UHPLC-MS/MS method for pharmacokinetic and bioavailability determination of five bioactive components in raw and various processed products of Polygala tenuifolia in rat plasma.

CONTEXT: Sibiricose A5 (A5), sibiricose A6 (A6), 3,6'-disinapoyl sucrose (DSS), tenuifoliside A (TFSA) and 3,4,5-trimethoxycinnamic acid (TMCA) are the main active components of Polygala tenuifolia Willd. (Polygalaceae) (PT) that are active against Alzheimer's disease. OBJECTIVE: To compare the pharmacokinetics and bioavailability of five active components in the roots of raw PT (RPT), liquorice-boiled PT (LPT) and honey-stir-baked PT (HPT). MATERIALS AND METHODS: The median lethal dose (LD50) was evaluated through acute toxicity test. The pharmacokinetics of five components after oral administration of extracts of RPT, LPT, HPT (all equivalent to 1.9 g/kg of RPT extract for one dose) and 0.5% CMC-Na solution (control group) were investigated, respectively, in Sprague-Dawley rats (four groups, n = 6) using UHPLC-MS/MS. In addition, the absolute bioavailability of A5, A6, DSS, TFSA and TMCA after oral administration (7.40, 11.60, 16.00, 50.00 and 3.11 mg/kg, respectively) and intravenous injection (1/10 of the corresponding oral dose) in rats (n = 6) was studied. RESULTS: The LD50 of RPT, LPT and HPT was 7.79, 14.55 and 15.99 g/kg, respectively. AUC 0- t of RPT, LPT and HPT were as follows: A5 (433.18 ± 65.48, 680.40 ± 89.21, 552.02 ± 31.10 ng h/mL), A6 (314.55 ± 62.73, 545.76 ± 123.16, 570.06 ± 178.93 ng h/mL) and DSS (100.30 ± 62.44, 232.00 ± 66.08, 197.58 ± 57.37 ng h/mL). The absolute bioavailability of A5, A6, DSS, TFSA and TMCA was 3.25, 2.95, 2.36, 1.17 and 42.91%, respectively. DISCUSSION AND CONCLUSIONS: The pharmacokinetic and bioavailability parameters of each compound can facilitate future clinical studies.

UHPLC-MS/MS method for simultaneous determination of Radix Polygalae glycolipids and organic acids in rat plasma and application in a pharmacokinetic study.

Radix Polygala (Yuanzhi in Chinese) is well-known in traditional Chinese medicine (TCM) and has been used for treatment of depression, brain protection, and memory improvement for thousands of years. This plant medicine is rich in saponins, glycolipids, and organic acids. The purpose of the current study was to develop a rapid, accurate, and sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of the following seven active components of Radix Polygala extracts in rat plasma: sibiricose A5 (A5); sibiricose A6 (A6); 3,6'-disinapoyl sucrose (DSS); tenuifoliside A (TFSA); tenuifoliside B (TFSB); tenuifoliside C (TFSC); and 3,4,5-trimethoxycinnamic acid (TMCA). Then, the pharmacokinetics were studied following oral administration. Plasma samples were precipitated with methanol. Chromatographic separation was successfully performed on a thermo C18 column (100 × 3.0 mm, 3 µm) with a mobile phase consisting of acetonitrile and 10 mmol/L of an ammonium acetate aqueous solution. Seven analytes were detected by multiple reaction monitoring (MRM) with an electrospray ionization source in the positive mode. The transitions of m/z were 517.1/174.9, 547.0/204.9, 753.2/205.2, 681.3/443.3, 667.2/205.1, 767.4/529.2, 236.8/103.2, and 136.9/92.9 for A5, A6, DSS, TFSA, TFSB, TFSC, TMCA, and salicylic acid (IS), respectively. The method validation showed good linearity in the range of 1-2000 ng/mL and LLOQs of 1 ng/mL for the 7 components in plasma. The accuracy, precision, and stability of QC samples were all within allowable ranges. In addition, no significant matrix effect was observed using this method. For the first time, the validated method has been successfully applied to the pharmacokinetic study of the seven components of Radix Polygala extracts in rat plasma. Moreover, this method may be applied for detecting prescriptions that contain Radix Polygala or other plant medicines that include one or more components above. The results of the pharmacokinetic study of the seven ingredients will provide important guidance to clinical medicine regarding Radix Polygala and prescriptions include Radix Polygala.

Determination of the botanical origin of honey by front-face synchronous fluorescence spectroscopy.

Front-face synchronous fluorescence spectroscopy combined with chemometrics is used to classify honey samples according to their botanical origin. Synchronous fluorescence spectra of three monofloral (linden, sunflower, and acacia), polyfloral (meadow mix), and fake (fake acacia and linden) honey types (109 samples) were collected in an excitation range of 240-500 nm for synchronous wavelength intervals of 30-300 nm. Chemometric analysis of the gathered data included principal component analysis and partial least squares discriminant analysis. Mean cross-validated classification errors of 0.2 and 4.8% were found for a model that accounts only for monofloral samples and for a model that includes both the monofloral and polyfloral groups, respectively. The results demonstrate that single synchronous fluorescence spectra of different honeys differ significantly because of their distinct physical and chemical characteristics and provide sufficient data for the clear differentiation among honey groups. The spectra of fake honey samples showed pronounced differences from those of genuine honey, and these samples are easily recognized on the basis of their synchronous fluorescence spectra. The study demonstrated that this method is a valuable and promising technique for honey authentication.

Simultaneous analysis of polygala acid, senegenin and 3,6'-disinapoylsucrose in rat plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study after oral administration.

A rapid and specific LC-MS/MS method has been developed for the simultaneous analysis of polygala acid, senegenin and 3,6'-disinapoylsucrose (DSS) in rat plasma. The method was applied to the pharmacokinetics studies of polygala acid, senegenin and DSS. The analysis was carried out on an Agilent Eclipse plus C18 reversed-phase column (100 × 4.6 mm, 3.5 µm) by gradient elution with methanol and ammonia (0.01%, v/v). The flow rate was 0.4 mL/min. All analytes including internal standard (IS) were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for polygala acid and DSS ranging from 2.5 to 2000 ng/mL, and senegenin ranging from 5 to 2000 ng/mL. The intra- and inter-day precisions (relative standard deviation) were <11.34 and 8.99%. The extraction recovery ranged from 70.89 ± 4.60 to 88.49 ± 3.26%, and that for the IS was 77.23 ± 3.68%. Stability studies showed that polygala acid, senegenin and DSS are stable during the preparation and analytical process. The validated method was successfully used to determine the concentration-time profiles of polygala acid, senegenin and DSS.

Intestinal transport of 3,6'-disinapoylsucrose, a major active component of Polygala tenuifolia, using Caco-2 cell monolayer and in situ rat intestinal perfusion models.

3,6'-Disinapoylsucrose is a major active component of the herb Polygala tenuifolia which has long been used for relieving tranquilization, uneasiness of the mind, and improving learning and memory. Our previous study found that 3,6'-disinapoylsucrose had a very low oral bioavailability. Its mechanisms of absorption in the small intestine have so far been unclear. In the present study, the absorption mechanisms of 3,6'-disinapoylsucrose were investigated by using the Caco-2 cell monolayer and in situ rat intestinal perfusion models. The 3,6'-disinapoylsucrose concentration was determined by an LC/MS/MS method. In a Caco-2 cell transport study, the results showed that 3,6'-disinapoylsucrose had very limited intestinal permeability with average apparent permeability coefficient values around (1.11-1.34) × 10(-7) cm/s from the apical (A) to the basolateral (B) side and (1.37-1.42) × 10(-7) cm/s from B to A, at concentrations of 5, 20, and 33 µM. No concentration dependence in the 3,6'-disinapoylsucrose transport was observed. The apparent permeability coefficient value of 3,6'-disinapoylsucrose (5 µM) from A to B greatly increased to 4.49 × 10(-7) and 1.81 × 10(-7) cm/s, respectively, when the cells were preincubated with EDTA (17 mM) and sodium caprate (5.14 mM). No significant effect on the 3,6'-disinapoylsucrose transport by the inhibitors including verapamil, cyclosporine A, and sodium azide was observed. Similar results were found in the small intestinal perfusion study. The apparent permeability coefficient value of 3,6'-disinapoylsucrose greatly increased from 3.97 × 10(-6) to 23.4 × 10(-6) and 20.0 × 10(-6) cm/s in the presence of EDTA (17 mM) and sodium caprate (5.14 mM), respectively, in perfusion buffer. An in vitro stability evaluation of 3,6'-disinapoylsucrose in the gastrointestinal tract showed that it was relatively stable both in the stomach and small intestine contents, while it was found to be more instable in the colon contents. All of the above results indicate that 3,6'-disinapoylsucrose might be transported across the intestinal mucosa by paracellular passive penetration and paracellular enhancers could increase the intestinal permeability of this compound and thus slightly improve its oral bioavailability.

Sodium ferric gluconate (SFG) in complex with sucrose for IV infusion: bioequivalence of a new generic product with the branded product in healthy volunteers.

OBJECTIVE: Parenteral sodium ferric gluconate in complex (Ferrlecit [branded SFG]) is used to treat patients with iron deficiency anemia undergoing chronic hemodialysis and receiving supplemental epoetin. This comparative pharmacokinetic study (GeneraMedix, Inc., Study 17909) evaluates whether the recently approved generic product Nulecit (generic SFG) and the branded product Ferrlecit (branded SFG) are bioequivalent. METHODS: In this open-label study, 240 healthy volunteers in a fasting state were assigned randomly to a single 10-min intravenous (IV) infusion of 125 mg of generic or branded SFG. Total and transferrin-bound iron concentrations were determined for the 36-h period after infusion and corrected for pretreatment levels. Maximum concentration (Cmax) and area under the concentration-time curve of 0 to 36 h (AUC[0-36]) were compared between the two products. Demonstration of bioequivalence required that the 90% confidence intervals of each parameter evaluated for generic SFG were within 80% to 125% of the corresponding values for branded SFG. RESULTS: Uncorrected and baseline-corrected mean serum concentrations of total serum iron during the 36-h assessment period were similar for generic and branded SFG. For total serum iron, the geometric mean ratios of corrected Cmax and AUC[0-36] were 100%. For transferrin-bound iron, the geometric mean ratios were 87% for corrected Cmax and 92% for corrected AUC[0-36]. All associated 90% confidence intervals were within the range of 80% to 125%. CONCLUSIONS: A new generic SFG in complex for IV infusion is bioequivalent to the branded SFG in complex for IV infusion. The generic SFG is AB rated by the FDA and considered therapeutically equivalent to the branded product.

Absorbable phenylpropenoyl sucroses from Polygala tenuifolia.

Three phenylpropenoyl sucroses--sibiricose A5, A6 and 3',6-disinapoyl sucrose--were isolated from the 30% EtOH extract of Polygala tenuifolia, which displayed antidepressant-like action. HPLC analysis indicated that the three phenylpropenoyl sucroses could be absorbed into serum. From the serum pharmacochemistry point of view, these three phenylpropenoyl sucroses might prevent or relieve depression.

Liposomal approach towards the development of a longer-acting factor VIII.

Prevention of spontaneous bleeding in patients with severe haemophilia A usually requires therapeutic infusions every 2-3 days because of the short half-life of factor VIII (FVIII). Longer-acting FVIII products that require less frequent infusions would be beneficial and might obviate the need for central catheters in most patients. Liposomal formulation can enhance the efficacy of some therapeutic products. The incorporation of high-molecular weight polyethylene glycol (PEG) can extend the circulatory half-life of the liposome. These combined approaches led to the development of BAY 79-4,980, a PEG-containing liposomal version of Kogenate FS (rFVIII-FS). Results from preclinical models and early clinical trials have shown that BAY 79-4,980 prolongs the time to the next bleed. Further clinical evaluation of the efficacy and long-term safety of BAY 79-4,980 are planned.

Inhibition by natural dietary substances of gastrointestinal absorption of starch and sucrose in rats and pigs: 1. Acute studies.

Rapid gastrointestinal absorption of refined carbohydrates (CHO) is linked to perturbed glucose-insulin metabolism that is, in turn, associated with many chronic health disorders. We assessed the ability of various natural substances, commonly referred to as "CHO blockers," to influence starch and sucrose absorption in vivo in ninety-six rats and two pigs. These natural enzyme inhibitors of amylase/sucrase reportedly lessen breakdown of starches and sucrose in the gastrointestinal tract, limiting their absorption. To estimate absorption, groups of nine SD rats were gavaged with water or water plus rice starch and/or sucrose; and circulating glucose was measured at timed intervals thereafter. For each variation in the protocol a total of at least nine different rats were studied with an equal number of internal controls on three different occasions. The pigs rapidly drank CHO and inhibitors in their drinking water. In rats, glucose elevations above baseline over four hours following rice starch challenge as estimated by area-under-curve (AUC) were 40%, 27%, and 85% of their internal control after ingesting bean extract, hibiscus extract, and l-arabinose respectively in addition to the rice starch. The former two were significantly different from control. L-Arabinose virtually eliminated the rising circulating glucose levels after sucrose challenge, whereas hibiscus and bean extracts were associated with lesser decreases than l-arabinose that were still significantly lower than control. The glucose elevations above baseline over four hours in rats receiving sucrose (AUC) were 51%, 43% and 2% of control for bean extract, hibiscus extract, and L-arabinose, respectively. Evidence for dose-response of bean and hibiscus extracts is reported. Giving the natural substances minus CHO challenge caused no significant changes in circulating glucose concentrations, indicating no major effects on overall metabolism. A formula combining these natural products significantly decreased both starch and sucrose absorption, even when the CHO were given simultaneously. These results support the hypothesis that the enzyme inhibitors examined here at reasonable doses can safely lower the glycemic loads starch and sucrose.

Inhibition by natural dietary substances of gastrointestinal absorption of starch and sucrose in rats 2. Subchronic studies.

Acute oral consumption of various natural inhibitors of amylase (bean and hibiscus extracts) and sucrase (L-arabinose) reduce absorption of starch and sucrose respectively in rats and pigs measured by lessened appearance of circulating glucose levels. The present subchronic study was designed to determine whether these selected inhibitors of gastrointestinal starch and sucrose absorption (so-called "carb blockers") remain effective with continued use and to assess their metabolic influences after prolonged intake. Sprague-Dawley rats were gavaged twice daily over nine weeks with either water or an equal volume of water containing a formula that included bean and hibiscus extracts and L-arabinose. To estimate CHO absorption, control and treated Sprague-Dawley rats were gavaged with either water alone or an equal volume of water containing glucose, rice starch, sucrose, or combined rice starch and sucrose. Circulating glucose was measured at timed intervals over four hours. The ability to decrease starch and sucrose absorption use. No toxic effects (hepatic, renal, hematologic) were evident. Blood chemistries revealed significantly lower circulating glucose levels and a trend toward decreased HbA1C in the nondiabetic rats receiving the natural formulation compared to control. Subchronic administration of enzyme inhibitors was also associated with many metabolic changes including lowered systolic blood pressure and altered fluid-electrolyte balance. We postulate that proper intake of natural amylase and sucrase inhibitors may be useful in the prevention and treatment of many chronic disorders associated with perturbations in glucose-insulin homeostasis secondary to the rapid absorption of refined CHO.

Soluble dietary fibre fraction of Trigonella foenum-graecum (fenugreek) seed improves glucose homeostasis in animal models of type 1 and type 2 diabetes by delaying carbohydrate digestion and absorption, and enhancing insulin action.

Trigonella foenum-graecum (fenugreek) seeds have been documented as a traditional plant treatment for diabetes. In the present study, the antidiabetic properties of a soluble dietary fibre (SDF) fraction of T. foenum-graecum were evaluated. Administration of SDF fraction (0 x 5 g/kg body weight) to normal, type 1 or type 2 diabetic rats significantly improved oral glucose tolerance. Total remaining unabsorbed sucrose in the gastrointestinal tract of non-diabetic and type 2 diabetic rats, following oral sucrose loading (2 x 5 g/kg body weight) was significantly increased by T. foenum-graecum (0 x 5 g/kg body weight). The SDF fraction suppressed the elevation of blood glucose after oral sucrose ingestion in both non-diabetic and type 2 diabetic rats. Intestinal disaccharidase activity and glucose absorption were decreased and gastrointestinal motility increased by the SDF fraction. Daily oral administration of SDF to type 2 diabetic rats for 28 d decreased serum glucose, increased liver glycogen content and enhanced total antioxidant status. Serum insulin and insulin secretion were not affected by the SDF fraction. Glucose transport in 3T3-L1 adipocytes and insulin action were increased by T. foenum-graecum. The present findings indicate that the SDF fraction of T. foenum-graecum seeds exerts antidiabetic effects mediated through inhibition of carbohydrate digestion and absorption, and enhancement of peripheral insulin action.

Effect of intravenous iron supplementation on hepatic cytochrome P450 3A4 activity in hemodialysis patients: a prospective, open-label study.

BACKGROUND: Cytochrome P450 (CYP) 3A4 is an enzyme with activity dependent on the reduction of heme iron that is responsible for the metabolism of many drugs. CYP3A4 activity is reduced in hemodialysis (HD) patients and thus may be related to functional iron deficiency. OBJECTIVE: The purpose of this study was to investigate the effect of IV iron supplementation on hepatic is CYP3A4 activity in HD patients. METHODS: This prospective, open-label study was conducted in 12 iron-deficient (transferrin saturation <20% or ferritin <100 ng/L) HD patients on stable medication regimens. To probe for hepatic CYP3A4 activity, an erythromycin breath test (ERMBT) was administered before and after 1 g IV iron sucrose (administered as a 100-mg dose [20 mg/mL]), at each of 10 consecutive HD sessions). CYP3A4 activity was estimated by the percentage of administered (14)C exhaled in a single-breath collection after the test dose of erythromycin underwent demethylation by CYP3A4. The ERMBT was also administered to 7 age-, sex-, and race-matched healthy controls. RESULTS: Twelve HD patients (6 Hispanic, 3 white, 3 Native American; 8 men, 4 women; mean [SEM] age, 56.2 [5.0] years; mean [SEM] weight, 77.0 [5.6] kg; and 7 controls (4 men, 3 women; mean [SEM] age, 51.3 [5.0] years; mean [SEM] weight, 77.5 [7.4] kg) were enrolled in the study. In the total HD population studied, mean (SEM) CYP3A4 activity did not change significantly after IV iron replacement (1.46 [0.27] vs 1.57 [0.24] (14)C exhaled/h). A subgroup of 7 HD patients had significantly lower CYP3A4 activity before IV iron replacement compared with the other 5 HD patients and controls (mean [SEM] 0.86 [0.24] vs 2.30 [0.26] and 2.10 [0.26] (14)C exhaled/h; P < 0.01). After IV iron replacement, mean (SEM) CYP3A4 activity increased in these 7 HD patients (120.1% [67.1%]); P = 0.04) and it was not statistically different from that of controls (1.50 [0.36] vs 2.10 [0.26]). CONCLUSIONS: Overall, IV iron administration had no significant effect on hepatic CYP3A4 activity. However, in a subset of HD patients with low baseline CYP3A4 activity indicated by low ERMBT values, IV iron supplementation was associated with a potentially clinically relevant increase in hepatic CYP3A4 activity. Further studies are needed to clarify mechanisms and clinical implications of this interaction.

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure.

We have analysed the incorporation of [(3)H]sucrose and [(3)H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [(3)H]sucrose or [(3)H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2-3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [(3)H]Sucrose uptake was twice that of [(3)H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [(3)H]sucrose and [(3)H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [(3)H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [(3)H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [(3)H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum.

Changes in sucrose and glycerol content in garlic shoot tips during freezing using PVS3 solution.

Changes in moisture content (MC), sucrose and glycerol concentration in garlic shoot tips were monitored during loading and unloading with PVS3 solution. Upon PVS3 treatment, shoot tip MC decreased rapidly and sucrose and glycerol concentrations increased rapidly during the first 30 min. Sucrose and glycerol concentrations increased more slowly thereafter. Shoot tip MC in after PVS3 treatment was affected by their size, but not by sucrose concentration of the preculture medium. As the size of shoot tips increased, so their MC increased after PVS3 treatment. However, sucrose and glycerol concentrations decreased after PVS3 incubation, and concentrations in dehydrated shoot tips were much lower than those measured in non-air dried controls. During unloading with 1.2 M sucrose medium, shoot tip MC increased rapidly during the first 10 min, whereas glycerol concentration decreased steadily over 90 min. Loading and unloading of PVS3 solution in garlic shoot tips follows the principle of solute bulk flow.

A novel method to calculate the extent and amount of drug transported into CSF after intranasal administration.

The aim of this paper is to establish a novel method to calculate the extent and amount of drug transported to brain after administration. The cerebrospinal fluid (CSF) was chosen as the target region. The intranasal administration of meptazinol hydrochloride (MEP) was chosen as the model administration and intravenous administration was selected as reference. According to formula transform, the extent was measured by the equation of X(A)CSF, infinity/X0 = Cl(CSF) AUC(0-->infinity)CSF/X0 and the drug amount was calculated by multiplying the dose with the extent. The drug clearance in CSF (Cl(CSF)) was calculated by a method, in which a certain volume of MEP solution was injected directly into rat cistern magna and then clearance was assessed as the reciprocal of the zeroth moment of a CSF level-time curve normalized for dose. In order to testify the accurateness of the method, 14C-sucrose was chosen as reference because of its impermeable characteristic across blood-brain barrier (BBB). It was found out that the MEP concentrations in plasma and CSF after intranasal administration did not show significant difference with those after intravenous administration. However, the extent and amount of MEP transported to CSF was significantly lower compared with those to plasma after these two administrations. In conclusion, the method can be applied to measure the extent and amount of drug transported to CSF, which would be useful to evaluate brain-targeting drug delivery.

Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study.

Exopectinase production by Aspergillus niger was compared in submerged fermentation (SmF) and solid-state fermentation (SSF). SSF was carried out using polyurethane foam (PUF) as the solid support. The purpose was to study the effect of sucrose addition (0 or 40 g/l) and water activity level (A(w)=0.99 or 0.96) on the level of enzyme activity induced by 15 g/l of pectin. Mycelial growth, as well as extracellular protease production, was also monitored. Sucrose addition in SmF resulted in catabolite repression of exopectinase activity. However, in SSF, an enhancement of enzyme activity was observed. Protease levels were minimal in SSF experiments with sucrose and maximal in SmF without sucrose. Exopectinase yields (IU/g X) were negligible in SmF with sucrose. The high levels of exopectinase with sucrose and high A(w) in SSF can be explained by a much higher level of biomass production without catabolite repression and with lower protease contamination.

Brain temperature modifies glutamate neurotoxicity in vivo.

The purpose of this study was to examine the effects of mild hypothermia and hyperthermia on glutamate excitotoxicity. Glutamate-induced cortical lesions were produced in hypothermic (32 degrees C), normothermic (37 degrees C), and hyperthermic (40 degrees C) rats by perfusion of a 0.5 M glutamate solution via a microdialysis probe. The volume of the lesion 7 days after glutamate perfusion was quantified histologically by image analysis. This histological assessment was performed in two experiments; in one, each of the target temperatures was induced before glutamate perfusion, and in the other, each of the target temperatures was induced after stopping the glutamate perfusion. We also examined the effect of temperature on the diffusion of exogenously delivered material in the extracellular space using autoradiography of the perfused glutamate solution containing 14C-labeled sucrose. In the two experiments in which each of the target temperatures was induced before or after glutamate perfusion, the volume of damage was reduced by mild hypothermia and enlarged by mild hyperthermia. The volume of 14C diffusion also increased as brain temperature increased. These results provide evidence that small variations of brain temperature modify glutamate excitotoxicity. The results also suggest that the change in glutamate diffusion in the extracellular space is one mechanism by which mild hypothermia and hyperthermia exert their protective and harmful effects respectively.

Hydrocortisone reinforces the blood-brain properties in a serum free cell culture system.

The increasing number of newly developed drugs demands for functional in vitro models of the blood-brain barrier to determine their brain uptake. Cultured cerebral capillary endothelial cells are considered to be such a model, however in serum containing media they exhibit low electrical resistances and high permeabilities compared to the in vivo situation. Here we report the establishment of a serum-free cell culture model. Withdrawal of serum already caused a twofold increase of transendothelial resistance (TER), which in presence of serum is about 100-150 Omega x cm2. We tested several supplements and found that hydrocortisone is a potent stimulator for the formation of barrier properties. TERs up to 1000 Omega x cm2 were measured in the presence of physiological relevant hydrocortisone concentrations. In correspondence to the TER increase hydrocortisone decreased cell monolayer permeability for sucrose down to 5x10(-7) cm/s, which is close to the in vivo value of 1.2x10(-7) cm/s and by a factor of five lower compared to cultures without hydrocortisone and in presence of serum.

Disposition of ingested olestra in weanling mini-pigs.

The disposition of ingested olestra in Hanford mini-pigs was examined by following a single oral gavage dose of radiolabelled (U-14C-sucrose) olestra Eight dosed animal (four/sex) and one undosed animal were killed 1, 3 and 7 days after dosing, and tissues were collected and counted. Urine and faeces were collected continuously and counted. Tissue lipids were extracted and analysed for intact radiolabelled olestra by size exclusion chromatography. Sucrose will be excreted in urine if olestra is absorbed and metabolized. Mean recovery of radiolabel was 96.6% of the administered dose. Of the recovered radiolabel, more than 99.4%, on average, was not absorbed and found in faeces, or cage and animal wash solutions. The absorbed radiolabel (0.6%), was distributed across the carcass, all tissues and blood, or excreted in urine. This radiolabel primarily came from the metabolism of glucose and fructose resulting from the hydrolysis of the trace levels of penta- and lower sucrose esters present in the test material. No radiolabel was found in the olestra-containing fraction of liver lipids, the primary measure of absorbed and non-metabolized olestra, at a detection limit of 0.0002% of dose. A conservative estimate of the amount of 14C-sucrose excreted in the urine was 0.0012%. The total absorption of intact olestra was thus less than 0.0014% of the dose, the sum of the two measures. These results indicate that intact olestra is essentially not absorbed by the weanling mini-pig, an animal with a young developing gastrointestinal tract similar to that of young children (2-5 yr).