but kidneys perfused with 300 mg/L trended toward lower metformin clearance (p=0.06). Creatinine clearance was not different between treatment groups. During NMP of porcine kidneys, metformin clearance peaked at 90 min of NMP (18.2±13.7 mL/min/100 g). Thereafter, metformin clearance declined, while creatinine clearance remained stable. This observation can be explained by saturation of metformin transporters with a Michaelis-Menten constant (95% CI) of 23.0 (10.0 to 52.3) mg/L.ConclusionsMetformin was secreted during NMP of both rat and porcine kidneys. Excretion of metformin decreased under increasing concentrations of metformin, which might be explained by saturation of metformin transporters rather than a self-inhibitory effect. It remains unknown whether a self-inhibitory effect contributes to metformin accumulation in humans with longer exposure times.

Background. Preconditioning of donor livers before organ retrieval may improve organ quality after transplantation. We investigated whether preconditioning with metformin reduces preservation injury and improves hepatobiliary function in rat donor livers during ex situ normothermic machine perfusion (NMP) and after orthotopic liver transplantation. Methods. Lewis rats were administered metformin via oral gavage, after which a donor hepatectomy was performed followed by a standardized cold storage period of 4 hours. Graft assessment was performed using NMP via double perfusion of the hepatic artery and portal vein. In an additional experiment, rat donor livers preconditioned with metformin were stored on ice for 4 hours and transplanted to confirm postoperative liver function and survival. Data were analyzed and compared with sham-fed controls. Results. Graft assessment using NMP confirmed that preconditioning significantly improved ATP production, markers for hepatobiliary function (total bile production, biliary bilirubin, and bicarbonate), and significantly lowered levels of lactate, glucose, and apoptosis. After orthotopic liver transplantation, metformin preconditioning significantly reduced transaminase levels. Conclusions. Preconditioning with metformin lowers hepatobiliary injury and improves hepatobiliary function in an in situ and ex situ model of rat donor liver transplantation.

Normothermic machine perfusion (NMP) enables viability assessment of donor livers prior to transplantation. NMP is frequently performed by using human blood products including red blood cells (RBCs) and fresh frozen plasma (FFP). Our aim was to examine the efficacy of a novel machine perfusion solution based on polymerized bovine hemoglobin‐based oxygen carrier (HBOC)‐201. Twenty‐four livers declined for transplantation were transported by using static cold storage. Upon arrival, livers underwent NMP for 6 hours using pressure‐controlled portal and arterial perfusion. A total of 12 livers were perfused using a solution based on RBCs and FFPs (historical cohort), 6 livers with HBOC‐201 and FFPs, and another 6 livers with HBOC‐201 and gelofusine, a gelatin‐based colloid solution. Compared with RBC + FFP perfused livers, livers perfused with HBOC‐201 had significantly higher hepatic adenosine triphosphate content, cumulative bile production, and portal and arterial flows. Biliary secretion of bicarbonate, bilirubin, bile salts, and phospholipids was similar in all 3 groups. The alanine aminotransferase concentration in perfusate was lower in the HBOC‐201–perfused groups. In conclusion, NMP of human donor livers can be performed effectively using HBOC‐201 and gelofusine, eliminating the need for human blood products. Perfusing livers with HBOC‐201 is at least similar to perfusion with RBCs and FFP. Some of the biomarkers of liver function and injury even suggest a possible superiority of an HBOC‐201–based perfusion solution and opens a perspective for further optimization of machine perfusion techniques. Liver Transplantation 24 528–538 2018 AASLD.

BACKGROUNDComparisons of peri-operative complications associated with paediatric (16 years) and adult anaesthesia are poorly available, especially in which cardiac surgery, organ transplantation and neurosurgery are involved.OBJECTIVEThe aim of this study was to evaluate the nature and incidence of peri-operative complications that might be due to anaesthesia and to identify independent risk factors for complications in children and adults, including those undergoing cardiac surgery, organ transplantation and neurosurgery.DESIGNRetrospective cohort study.SETTINGThe study was performed at the University Medical Centre Groningen in the 4 years between 1 January 2010 and the 31 December 2013.MAIN OUTCOME MEASURESComplications and their severity were graded according to the standard complication score (20 items) of the Dutch Society of Anaesthesia. Univariate and multivariate regression analysis was used to identify independent risk factors for the reported complications.RESULTSA total of 81267 anaesthetic cases were included. In the paediatric cohort, there were 410 (2.9%) complications and 1675 (2.5%) in the adults. In both cohorts age, American Society of Anaesthesiologists classification and emergency treatment were independent risk factors for complications. With respect to age, infants less than 1 year were at the highest risk, whereas in the adult cohort, increased age was related to a greater number of complications. The incidences of the specific complications were different between both cohorts. Upper airway obstruction was more frequently observed in paediatric patients (26%), whereas in the adults, complications with the highest incidence concerned conversion of regional-to-general anaesthesia (25%) and hypotension (17%).CONCLUSIONRisk factors for all peri-operative complications were similar for paediatric and adult anaesthesia. However, the incidence of specific complications differed between both age categories.

BACKGROUND:The mechanism through which oxygenated hypothermic machine perfusion (HMP) improves viability of human extended criteria donor (ECD) livers is not well known. Aim of this study was to examine the benefits of oxygenated HMP after static cold storage (SCS).METHODS:Eighteen ECD livers that were declined for transplantation underwent ex situ viability testing using normothermic (37 °C) machine perfusion (NMP) after traditional SCS (0 °C-4 °C) for 7 to 9 hours. In the intervention group (n = 6), livers underwent 2 hours of oxygenated HMP (at 12 °C) after SCS and before NMP. Twelve control livers underwent NMP without oxygenated HMP after SCS.RESULTS:During HMP, hepatic ATP content increased greater than 15-fold, and levels remained significantly higher during the first 4 hours of NMP in the HMP group, compared with controls. Cumulative bile production and biliary secretion of bilirubin and bicarbonate were significantly higher after HMP, compared with controls. In addition, the levels of lactate and glucose were less elevated after HMP compared with SCS preservation alone. In contrast, there were no differences in levels of hepatobiliary injury markers AST, ALT, LDH, and gamma-GT after 6 hours of NMP. Hepatic histology at baseline and after 6 hours of NMP revealed no differences in the amount of ischemic necrosis between both groups.CONCLUSIONS:Two hours of oxygenated HMP after traditional SCS restores hepatic ATP levels and improves hepatobiliary function but does not reduce (preexisting) hepatobiliary injury in ECD livers.

BACKGROUND:Experimental studies have suggested that end-ischaemic dual hypothermic oxygenated machine perfusion (DHOPE) may restore hepatocellular energy status and reduce reperfusion injury in donation after circulatory death (DCD) liver grafts. The aim of this prospective case-control study was to assess the safety and feasibility of DHOPE in DCD liver transplantation.METHODS:In consecutive DCD liver transplantations, liver grafts were treated with end-ischaemic DHOPE. Outcome was compared with that in a control group of DCD liver transplantations without DHOPE, matched for donor age, donor warm ischaemia time, and recipient Model for End-stage Liver Disease (MELD) score. All patients were followed for 1 year.RESULTS:Ten transplantations involving liver grafts treated with DHOPE were compared with 20 control procedures. There were no technical problems. All 6-month and 1-year graft and patient survival rates were 100 per cent in the DHOPE group. Six-month graft survival and 1-year graft and patient survival rates in the control group were 80, 67 and 85 per cent respectively. During DHOPE, median (i.q.r.) hepatic adenosine 5'-triphosphate (ATP) content increased 11-fold, from 6 (3-10) to 66 (42-87) µmol per g protein (P = 0·005). All DHOPE-preserved livers showed excellent early function. At 1 week after transplantation peak serum alanine aminotransferase (ALT) and bilirubin levels were twofold lower in the DHOPE group than in the control group (ALT: median 966 versus 1858 units/l respectively, P = 0·006; bilirubin: median 1·0 (i.q.r. 0·7-1·4) versus 2·6 (0·9-5·1) mg/dl, P = 0·044). None of the ten DHOPE-preserved livers required retransplantation for non-anastomotic biliary stricture, compared with five of 20 in the control group (P = 0·140).CONCLUSION:This clinical study of end-ischaemic DHOPE in DCD liver transplantation suggests that the technique restores hepatic ATP, reduces reperfusion injury, and is safe and feasible. RCTs with larger numbers of patients are warranted to assess the efficacy in reducing post-transplant biliary complications.

Bile duct injury may occur during liver procurement and transplantation, especially in livers from donation after circulatory death (DCD) donors. Normothermic machine perfusion (NMP) has been shown to reduce hepatic injury compared to static cold storage (SCS). However, it is unknown whether NMP provides better preservation of bile ducts. The aim of this study was to determine the impact of NMP on bile duct preservation in both DCD and non‐DCD livers. DCD and non‐DCD livers obtained from Lewis rats were preserved for 3 hours using either SCS or NMP, followed by 2 hours ex vivo reperfusion. Biomarkers of bile duct injury (gamma‐glutamyltransferase and lactate dehydrogenase in bile) were lower in NMP‐preserved livers compared to SCS‐preserved livers. Biliary bicarbonate concentration, reflecting biliary epithelial function, was 2‐fold higher in NMP‐preserved livers (P < 0.01). In parallel with this, the pH of the bile was significantly higher in NMP‐preserved livers (7.63 ± 0.02 and 7.74 ± 0.05 for non‐DCD and DCD livers, respectively) compared with SCS‐preserved livers (7.46 ± 0.02 and 7.49 ± 0.04 for non‐DCD and DCD livers, respectively). Scanning and transmission electron microscopy of donor extrahepatic bile ducts demonstrated significantly decreased injury of the biliary epithelium of NMP‐preserved donor livers (including the loss of lateral interdigitations and mitochondrial injury). Differences between NMP and SCS were most prominent in DCD livers. Compared to conventional SCS, NMP provides superior preservation of bile duct epithelial cell function and morphology, especially in DCD donor livers. By reducing biliary injury, NMP could have an important impact on the utilization of DCD livers and outcome after transplantation. Liver Transplantation 22 994–1005 2016 AASLD

Background. Ex situ normothermic machine perfusion (NMP) can be performed after traditional static cold preservation to assess graft function and viability before transplantation. It is unknown whether this results in activation of coagulation and fibrinolysis, as may occur upon graft reperfusion in vivo. Methods. Twelve donor livers declined for transplantation underwent 6 hours of end-ischemic NMP using a heparinized plasma-based perfusion fluid. Concentration of prothrombin fragment F1 + 2 (marker of coagulation activation), D-dimer, plasmin-antiplasmin complex, tissue plasminogen activator and plasminogen activator inhibitor-1 (markers for fibrinolysis) and alanine aminotransferase (ALT) (marker of ischemia-reperfusion [I/R] injury) were measured in perfusion fluid at regular intervals. Liver biopsies were examined for the presence of fibrin, using light microscopy after Maurits, Scarlet and Blue staining. Results. No significant increase in prothrombin F1 + 2 was noted during NMP. D-dimer and plasmin-antiplasmin complex levels increased soon after start of NMP and D-dimer concentrations correlated significantly with levels of tissue plasminogen activator. In livers displaying good function during NMP, perfusate levels of ALT and D-dimers were low (<= 3500 ng/mL), whereas significantly higher D-dimer levels (>3500 ng/mL) were in found in livers with poor graft function. Activation of fibrinolysis correlated significantly with the degree of I/R injury, as reflected by ALT levels. Conclusions. End-ischemic ex situ NMP results in activation of fibrinolysis, but not of coagulation. Markers of fibrinolysis activation correlate significantly with markers of I/R injury. High concentrations of D-dimer early after start of NMP can be considered a marker of severe I/R injury and a predictor of poor liver graft function.

Introduction: Normothermic machine perfusion (NMP) enables viability assessment of donor livers prior to transplantation. Up until now, NMP has been performed using human blood products including red blood cells (RBC). The aim was to examine the efficacy of a new machine perfusion solution based on polymerized bovine hemoglobin (Hemopure). Methods: Twenty-four livers declined for transplantation were included. After a median static cold preservation period of 8.1 hr, livers underwent NMP for 6 hr using pressure-controlled portal and arterial perfusion. Group 1 (n = 12) underwent NMP using a solution based on RBC and fresh frozen plasma (FFP), group 2 (n = 6) was perfused with Hemopure and FFP, and group 3 (n = 6) with Hemopure and Gelofusine, a gelatin-based colloid solution. Graft function and viability was compared. Results: Portal flow was significantly higher in livers perfused with Hemopure (median at 6 hr NMP in group 1: 848 ml/min [IQR: 663-1392], group 2: 1890 ml/min [1530-2173], group 3: 1830 ml/min [1713-2030]; p < 0.05). Cumulative bile production was significantly higher in livers perfused with Hemopure (group 1: 8.2 ml/kg liver [6.0–23.6]; group 2: 27.0 ml/kg liver [15.5–71.7], group 3: 29.0 ml/kg liver [22.4–44.9]; p < 0.05). Intrahepatic ATP content was higher in livers perfused with Hemopure. Peak oxygen consumption was similar in all three groups. Conclusion: Ex situ NMP of human donor livers can be performed effectively using a novel acellular perfusion solution based on Hemopure, eliminating the need for human blood products. Livers perfused with a Hemopure-based solution produced significantly more bile than RBC-perfused livers.

Background: Pre- or reconditioning of donor livers can improve organ quality prior to transplantation. The aim of this study was to investigate whether metformin as pre- or reconditioning agent is able to reduce preservation injury in rat donor livers and improve hepatobiliary function during ex situ normothermic machine perfusion (NMP). Methods: To study the preconditioning effects of metformin, metformin was administered via oral gavage 12 and 2 hours before the hepatectomy. To assess the reconditioning effects of metformin, in 2 other groups, metformin was added to the NMP perfusion fluid in two different concentrations (30 and 300 mg/L). In the reference group, no pre- or reconditioning was carried out. In all groups, rat donor livers were preserved for 4 hours in preservation fluid on melting ice. Thereafter, NMP was performed for viability assessment. Results: Preconditioning improved ATP production and hepatobiliary function (assessed by total bile production, biliary bilirubin and bicarbonate) and significantly lowered levels of lactate and glucose during NMP. On the other hand, metformin preconditioning did not reduce markers for hepatobiliary injury such as AST, ALT, LDH, caspase-3 activity, TBARS or biliary gamma-GT and LDH. Reconditioning with metformin did not improve hepatobiliary function or reduce injury markers during NMP. Conclusion: Preconditioning of rat donor livers with metformin improves hepatobiliary function but does not reduce preservation injury as assessed during 3 hours of NMP. Reconditioning with metformin showed no beneficial effects.

Frozen section analysis (FS) during cancer surgery is widely used to assess resection margins. However, in hilar cholangiocarcinoma (HCCA), FS may be less reliable because of the specific growth characteristics of the tumor. The aim of this study was to determine the accuracy and consequences of intraoperative FS of the proximal bile duct margins in HCCA. Between 1990 and 2014, 67 patients underwent combined extrahepatic bile duct resection and partial liver resection for HCCA with the use of FS. Sensitivity and specificity of FS was 68% and 97%, respectively. Seventeen of 67 patients (25%) displayed a positive bile duct margin at FS. The false-negative rate was 16% (eight patients). Ten patients (15%) with a positive bile duct margin underwent an additional resection in an attempt to achieve negative margins, which succeeded in three patients (4%). However, only one of these three patients did not have concomitant lymph node metastases, which are associated with a poor prognosis by itself. The use of FS of the proximal bile duct is of limited clinical value because of the relatively low sensitivity, high risk of false-negative results, and the low rate of secondary obtained tumor-free resection margins. Supported by the literature, a new approach to the use of FS in HCCA should be adopted, reserving the technique only for cases in which a substantial additional resection is possible.

Liver transplantation for hilar cholangiocarcinoma (hCCA) has regained attention since the Mayo Clinic reported their favorable results with the use of a neo-adjuvant chemoradiation protocol. However, debate remains whether the success of the protocol should be attributed to the neo-adjuvant therapy or to the strict selection criteria that are being applied. The aim of this study was to investigate the value of patient selection alone on the outcome of liver transplantation for hCCA. In this retrospective study, patients that were transplanted for hCCA between1990 and 2010 in Europe were identified using the European Liver Transplant Registry (ELTR). Twenty-one centers reported 173 patients (69%) of a total of 249 patients in the ELTR. Twenty-six patients were wrongly coded, resulting in a study group of 147 patients. We identified 28 patients (19%) who met the strict selection criteria of the Mayo Clinic protocol, but had not undergone neo-adjuvant chemoradiation therapy. Five-year survival in this subgroup was 59%, which is comparable to patients with pretreatment pathological confirmed hCCA that were transplanted after completion of the chemoradiation protocol at the Mayo Clinic. In conclusion, although the results should be cautiously interpreted, this study suggests that with strict selection alone, improved survival after transplantation can be achieved, approaching the Mayo Clinic experience.

End-ischemic oxygenated hypothermic machine perfusion (HMP) can restore cellular ATP energy stores in donor livers prior to transplantation. Given the significant arterial supply of oxygen to cholangiocytes and the ATP-driven bile production, HMP was hypothesized to improve the quality of bile by improving hepatic and cholangiocytic secretory function. Eighteen human donor livers declined for transplantation were preserved with static cold storage (SCS; median duration 8.1 hr) . Six of these livers underwent 2 hr oxygenated HMP, followed by 6 hr normothermic machine perfusion (NMP) for viability assessment (HMP group). Controls were 12 livers that underwent 6 hr NMP immediately after SCS (control group). Bile composition and gene expression of hepatocyte and cholangiocyte bile transporters (BSEP, MDR3, CYP7A1, AE2 and CFTR) were compared. Cumulative bile production during NMP was significantly higher in HMP preserved livers, compared to controls (at 6 hr NMP: 33.8 vs. 8.2 ml/kg liver; p = 0.019). Biliary secretion of bile salts, phospholipids and HCO3− during NMP in bile was higher in the HMP group, compared to controls (bile salts: 0.060 vs. 0.003 mmol/kg liver, p = 0.052; phospholipids: 0.019 vs. 0.003 mmol/kg liver, p = 0.053; bicarbonate during last 30 min: 477 vs. 175 mmol/kg/30 min liver respectively, p = 0.089). Gene expression of transporters was not different between groups. A short period of 2 hrs HMP after conventional SCS results in significantly higher bile output after subsequent warm reperfusion and increased biliary secretion of bile salts, phospholipids and bicarbonate. This increased secretory function is likely explained by increased function of the ATP-dependent bile transporters.

Background: Livers with moderate (30-60%) macrovesicular steatosis have been associated with poor outcome after transplantation. Aim of this study was to examine the outcome after transplantation of livers with moderate macrovesicular steatosis when the cold ischemia time (CIT) is kept very short.Methods: Postoperative outcome of 19 recipients of a moderate steatotic liver were compared with a matched control group of 95 recipients of a nonsteatotic liver graft (1:5 ratio). We studied graft/patient survival rates, incidences of primary nonfunction, postoperative complications (classified according to the Clavien-Dindo classification), first-week postoperative hepatic injury serum markers (AST/ALT), and liver function tests (PT time/bilirubin/lactate). In addition, we studied reversal of graft steatosis in follow-up biopsies.Results: Median CIT in livers with moderate steatosis and in controls was below 8 h in both groups. Although short-and long-term patient/graft survival rates and results of liver function tests were similar, serum markers of hepatic injury and postoperative complications (especially grade IVa) were significantly higher in recipients of a moderate steatotic liver. Reversal of steatosis was seen in 9 of the 11 (82%) recipients with follow-up liver biopsies.Conclusion: Despite the association with severe postoperative complications, moderate macrovesicular steatotic livers can be used successfully for transplantation if the CIT is kept very short.