Effects of hepatic ischemia-reperfusion injury on the blood-brain barrier permeability to [14C] and [13C]sucrose.

Hepatic encephalopathy that is associated with severe liver failure may compromise the blood-brain barrier (BBB) integrity. However, the effects of less severe liver diseases, in the absence of overt encephalopathy, on the BBB are not well understood. The goal of the current study was to investigate the effects of hepatic ischemia-reperfusion (IR) injury on the BBB tight junction permeability to small, hydrophilic molecules using the widely used [14C]sucrose and recently-proposed alternative [13C]sucrose as markers. Rats were subjected to 20 min of hepatic ischemia or sham surgery, followed by 8 h of reperfusion before administration of a single bolus dose of [14C] or [13C]sucrose and collection of serial (0-30 min) blood and plasma and terminal brain samples. The concentrations of [14C] and [13C]sucrose in the samples were determined by measurement of total radioactivity (nonspecific) and LC-MS/MS (specific), respectively. IR injury significantly increased the blood, plasma, and brain concentrations of both [14C] and [13C]sucrose. However, when the brain concentrations were corrected for their respective area under the blood concentration-time curve, only [14C]sucrose showed significantly higher (30%) BBB permeability values in the IR animals. Because [13C]sucrose is a more specific BBB permeability marker, these data indicate that our animal model of hepatic IR injury does not affect the BBB tight junction permeability to small, hydrophilic molecules. Methodological differences among studies of the effects of liver diseases on the BBB permeability may confound the conclusions of such studies.

Evaluation of [14C] and [13C]Sucrose as Blood-Brain Barrier Permeability Markers.

Nonspecific quantitation of [14C]sucrose in blood and brain has been routinely used as a quantitative measure of the in vivo blood-brain barrier (BBB) integrity. However, the reported apparent brain uptake clearance (Kin) of the marker varies widely (∼100-fold). We investigated the accuracy of the use of the marker in comparison with a stable isotope of sucrose ([13C]sucrose) measured by a specific liquid chromatography-tandem mass spectrometry method. Rats received single doses of each marker, and the Kin values were determined. Surprisingly, the Kin value of [13C]sucrose was 6- to 7-fold lower than that of [14C]sucrose. Chromatographic fractionation after in vivo administration of [14C]sucrose indicated that the majority of the brain content of radioactivity belonged to compounds other than the intact [14C]sucrose. However, mechanistic studies failed to reveal any substantial metabolism of the marker. The octanol:water partition coefficient of [14C]sucrose was >2-fold higher than that of [13C]sucrose, indicating the presence of lipid-soluble impurities in the [14C]sucrose solution. Our data indicate that [14C]sucrose overestimates the true BBB permeability to sucrose. We suggest that specific quantitation of the stable isotope (13C) of sucrose is a more accurate alternative to the current widespread use of the radioactive sucrose as a BBB marker.

Development and validation of a sensitive UPLC-MS/MS method for the quantitation of [(13)C]sucrose in rat plasma, blood, and brain: Its application to the measurement of blood-brain barrier permeability.

Accurate and reproducible measurement of blood-brain barrier (BBB) integrity is critical in the assessment of the pathophysiology of the central nervous system disorders and in monitoring therapeutic effects. The widely-used low molecular weight marker [(14)C]sucrose is non-specific in the absence of chromatographic separation. The purpose of this study was to develop and validate a sensitive and reproducible LC-MS/MS method for the analysis of stable isotope-modified [(13)C12]sucrose in brain, plasma, and blood to determine BBB permeability to sucrose. After addition of internal standard (IS, [(13)C6]sucrose), the marker and IS were recovered from diluted rat blood, plasma, and brain homogenate by protein precipitation using acetonitrile. The recovery of the marker and IS was almost quantitative (90-106%) for all three matrices. The recovered samples were directly injected into an isocratic UPLC system with a run time of 6 min. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r(2)≥0.99) in the concentration ranges tested for the diluted blood and plasma (10-1000 ng/mL) and brain homogenate (1-200 ng/mL). The lower limit of quantitation of the assay was 0.5 pg injected on column. The assay was validated (n=5) based on acceptable intra- and inter-run accuracy and precision values. The method was successfully used for the measurement of serial blood and plasma and terminal brain concentrations of [(13)C12]sucrose after a single intravenous dose (10 mg/kg) of the marker to rats. As expected, the apparent brain uptake clearance values of [(13)C12]sucrose were low in healthy rats. The method may be useful for determination of the BBB integrity in animal models.

Effects of Pringle maneuver and partial hepatectomy on the pharmacokinetics and blood-brain barrier permeability of sodium fluorescein in rats.

Liver diseases are known to affect the function of remote organs. The aim of the present study was to investigate the effects of Pringle maneuver, which results in hepatic ischemia-reperfusion (IR) injury, and partial hepatectomy (Hx) on the pharmacokinetics and brain distribution of sodium fluorescein (FL), which is a widely used marker of blood-brain barrier (BBB) permeability. Rats were subjected to Pringle maneuver (total hepatic ischemia) for 20 min with (HxIR) or without (IR) 70% hepatectomy. Sham-operated animals underwent laparotomy only. After 15 min or 8h of reperfusion, a single 25-mg/kg dose of FL was injected intravenously and serial (0-30 min) blood and bile and terminal brain samples were collected. Total and free (ultrafiltration) plasma, total brain homogenate, and bile concentrations of FL and/or its glucuronidated metabolite (FL-Glu) were determined by HPLC. Both IR and HxIR caused significant reductions in the biliary excretions of FL and FL-Glu, resulting in significant increases in the plasma AUC of the marker. Additionally, the free fraction of FL in plasma was significantly increased by HxIR. Although the brain concentrations of FL were increased by almost twofold in both IR and HxIR animals, the brain concentrations corrected by the free FL AUC (and not the total AUC) were similar in both groups at either time points. It is concluded that Pringle maneuver and/or partial hepatectomy substantially alters the hepatobiliary disposition, plasma AUC, plasma free fraction, and brain accumulation of FL without altering the BBB permeability to the marker.

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.

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 25mg/kg dose of FL was administered intravenously to 10-day PCA and sham-operated rats, and serial blood and bile (0-30min) and terminal (30min) 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.

Polyphenols content, cytotoxic, membrane stabilizing and thrombolytic activities of Sarcolobus globosus:a medicinal plant from Sundarban Forest / Contenido de polifenoles, citotoxicidad, estabilizante de membranas y actividades tromboliticas de Sarcolobus globosus: una planta medicinal de la Foresta Sundarban

The crude methanolic extract of the bark of Sarcolobus globosus (Family-apocynaceae) and its different organic soluble Kupchan fractions were screened for total phenol content (TPC), cytotoxic, membrane stabilizing and thrombolytic activities. The polyphenol content was determined colorimetrically using Folin-Ciocalteu method and expressed in gallic acid equivalent. The chloroform soluble Kupchan fraction (CSF) exhibited higher level of Total Polyphenol Contents (TPC, 54.21 gm of GAE/100 gm of dried extract). In the brine shrimp lethality bioassay, the crude methanolic extract (MEBP) exhibited significant cytotoxicity. The membrane stabilizing activity was assessed by using erythrocyte in hypotonic solution and was compared with acetyl salicylic acid. The hexane soluble Kupchan fraction (HSF) produced 52.73 percent inhibition of hemolysis of RBC as compared to 65.38 percent revealed by acetyl salicylic acid (0.10 mg/mL). In thrombolytic study screening, the crude methanolic extract demonstrated significant thrombolytic activity in human blood specimen.

El extracto crudo metanólico de la corteza de Sarcolobus globosus (Familia-apocynaceae) y sus diferentes fracciones solubles Kupchan fueron identificadas para contenido total de fenoles (CTF), actividades citotóxicas, estabilizantes de membrana y trombolíticas. El contenido de polifenoles fue determinado colorimétricamente usando el método Folin-Ciocalteu y expresados en equivalentes a ácido gálico. La fracción Kupchan soluble en cloroformo (FSC) exhibió los mayores niveles de Contenido Total de Polifenoles (CFT, 54,21 gm of GAE/100gm de extracto seco). En el bioensayo de letalidad (Artemia salina), el extracto metanólico crudo (EMC) exhibió una siginificativa citotoxicidad. La actividad estabilizadora de membrana fue estimada usando eritrocitos en un medio hipotónico y fue comparado con el ácido acetil salicílico. La fracción Kupchan soluble en hexano (FSH) produjo un 52,73 por ciento de inhibición de la hemólisis de los glóbulos rojos comparado con un 65,38 por ciento revelado por el ácido acetil salicílico (0,1 mg/mL). En las determinaciones trombolíticas, el extracto metanólico crudo demostró una significativa actividad trombolítica en una muestra de sangre humana.