Rapid Determination of Reduced and Oxidized Glutathione Levels Using a New Thiol-Masking Reagent and the Enzymatic Recycling Method: Application to the Rat Liver and Bile Samples

A microtiter plate assay for quantitation of reduced (GSH) and oxidized (GSSG) glutathione in the rat liver tissue and bile is described. The assay is based on the established enzymatic recycling method and a new thiol-masking reagent, 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate (M4VP). Samples were first processed by homogenization with (liver) or addition of (bile) sulfosalicylic acid. The total glutathione and GSSG were then determined before and after rapid (\u3e= 2 min) and efficient (100%) masking of the GSH content of the samples with M4VP followed by the enzymatic recycling assay. The percentages of error and coefficient of variation of the assay were within the accepted guidelines, indicating the accuracy and precision of the assay in the range of 6.25-100 pmol GSH per microplate well and 2.17-140 pmol GSSG per well, with lower limit of quantitation of 6.25 and 2.17 pmol per well for GSH and GSSG, respectively. Furthermore, the recoveries of added GSH or GSSG from the liver and bile samples were accurate and precise. The assay was applied to measurement of GSH, GSSG, and GSH:GSSG ratio in the liver and serially collected bile samples in sham-operated and ischemic rat livers, demonstrating a depletion of glutathione and a decrease in the GSH:GSSG ratio as a result of ischemia. The developed assay is rapid, sensitive, accurate, and precise and is suitable for studies of the redox status of liver under physiologic and pathophysiologic conditions

Effects of In Vivo Hepatic Ischemia-Reperfusion Injury on the Hepatobiliary Disposition of Rhodamine 123 and its Metabolites in Isolated Perfused Rat Livers

Purpose. A few studies have shown that normothermic hepatic ischemia-reperfusion (IR) injury may affect the mRNA and/or protein levels of canalicular transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2). However, the effects of the injury on the functions of these canalicular transporters with respect to the biliary excretion of drugs remain largely unknown. Therefore, the purpose of this study was to investigate the effects of warm hepatic IR on the hepatobiliary disposition of rhodamine 123 (RH-123), a P-gp substrate, and its glucuronidated metabolite (RH-Glu), an Mrp2 substrate, in rats. Methods. Twenty four or 72 h following a 60-min partial ischemia or sham operation in rats, livers were isolated and perfused ex vivo with a constant concentration (~100 ng/mL) of RH-123. The concentration of RH-123 and its glucuronidated (RH-Glu) and deacylated (RH-110) metabolites were determined in the outlet perfusate, bile, and the liver tissue using HPLC, and relevant pharmacokinetic parameters were estimated. Results. Twenty-four-h IR caused a significant reduction in the hepatic extraction ratio of RH-123 (IR: 0.857 ± 0.078; Sham: 0.980 ± 0.017) and the biliary recovery of the parent drug and RH-Glu by 43% and 44%, respectively. The reductions in the biliary recovery were associated with significant reductions in the apparent biliary clearance of RH-123 and RH-Glu. Mass balance data showed that the formation of the glucuronidated or deacylated metabolite was not significantly affected by the 24-h IR injury. In contrast to the 24-h IR, the injury did not have any effect on the hepatobiliary disposition of RH-123 or its metabolites following 72 h of reperfusion. Conclusions. It is concluded that the pharmacokinetics of drugs that are subject to biliary excretion by the canalicular P-gp and Mrp2 transporters may be altered shortly after hepatic IR injury

Effects of Short-Term Portacaval Anastomosis on the Peripheral and Brain Disposition of the Blood-Brain Barrier Permeability Marker Sodium Fluorescein in Rats

Contradictory results have been reported with regard to the effects of various models of hepatic encephalopathy on the blood-brain barrier (BBB) permeability, which may be due partly to the use of brain concentrations of BBB markers without attention to their peripheral pharmacokinetics. The purpose of the current study was to investigate the effects of short-term portacaval anastomosis (PCA), a type B model of hepatic encephalopathy, on the peripheral pharmacokinetics and brain distribution of sodium fluorescein (FL), which is a small molecule marker of BBB passive permeability. A single 25 mg/kg dose of FL was administered intravenously to 10-day PCA and sham-operated rats, and serial blood and bile (0-30 min) and terminal (30 min) brain samples were collected, and the concentrations of FL and its glucuronidated metabolite (FL-Glu) were measured by HPLC. Additionally, the free fractions of FL (fu) in all the plasma samples were determined, and the effects of bile salts on fu were investigated in vitro. Passive permeability of BBB to FL was estimated by brain uptake clearance (Kin) based on both the brain concentrations of FL and plasma concentrations of free (unbound) FL. PCA caused a 26% increase in the fu of FL in plasma, which was due to competition of bile acids with FL for binding to plasma proteins. Additionally, PCA reduced the biliary excretion of FL-Glu by 55%. However, free Kin values (μl/min/g brain) for the sham (0.265 ± 0.034) and PCA (0.228 ± 0.038) rats were not significantly different. It is concluded that whereas 10-day PCA alters the peripheral pharmacokinetics of FL, it does not significantly affect the BBB permeability to the marker

A Hybrid Modified Ant Colony Optimization - Particle Swarm Optimization Algorithm for Optimal Node Positioning and Routing in Wireless Sensor Networks

Wireless Sensor Networks (WSNs) have been widely deployed in hostile locations for environmental monitoring. Sensor placement and energy management are the two main factors that should be focused due to certain limitations in WSNs. The nodes in a sensor network might not stay charged when energy draining takes place; therefore, increasing the operational lifespan of the network is the primary purpose of energy management. Recently, major research interest in WSN has been focused with the essential aspect of localization. Several types of research have also taken place on the challenges of node localization of wireless sensor networks with the inclusion of range-free and range-based localization algorithms. In this work, the optimal positions of Sensor Nodes (SNs) are determined by proposing a novel Hybrid M-ACO – PSO (HMAP) algorithm. In the HMAP method, the improved PSO utilizes learning strategies for estimating the relay nodes\u27 optimal positions. The M-ACO assures the data conveyance. A route discovers when it relates to the ideal route irrespective of the possibility of a system that includes the nodes with various transmission ranges, and the network lifetime improves. The proposed strategy is executed based on the energy, throughput, delivery ratio, overhead, and delay of the information packets

Hepatic immunosuppressive effects of systemically administered novel dextran–methylprednisolone prodrugs with peptide linkers in rats

The hepatic immunosuppressive activities of two novel dextran prodrugs of methylprednisolone (MP) containing one (DMP1) or five (DMP5) amino acids as linkers were studied in rats. At various times (0–2 weeks) after intravenous administration of single 5 mg/kg (MP equivalent) doses of each prodrug or MP succinate (MPS), livers were isolated and immunologically stimulated ex vivo with lipopolysaccharide. The concentrations of tumor necrosis factor (TNF)‐α in the outlet perfusate were then quantitated to assess immune response. Additionally, the concentrations of DMP1, DMP5, and/or MP were measured in the liver. MPS, DMP5, or DMP1 injections caused a maximum of 48.9%, 63.5%, or 85.7% decrease in the TNF‐α secretion into the perfusate, with the time above the 50% inhibitory effect being \u3c5, \u3c24, or 120 h, respectively. Additionally, the area under the effect–time curve for DMP1 was 11‐fold or fourfold higher than that after the administration of MPS or DMP5, respectively. Relatively high concentrations of DMP1 were present in the liver even at the last sampling time of 2 weeks. These data suggest that a single intravenous dose of DMP1 produces an intense and sustained immunosuppression in the liver for a relatively long time, which may be useful in liver transplantation

Effects of Hepatic Ischemia-Reperfusion Injury on the P-Glycoprotein Activity at the Liver Canalicular Membrane and Blood-Brain Barrier Determined by In Vivo Administration of Rhodamine 123 in Rats

Purpose To investigate the effects of normothermic hepatic ischemia-reperfusion (IR) injury on the activity of P-glycoprotein (P-gp) in the liver and at the blood-brain barrier (BBB) of rats using rhodamine 123 (RH-123) as an in vivo marker. Methods Rats were subjected to 90 min of partial ischemia or sham surgery, followed by 12 or 24 h of reperfusion. Following intravenous injection, the concentrations of RH-123 in blood, bile, brain, and liver were used for pharmacokinetic calculations. The protein levels of P-gp and some other transporters in the liver and brain were also determined by Western blot analysis. Results P-gp protein levels at the liver canalicular membrane were increased by twofold after 24 h of reperfusion. However, the biliary excretion of RH-123 was reduced in these rats by 26%, presumably due to IR-induced reductions in the liver uptake of the marker and hepatic ATP concentrations. At the BBB, a 24% overexpression of P-gp in the 24-h IR animals was associated with a 30% decrease in the apparent brain uptake clearance of RH-123. The pharmacokinetics or brain distribution of RH-123 was not affected by the 12-h IR injury. Conclusions Hepatic IR injury may alter the peripheral pharmacokinetics and brain distribution of drugs that are transported by P-gp and possibly other transporters

UTILIZING THE UNUSED GLASS SUBSTANCE IN STAND-IN OF CEMENT

Glass can be used in lots of forms in day-to-day existence. It's limited life time after utilize it is either stock stacked or delivered to landfills. Since glass is non-biodegradable, landfills don't offer an atmosphere friendly solution. Hence, there's strong have to utilize waste glasses. Many efforts happen to be designed to use waste glass in concrete industry like a substitute of coarse aggregate, fine aggregate and cement. Its performance like a coarse aggregate substitute has been discovered to become non-acceptable due to strength regression and expansion because of alkali-silica reaction. The study implies that there's strength loss because of fine aggregate substitution also. The purpose of the current work ended up being to use glass powder like a substitute of cement to evaluate the pozzolanic activity of proper glass powder in concrete and compare its performance along with other pozzolanic materials like silica fume and fly ash. A number of tests were conducted to review the result of 15% and 30% substitute of cement by silica fume, fly ash and glass powder on compressive durability and strength by means of capillary absorption. The particle size effect was evaluated by utilizing glass powder of size 150µm-100µm and glass powder of size under 100µm. The current study implies that waste glass, if ground finer than 100µm shows a pozzolanic behavior. It responds to lime at initial phase of hydration developing extra CSH gel therefore developing denser cement matrix. The first use of alkalis by glass particles mitigate alkali-silica reaction hence increase reliability of concrete

Effects of In Vivo Hepatic Ischemia-Reperfusion Injury on the Hepatobiliary Disposition of Rhodamine 123 and its Metabolites in Isolated Perfused Rat Livers

Purpose. A few studies have shown that normothermic hepatic ischemia-reperfusion (IR) injury may affect the mRNA and/or protein levels of canalicular transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2). However, the effects of the injury on the functions of these canalicular transporters with respect to the biliary excretion of drugs remain largely unknown. Therefore, the purpose of this study was to investigate the effects of warm hepatic IR on the hepatobiliary disposition of rhodamine 123 (RH-123), a P-gp substrate, and its glucuronidated metabolite (RH-Glu), an Mrp2 substrate, in rats. Methods. Twenty four or 72 h following a 60-min partial ischemia or sham operation in rats, livers were isolated and perfused ex vivo with a constant concentration (~100 ng/mL) of RH-123. The concentration of RH-123 and its glucuronidated (RH-Glu) and deacylated (RH-110) metabolites were determined in the outlet perfusate, bile, and the liver tissue using HPLC, and relevant pharmacokinetic parameters were estimated. Results. Twenty-four-h IR caused a significant reduction in the hepatic extraction ratio of RH-123 (IR: 0.857 ± 0.078; Sham: 0.980 ± 0.017) and the biliary recovery of the parent drug and RH-Glu by 43% and 44%, respectively. The reductions in the biliary recovery were associated with significant reductions in the apparent biliary clearance of RH-123 and RH-Glu. Mass balance data showed that the formation of the glucuronidated or deacylated metabolite was not significantly affected by the 24-h IR injury. In contrast to the 24-h IR, the injury did not have any effect on the hepatobiliary disposition of RH-123 or its metabolites following 72 h of reperfusion. Conclusions. It is concluded that the pharmacokinetics of drugs that are subject to biliary excretion by the canalicular P-gp and Mrp2 transporters may be altered shortly after hepatic IR injury

Liposomal Fasudil, a Rho-Kinase Inhibitor, for Prolonged Pulmonary Preferential Vasodilation in Pulmonary Arterial Hypertension

Current pharmacological interventions for pulmonary arterial hypertension (PAH) require continuous infusions, multiple inhalations, or oral administration of drugs that act on various pathways involved in the pathogenesis of PAH. However, invasive methods of administration, short duration of action, and lack of pulmonary selectivity result in noncompliance and poor patient outcomes. In this study, we tested the hypothesis that encapsulation of an investigational anti-PAH molecule fasudil (HA-1077), a Rho-kinase inhibitor, into liposomal vesicles results in prolonged vasodilation in distal pulmonary arterioles. Liposomes were prepared by hydration and extrusion method and fasudil was loaded by ammonium sulfate-induced transmembrane electrochemical gradient. Liposomes were then characterized for various physicochemical properties. Optimized formulations were tested for pulmonary absorption and their pharmacological efficacy in a monocrotaline (MCT) induced rat model of PAH. The entrapment efficiency of optimized liposomal fasudil formulations was between 68.1 ± 0.8% and 73.6 ± 2.3%, and the cumulative release at 37 °C was 98–99% over a period of 5 days. Compared to intravenous (IV) fasudil, a ~ 10 fold increase in the terminal plasma half-life was observed when liposomal fasudil was administered as aerosols. The t1/2 of IV fasudil was 0.39 ± 0.12 h. and when given as liposomes via pulmonary route, the t1/2 extended to 4.71 ± 0.72 h. One h after intratracheal instillation of liposomal fasudil, mean pulmonary arterial pressure (MPAP) was reduced by 37.6 ± 5.7% and continued to decrease for about 3 h, suggesting that liposomal formulations produced pulmonary preferential vasodilation in MCT induced PAH rats. Overall, this study established the proof-of-principle that aerosolized liposomal fasudil is a feasible option for a non-invasive, controlled release and pulmonary preferential treatment of PAH

Attenuation of Acute Rejection in a Rat Liver Transplantation Model by a Liver-Targeted Dextran Prodrug of Methylprednisolone

Background. The use of methylprednisolone (MP) and other corticosteroids for the treatment of acute liver allograft rejection is associated with severe toxicities in nontarget tissues. Therefore, selective delivery of NIP to the liver may improve its efficacy and alleviate its side effects. We investigated the effects of a novel liver-targeted dextran prodrug of MP (DMP) in an orthotopic rat liver transplantation (OLT) model. Methods. The model consisted of a high responder rejection strain combination (Dark Agouti donors and Lewis recipients). Liver recipients were intravenously administered saline or a single subtherapeutic dose of MP (5 mg/kg) as the parent drug (MP) or its prodrug (DMP). Different groups were then monitored for graft survival or euthanized 5 or 9 days posttransplantation. Plasma chemistry, including alkaline phosphatase and bilirubin, allograft histology, and survival duration were determined. Results. Untreated recipients exhibited elevated plasma levels of liver injury markers, progressive portal and venous inflammation and cellular infiltration in liver allografts, and a mean graft survival time (MST) of 10.5 days. MP treatment did not alter any of these parameters. In contrast, a single dose of DMP resulted in a decrease in plasma levels of liver injury markers, a decrease in histological grade of rejection on day 5, and a substantial increase in MST (27.5 days). Conclusions. These results demonstrate attenuation of acute rejection following local (allograft) immunosuppression with a single subtherapeutic dose of NIP delivered as a liver-targeted prodrug. Dextran prodrugs may be useful for selective delivery of immunosuppressants to the liver following liver transplantation