Ex Vivo Reperfusion Model to Evaluate Utility of Machine Preservation for Porcine Liver Donated After Cardiac Death.

BACKGROUND: Machine perfusion (MP) techniques are expected to prove useful for preserving the organ viability and recovering organ function for organ transplantation. Furthermore, an accurate assessment of organ viability using MP is important for expanding the donor criteria. In this study, an ex vivo reperfusion model (ERM) simulating transplantation using diluted autologous blood under normothermic conditions was evaluated for its utility of MP under subnormothermic conditions for livers donated after cardiac death (DCD). METHODS: The liver preservation methods for DCD porcine livers were evaluated using the ERM. This investigation was performed using a novel perfusion system developed by our research group. Porcine livers were procured with a warm ischemia time (WIT) of 60 minutes. The organs were then preserved using subnormothemic machine perfusion (SNMP) or static cold storage (CS) for 4 hours. We also compared these tissues with SNMP livers procured under a WIT of 0 minutes. After the preservation, the livers were reperfused for 2 hours using the ERM with diluted autologous blood oxygenated by a membrane oxygenator under NMP conditions. Reperfusion was evaluated based on perfusion flow dynamics and outflow of deviating enzymes. RESULTS: In the early stages of reperfusion, pressure in the blood vessels increased sharply in the CS group. Furthermore, the amount of aspartate aminotransferase accumulation was lower in the SNMP group than in the other groups. These results suggest ischemia-reperfusion injury is suppressed in SNMP conditions. CONCLUSION: An ERM has use in evaluating the utility of MP for the DCD liver.

Optimum Perfusate Volume of Purified Subnormothermic Machine Perfusion for Porcine Liver Donated After Cardiac Death.

INTRODUCTION: Subnormothermic machine perfusion (SNMP) shows some advantages for the preservation of grafts donated after cardiac death (DCD) and improvements in machine perfusion (MP) technology are important to enhance organ preservation outcomes for liver transplantation. In this study, we focused on purified subnormothermic machine perfusion (PSNMP) and volumes of perfusate removed to substitute for purification and replaced by modified University of Wisconsin-gluconate after the start of perfusion and investigated, in particular, the optimum perfusate purification volume. Several purification volumes under SNMP were compared. In addition, the perfusate purification during MP was indicated as a potential technique to enhance the organ quality of DCD grafts and extended-criteria donors. METHODS: The PSNMP at several volumes (0.5 L, 1.5 L, and 3 L) were compared with regular SNMP without any purification treatment (untreated control). In the PSNMP group, all perfusate was removed to substitute for purification of the perfusate by modified University of Wisconsin-gluconate solution after the start of perfusion. After removing the perfusate, new perfusate with the same components was perfused to preserve the porcine livers obtained under warm ischemia for 60 minutes using SNMP at 22°C porcine liver for 4 hours. RESULTS: The concentrations of aspartate aminotransferase and lactate dehydrogenase in the untreated group were significantly higher during perfusion compared to those of the intervention group. There are no significant differences among the volume conditions of the purification groups. CONCLUSIONS: The optimal volume of perfusate purification was confirmed with a simple experimental comparison between untreated and PSNMP conditions.

Regulated Oxygenation of Rewarming Machine Perfusion for Porcine Donation After Cardiac Death Liver Transplantation.

INTRODUCTION: Machine perfusion (MP) is particularly expected to preserve and resuscitate an organ obtained from extended criteria donors or donation after cardiac death to expand the donated organ pool for organ transplantation. This method requires to be investigated an optimal preservation condition. The aim of this study is investigation of the optimal oxygenation conditions under rewarming MP (RMP). METHODS: Porcine livers were perfused with an RMP system developed by our research group. All livers were procured under warm ischemia time of 60 minutes, and preserved in static cold storage for 2 hours, and perfused for 2 hours using the RMP. For group 1, the livers were supplied with oxygen constantly through perfusate. For group 2, the livers were supplied with oxygen increasingly with controlling flow rates and oxygen concentration. Effluent enzymes were obtained during perfusion preservation. RESULTS: The average levels of alanine aminotransferase were lower in group 2 than in group 1 during RMP, and also decreasing the hepatic artery pressures after 60 minutes. CONCLUSIONS: Regulated oxygenation of RMP has possibility to improve the graft preservation for liver transplantation.

Reduction of analysis time in gas chromatography. Application of low-pressure gas chromatography-tandem mass spectrometry to the determination of pesticide residues in vegetables.

An alternative to conventional capillary gas chromatography (GC) is evaluated as a new approach to determine pesticide residues in vegetables. Low-pressure gas chromatography-tandem mass spectrometry (LP-GC-MS-MS) is proposed after a fast and simple extraction of the vegetable samples with dichloromethane and without clean up. The use of the above-mentioned GC technique reduced the total time required to determine 72 pesticides to less than half the present time (31 min), increasing the capability of a monitoring routine laboratory. The use of guard column and plug of carbofrit into the glass liner in combination with LP-GC was evaluated. The method was validated with limits of quantitation low enough to determine the pesticide residues at concentrations below the maximum residue levels stated by legislation. In order to assess its applicability to the analysis of real samples, 25 vegetable samples previously determined using conventional-capillary GC-MS-MS were analysed by LP-GC-MS-MS. The results obtained with the compared techniques showed differences lower than 0.01 mg kg(-1).

Determination of heavy metals in crayfish by ICP-MS with a microwave-assisted digestion treatment.

Contamination of the American red crayfish, from the Guadiamar riverside, is due to the disastrous toxic spill that occurred on 25 April 1998 in the mining area of Aznalcóllar-Los Frailes, Spain. A high concentration of heavy metals in the waters from the mine pool and their spill to the river Guadiamar was the cause of the destruction of a great number of animal and vegetable organisms. An ICP-MS method for the total determination of heavy metals (Cu, Zn, Pb, and Cd) in whole bodies of American red crayfish (Procambarus clarkii) was used. Metals were extracted from the matrix in a closed-vessel microwave digestion system with nitric acid and hydrogen peroxide. A study of the uncertainty of the method for the determination of metals was carried out; at a concentration of 5 microg/L, the uncertainty was below 34%.

Nrf2-deficient female mice develop lupus-like autoimmune nephritis.

BACKGROUND: NF-E2-related factor 2 (Nrf2) is a basic leucine zipper transcriptional activator essential for the coordinate transcriptional induction of antioxidant enzymes and phase II drug metabolizing enzymes through the antioxidant response element/electrophile response element. The Nrf2-deficient mice were found to develop normally under standard laboratory conditions. However, upon closer examination, we found that aged female Nrf2-deficient mice displayed a shortened lifespan and developed severe glomerulonephritis. The present study investigated the glomerulonephritis findings in Nrf2-deficient mice. METHODS: To evaluate glomerular lesions of Nrf2-deficient mice, histological and functional analyses were performed. The amounts of serum immunoglobulins, anti-double-stranded (ds) DNA antibody, and lipid peroxidation using thiobarbituric acid reactive substances (TBARS) also were measured. RESULTS: Nrf2-deficient female mice over 60 weeks of age developed severe nephritis characterized by cellular proliferation, lobular formation, crescent formation, and subepithelial electron-dense deposits. In immunofluorescent assays, Nrf2-deficient female mice showed mesangial deposits and massive granular deposits of IgG, IgM, and C3 along the capillary walls. Higher serum levels of IgG, anti-dsDNA antibody, lower creatinine clearance, and slight splenomegaly also were found in Nrf2-deficient female mice. A higher concentration of TBARS also was found in Nrf2-deficient female mice. CONCLUSIONS: These data indicate that the aged Nrf2-deficient female mice develop lupus-like autoimmune nephritis and suggest that nrf2 is one of the genes determining susceptibility to autoimmune disease. Analysis of nephritis in the Nrf2-deficient female mouse may clarify the mechanisms leading to the development of lupus disease.

Influence of magnesium deficiency on the bioavailability and tissue distribution of iron in the rat.

We investigated the effect of dietary magnesium (Mg) deficiency on the nutritive utilization and tissue distribution of iron (Fe). Wistar rats were fed an Mg-deficient diet (56 mg/kg) for 70 days. Absorbed Fe, Fe balance, number of the erythrocytes [red blood cells (RBC)] and leukocytes white blood cells (WBC)], hemoglobin (Hb), and Fe content were determined in samples of plasma, whole blood, skeletal muscle, heart, kidney, liver, spleen, femoral bone, and sternum obtained on experimental days 21, 35, and 70. The Mg-deficient diet significantly increased Fe absorption and Fe balance from week 5 until the end of the experimental period. This effect was accompanied by a significant decrease in the concentration of RBC and Hb from day 35, which caused the decrease in whole blood Fe seen on day 70. However, WBC were significantly increased from day 21 until the end of the experimental period. Mg deficiency significantly increased plasma and liver Fe at all three time points investigated. Spleen, heart, and kidney Fe were significantly increased only at the end of the study. However, on day 70, Fe concentration in the sternum had decreased significantly. No changes were found in skeletal muscle or femur Fe content. Mg deficiency led to increased intestinal absorption of Fe and decreased RBC counts, possibly as a result of increased fragility of the erythrocytes. Intestinal interactions between Fe and Mg, together with activation of erythropoiesis as a result of hemolysis, favored intestinal absorption of Fe. This situation gave rise to an increase in plasma Fe levels, which in turn favored Fe uptake and storage by different organs, especially the liver and spleen. However, despite the increased Fe content seen in the tissues of rats fed the Mg-deficient diet, these animals were unable to compensate for the hemolysis caused by this nutritional deficiency.

Effect of magnesium deficiency on enterocyte Ca, Fe, Cu, Zn, Mn and Se content.

In previous studies based on indirect procedures, we reported that Mg deficit increased the bioavailability of a number of elements such as calcium, zinc, iron, copper, manganese and decreased selenium absorption. The present study was designed to verify these findings by direct methods. We investigated the effect of dietary magnesium deficiency on enterocyte Ca, Fe, Zn, Cu, Mn and Se concentrations. Male Wistar rats were fed a Mg-deficient diet (129 mg Mg/kg food) for 70 days. Whole enterocytes from the upper jejunum were isolated and Ca, Fe, Zn, Cu, Mn and Se were determined. The results were compared with findings in a control group that was pair-fed with an identical diet except that it covered this species's nutritional requirements for Mg (480 mg Mg/kg food). The Mg-deficient diet significantly increased enterocyte content of Ca, Fe, Zn, Cu and Mn; however, we found no significant changes in the Se content of these cells. These data support the results obtained by indirect methods.

Magnesium-manganese interactions caused by magnesium deficiency in rats.

OBJECTIVE: We investigated the effect of dietary magnesium (Mg) deficiency on the nutritive utilization and tissue distribution of manganese (Mn). METHODS: Wistar rats were fed a Mg-deficient diet (56 mg/kg) for 70 day. Absorbed Mn, Mn balance and Mn content in plasma, whole blood, skeletal muscle, heart, kidney, liver, femoral bone and sternum were determined after 21, 35 and 70 days. RESULTS: The Mg-deficient diet significantly increased Mn apparent absorption and Mn balance from week five until the end of the experimental period. This effect was accompanied by a significant increase in the concentration of Mn in heart at all three time points. Whole blood, skeletal muscle and kidney Mn were significantly increased from day 35, and femur Mn content was increased only at the end of the study (day 70). However, Mn concentration in the sternum decreased significantly from day 35. No changes were found in liver Mn content. CONCLUSION: Mg deficiency increased Mn absorbed, and this favored the deposition of Mn in all tissues studied except the liver and trabecular bone. The lack of response by the liver to increased Mn absorption may have led to the redistribution of this ion to other tissues.

Effect of magnesium deficiency on vitamin B2 and B6 status in the rat.

OBJECTIVE: The present study was undertaken to investigate the effect of dietary magnesium (Mg) deficiency on vitamin B2 (riboflavin) and B6 (pyridoxine) status in rats. METHODS: We fed Wistar rats an Mg-deficient diet (56 mg magnesium per kg food) for 70 days. On days 21, 35 and 70 we measured Mg and manganese (Mn) in plasma and whole blood, alkaline phosphatase in plasma, erythrocyte glutathione reductase activity coefficient (AC-EGR) for riboflavin status, and erythrocyte aspartase transaminase activity coefficient (AC-EAST) for vitamin B6 status. RESULTS: Intake of the Mg-deficient diet significantly decreased plasma and whole blood levels of Mg and plasma alkaline phosphatase activity on all sampling days. The Mg deficit had no effect on plasma concentrations of Mn, but significantly increased whole blood levels of this element on days 35 and 70. The Mg-deficient diet had no effect on AC-EGR, and thus appeared not to affect riboflavin status. However, AC-EAST was significantly increased on day 70, implying that the animals were at risk for vitamin B6 deficiency. CONCLUSION: Mg deficiency impairs vitamin B6 status by depleting intracellular Mg and thus inhibits the activity of alkaline phosphatase, a metalloenzyme required for the uptake of pyridoxal phosphate by tissues. Although part of the intraerythrocyte loss of Mg is countervailed by Mn, which can act as a substitute activator of alkaline phosphatase, the degree of compensation is insufficient to fully offset the decrease in activity caused by Mg depletion.

Risk factors in the pregnancy of patients with systemic lupus erythematosus: association of hypocomplementaemia with poor prognosis.

Fetal wastage is still high in the pregnancies of patients with systemic lupus erythematosus (SLE). We examined retrospectively the cases of 38 patients with inactive SLE in whom pregnancy was either desired or had already been obtained. The prevalence of antiphospholipid antibodies in the group with fetal loss was high. The antibodies were, however, also detected in five of 14 patients who had had a live birth. It was noted that low levels of serum complement activity (CH50 less than 25 U/ml) occurred in five of six patients with fetal loss, but in only two of 22 with a live birth. Serial studies also confirmed a close association between decreased serum complement activity and poor fetal prognosis in lupus pregnancy. Treatment with increased doses of prednisolone may help to achieve successful live births. Thus hypocomplementaemia may be associated with a worse prognosis for the fetus in the pregnancies of some patients with SLE in remission.