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Differential passage of [14C]sucrose and [3H]inulin across rat blood-brain barrier after cerebral ischemia.

The biophysical nature of blood-brain barrier (BBB) opening after ischemic or hemorrhagic stroke or traumatic brain injury is unresolved. Ultrastructural (electron micrograph) investigations of experimental BBB injury commonly indicate the abnormal presence of vesicles or tubular structures in cerebrovascular endothelial cells, suggesting the likelihood of convective, fluid-phase transport of blood substances into brain. We measured transfer constants (K(i)s) for the simultaneous passage of two intravenously delivered tracers ([14C]sucrose, mol wt=342; [3H]inulin approximately 5,000) across the intact BBB in the rat, and 24 h after global cerebral ischemia (16-20 min duration) or 24, 48 or 72 h after focal ischemia (2 h duration). In both ischemia models, the upward increment in K(i) (DeltaK(i)) for sucrose, indicating the extra injury-related tracer flux into brain, significantly exceeded that for inulin, as might be expected with faster diffusion of the smaller molecule through injury pores or channels. This inequality of DeltaK(i)s did not suggest a major role for convective, fluid-phase transport by endothelial vesicular or tubular structures and a predominance of diffusional transport was indicated.

Animals↗

Pitfalls in measuring inulin and para-amino-hippuric acid clearances.

Various carbohydrates and a variety of widely used medicines interfere with the generally used laboratory methods for determining inulin and para-aminohippuric acid (PAH). When these pitfalls are not recognized, false measurements of inulin and PAH clearances, which represent glomerular filtration rate and renal plasma flow respectively, are obtained. When performing these tests a careful history of dietary habits and oral drug therapy must be taken.

Ammonium Chloride↗

A new recycling technique for human placental cotyledon perfusion: application to studies of the fetomaternal transfer of glucose, inulin, and antipyrine.

A previously described technique has been modified to permit the continuously recirculating perfusion of the separate maternal and fetal circulations of an isolated cotyledon of human placenta. Viability of the perfused cotyledons was established by measurements of oxygen consumption (average, 0.18 ml/gm/hr), glucose utilization (average, 1.0 mg/gm/hr), and lactate production (less than 0.01 mumol/gm/hr), and integrity of the placental barrier by the failure of India ink, 125I-albumin, or 35S-sulfobromophthalein to cross from fetal to maternal circulation. Clearance of 3H-inulin from the fetal circuit, 0.0059 +/- 0.0005 (SE) ml/min/gm, corresponded to 2.5% of its clearance by the adult human kidney. Clearance of 14C-antipyrine was 0.013 +/- 0.003 ml/min/gm. After introduction into the fetal circuit, the observed appearance of both inulin and antipyrine in the maternal circuit closely paralleled curves predicted by a simple mathematical model. The use of a continuously recirculating perfusion system is technically feasible, and has advantages over the single-pass technique for studying transplacental transfer of metabolites with a low efficiency of extraction.

Animals↗

125I labelled inulin: a convenient marker for deposition of liposomal contents in vivo.

The utility of an iodinated derivative of inulin (125I-tyraminyl-inulin, 125ITI) for reporting in vivo tissue distributions of liposomal contents is described. It is shown, employing a rat model, that this probe satisfies the criteria that the free form is rapidly cleared from the circulation and excreted, whereas 125ITI encapsulated in large unilamellar vesicle (LUV) systems and subsequently taken up in various tissues exhibits a long (greater than 3 days) retention time. Further, high specific activities (greater than 1 muCi per microliter) are easily achievable, allowing low LUV dose levels (less than or equal to 2.5 mumole phospholipid/kg body weight) to be employed. Minimal tissue workups for quantitation of 125ITI distributions are required. It is concluded that from criteria of sensitivity, expense and simplicity, 125ITI is a most convenient probe for characterizing liposome deposition in vivo.

Animals↗

Pyrolysis of inulin, glucose, and fructose.

The pyrolytic behavior of inulin, a (2-->1)-linked fructofuranan, is described. Parallel investigations of the pyrolysis of glucose and of fructose were conducted to supplement the inulin results and to aid comparison with previous results from glucans. Effects of neutral and basic additives are emphasized. As with glucans, the addition of such additives (especially basic) increases the yields of the one-, two-, and three-carbon products (as well as of hexosaccharinolactones), while generally decreasing the yields of anhydro sugar and furan derivatives. The former products include glycolaldehyde, acetol, dihydroxy-acetone, acetic acid, formic acid, and lactic acid. Mechanistic speculations are made regarding the origins of these compounds, as well as of furan derivatives and saccharinic acid lactones. Parallels with alkaline degradation are considered.

Carbohydrate Sequence↗

Di-D-fructose dianhydrides from the pyrolysis of inulin.

Inulin was pyrolyzed in air to produce di-D-fructose dianhydrides (DFDAs) in approximately 26% yield, three of which were identified by MS, NMR, and comparison with literature data. The mass spectra of the per-O-trimethylsilyl derivatives of the DFDAs are discussed. A mechanism is proposed for the formation of DFDAs from inulin during pyrolysis.

Disaccharides↗

Carboxymethylation of inulin.

Inulin was carboxymethylated in aqueous alkaline medium with monochloroacetic acid as the reagent. The degree of substitution of the reaction product was determined by titration, LC analysis and 13C NMR spectroscopy. Carboxymethylinulin with a degree of substitution between 0.2 and 1 was obtained depending on the molar ratio of inulin-monochloroacetic acid. Increasing the concentration of the reaction mixture and lowering the reaction temperature resulted in higher selectivities towards carboxymethylinulin. Determination of the molecular weight distribution was performed by GPC and by multi-angle laser light scattering. Carboxymethylation caused little or no degradation of the chain length of the starting material.

Acetates↗

Stabilization of emulsions using polymeric surfactants based on inulin.

The use of polymeric surfactants for stabilization of emulsions is described. A brief account of general classification and description of polymeric surfactants is given. This is followed by a description of the adsorption and conformation of polymeric surfactants at interfaces. The theoretical approaches for studying polymer adsorption are briefly described. This is followed by a section on the experimental techniques that can be applied to study adsorption and conformation of polymers at the interface. Examples are given to illustrate the experimental techniques. A section is devoted to the interaction between droplets containing adsorbed polymer layers (steric stabilization). The last section gives results on oil-in-water (O/W) emulsions stabilised with a novel graft copolymeric surfactant based on inulin that has been modified by introducing alkyl groups. Two oils were used, namely Isopar M (isoparaffinic oil) and cyclomethicone. Emulsions prepared using the inulin-based surfactant have large droplets, but this could be significantly reduced by addition of a cosurfactant in the oil phase, namely Span 20. The stability of the emulsions was investigated in water, in 0.5, 1.0, 1.5 and 2 mol dm(-3) NaCl and in 0.5, 1.0, 1.5 and 2 mol dm(-3) MgSO(4). These emulsions were stable for more than 1 year up to 50 degrees C in NaCl concentrations up to 2 mol dm(-3) and 1 mol dm(-3) MgSO(4). This high stability in high electrolyte concentrations could be attributed to the nature of the hydrophilic (stabilizing) polyfructose chain. This was confirmed using cloud point measurements, which showed high hydration of the polyfructose chain in such high electrolyte concentrations. This ensured the long-term physical stability resulting from the strong steric repulsion between the polyfructose chains.

Adsorption↗

Spray freeze drying to produce a stable Delta(9)-tetrahydrocannabinol containing inulin-based solid dispersion powder suitable for inhalation.

The purpose of this study is to investigate whether spray freeze drying produces an inhalable solid dispersion powder in which Delta(9)-tetrahydrocannabinol (THC) is stabilised. Solutions of THC and inulin in a mixture of tertiary butanol (TBA) and water were spray freeze dried. Drug loads varied from 4 to 30 wt.%. Various powder characteristics of the materials were determined. Stability of THC was determined and compared with freeze dried material. The powders, dispersed with an inhaler based on air classifier technology, were subjected to laser diffraction analysis and cascade impactor analysis. Highly porous particles having large specific surface areas (about 90 m(2)/g) were produced. At high drug loads, THC was more effectively stabilised by spray freeze drying than by freeze drying. Higher cooling rates during spray freeze drying result in improved incorporation. Fine particle fractions of up to 50% were generated indicating suitability for inhalation. It was concluded that spray freeze drying from a water-TBA mixture is a suitable process to include lipophilic drugs (THC) in inulin glass matrices. High cooling rates during the freezing process result in effective stabilisation of THC. The powders can be dispersed into aerosols with a particle size appropriate for inhalation.

Administration, Inhalation↗

Structure of the enzymatically synthesized fructan inulin.

Construction, purification and characterization of a fusion protein of maltose-binding protein of Escherichia coli and the fructosyltransferase of Streptococcus mutans is described. With the purified protein, in vitro synthesis of inulin was performed. The obtained polysaccharide was characterized by high-performance size-exclusion chromatography (HPSEC) and static light scattering (SLS) in dilute aqueous and dimethyl sulfoxide solution. For all samples very high molecular weights between 60 x 10(6) and 90 x 10(6) g/mol and a remarkable small polydispersity index of 1.1 have been determined. Small root-mean-square radii of gyration point to a compact conformation in dilute solution. No difference between native and enzymatically synthesized inulin was observed by X-ray powder diffraction and thermoanalysis of solid samples.

ATP-Binding Cassette Transporters↗

Differences in chain length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1-FFT cDNAs.

A newly isolated cDNA clone, Cy3, encoding the fructan fructan 1-fructosyltransferase (1-FFT) from artichoke was expressed using tobacco protoplasts as expression system. Analysis of the inulin molecules synthesized upon incubation of protoplast extracts with a mixture of oligofructans (DP3-5) shows the production of inulins with a degree of polymerization (DP) of up to 23, whereas parallel experiments performed using a 1-FFT cDNA from Jerusalem artichoke led to the production of fructans with a DP of up to only 12. The results of in vitro fructan synthesis catalyzed by transiently expressed enzymes therefore reflect the difference of in vivo fructan composition of Jerusalem artichoke (M(DP) = 8-10) and artichoke (M(DP) = 65). These data suggest that the fructan pattern in a given species is mainly defined by the enzymatic characteristics of 1-FFT.

Amino Acid Sequence↗

Demonstration of a transcellular vesicle pathway for biliary excretion of inulin in rat liver.

We tested the hypothesis that the blood to bile transport of hydrophilic inert nonelectrolytes such as inulin is mediated in part by a transcellular pathway involving endosomelike vesicles (transcytosis). Forty minutes after intravenous injection of [3H]methoxyinulin into renal pedicle ligated rats, 0.8% of the radioactivity was recovered in liver homogenate and 85% +/- 3.6% of this radioactivity was associated with membrane bound vesicles. Subcellular fractionation studies and electron microscopy confirmed this association. If rats were treated with taurochenodeoxycholic acid, 5 mumol/100 g body wt, the hepatocellular uptake of [3H]methoxyinulin increased approximately twofold and [3H]methoxyinulin was again recovered in small subcellular vesicles. Furthermore, taurochenodeoxycholic acid also stimulated the biliary excretion of [3H]methoxyinulin, which peaked in bile at 20 min. Taurochenodeoxycholic acid had similar effects on the biliary excretion of horseradish peroxidase, a protein known to be transported from blood to bile by membrane vesicles. Thus under the conditions of these experiments, the dihydroxy bile acid taurochenodeoxycholic acid can stimulate the rate of vesicle-dependent transcellular transport into bile. If inulin clearance represents a maximal estimate of this process, only 6%-8% of total bile production in the rat under basal conditions would be mediated by vesicle-mediated transcytosis.

Animals↗

Determination of inulin in meat products by high-performance liquid chromatography with refractive index detection.

Inulin is a naturally occurring carbohydrate with beneficial nutritional and technological properties. A high-performance liquid chromatographic (HPLC) method was developed for the quantitative determination of these beta-fructans in meat products, containing this type of additive. The method includes extraction of inulin with hot water, followed by hydrolysis with inulinase enzyme, and determination of the released fructose by HPLC with refractive index detection. An internal standard of rhamnose was used to quantify fructose. The method incorporates a sample blank (without inulinase hydrolysis) for each specimen to subtract contributions of free fructose and fructose from sucrose. The results showed good precision with average RSDs of 2.4% for repeatability and 5.2% for reproducibility. Analytical recovery ranged from 102 to 106%. Satisfactory linearity (r=0.999) was obtained.

Chromatography, High Pressure Liquid↗

Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [14C]inulin in rat.

Parasynaptic communication, also termed volume transmission, has been suggested as an important means to mediate information transfer within the central nervous system. The purpose of the present study was to visualize by autoradiography the available channels for fluid movement within the extracellular space following injection of the inert extracellular marker [14C]inulin into the lateral ventricle or cisterna magna. Bolus injections of 5 microl of 1 microCi of [14C]inulin were made in awake rats via chronically implanted cannulae. After survival times ranging from 5 min to 4 h, brains were processed for in vivo autoradiography. At 5 min the tracer distributed throughout the ventricles, subarachnoid spaces and cisterns "downstream" of the injection sites. Penetration into the brain from these sites was complex with preferential entry along the ventral side of the brain, especially into the hypothalamus and brainstem. By 4 h virtually the entire brain was labeled irrespective of the site of tracer application. Sustained tracer entry from subarachnoid spaces suggests that some areas act as depots to trap circulating material. This mechanism may contribute to the pattern of deep penetration at later time-points. The spatial and temporal characteristics of fluid movement throughout the brain are instructive in the interpretation of many experimental procedures involving injection of molecules into the cerebrospinal fluid.

Animals↗

Rapid and simple determination of inulin in biological fluids by high-performance liquid chromatography with light-scattering detection.

We report a new high-performance liquid chromatography method developed for measuring inulin in plasma and urine using ion moderated partition chromatography and evaporative light-scattering detection. Samples are deproteinized with a zinc acetate and phosphotungstic acid solution and added with melezitose as an internal standard. The chromatographic separation is carried out in 16 min at a flow-rate of 0.6 ml/min using deionized water as the mobile phase. Within-run precision, measured at four different concentrations (0.050 mg/ml, 0.150 mg/ml, 0.300 mg/ml and 1.200 mg/ml), ranges from 1.7 to 3.4% in plasma and from 1.5 to 3.5% in urine. Similarly, between-run precision is in plasma from 2.0 to 4.3% and in urine from 2.0 to 4.4%. Analytical recovery ranges from 97.9 to 100.1% in plasma and from 99.1 to 99.7% in urine, respectively. Detection limit (signal-to-noise ratio=3) is 5 microg/ml both in plasma and urine. The method is simple, sensitive, without interference due to hexoses or drugs commonly taken by patients with renal diseases, and offers the advantage of measuring inulin without previous hydrolysis of the molecule.

Adolescent↗

Synthesis and characterisation of inulin-azo hydrogels designed for colon targeting.

The present paper describes the synthesis and characterisation of new hydrogel systems designed for colon targeting. The gels were composed of methacrylated inulin (MA-IN), copolymerized with the aromatic azo agent bis(methacryloylamino)azobenzene (BMAAB) and 2-hydroxyethyl methacrylate (HEMA) or methacrylic acid (MA). The gels were assessed by studying the influence of various parameters on the dynamic and equilibrium degree of swelling. It was shown that the uptake of water in the gels was inversely proportional to the MA-IN feed concentration, the degree of substitution of the inulin backbone, and the concentration of BMAAB. The latter can probably be explained by the hydrophobic nature and rigidity of the aromatic azo agent. Incorporation of the hydrophilic monomers HEMA or MA also reduced the equilibrium degree of swelling. An increasing network density and hydrogen bonding propensity, can suggested to be responsible for this observation. It was shown that water uptake in the hydrogels was controlled by both relaxation and diffusion mechanisms (anomalous behaviour). When the release of the model drug prednisolone was studied in phosphate buffer, it was shown that >80% of the drug was released during the first 3 h from hydrogels of MA-IN:HEMA. Although drug release decreased significantly from MA-IN:HEMA:BMAAB hydrogels, it remained too high: approximately 50% of the drug was released after 5 h. The same observation was made for hydrogels containing MA instead of HEMA. These results clearly point out the difficulty in finding the optimal balance between swelling to allow degradation in the colon (high swelling of the gels) and low premature drug release before the colonic environment is reached (low swelling properties).

Anti-Inflammatory Agents↗

Use of gamma-inulin/liposomes/Vitamin E adjuvant combination in contraceptive vaccines.

The adjuvanticity of two gamma inulin/liposomes/Vitamin E combinations was evaluated in the mouse, in contraceptive vaccines with sperm protein extracts or a synthetic HE2 peptide ("Human Epididymis gene product"; residues 15-28) as antigen. The HE2 peptide was not conjugated to a protein carrier, to ensure that the antibodies elicited were specific against the HE2 peptide. The adjuvant combinations were designed to increase adjuvanticity, as their components have complementary mechanisms, and their performance was compared to Freund's adjuvant. Antibody production against native sperm structures was determined in sera by ELISA immunoassay and immunohistology. Toxicity of adjuvants was determined by histopathological study and treated mice were monitored for signs of pain or distress. Our results show that the gamma inulin (1-2 microm particle size)/liposomes/Vitamin E combination, with sperm protein extracts, is better than Freund's adjuvant because it elicits good antibody titres without any toxicity. When the synthetic HE2 peptide is used as antigen, the gamma inulin (1-2 microm particle size)/liposomes/Vitamin E combination is less effective than Freund's adjuvant; nevertheless, the anti-HE2 antibodies elicited are highly specific and recognize native structures in sperm.

Adjuvants, Immunologic↗

[Alcoholic fermentation of inulin by various strains of yeasts].

Strains of fourteen species of yeasts able to ferment inulin without previous chemical or physical hydrolysis were studied on semi-synthetic medium by evaluation of CO2 production under anaerobic conditions. Among them, Kluyveromyces cicerisporus, Candida macedoniensis and Candida utilis showed the best kinetic characteristics of fermentation. Experiments were carried out to specify the action of different parameters such as temperature, pH and exogenous ethanol concentration. The results obtained on semi-synthetic medium were confirmed on Jerusalem artichoke juice. The optimal temperature for fermentation was 35 degrees C for the three strains, but K. cicerisporus alone conserved its fermentative capacity at 40 degrees C, at low pH (3.5) and with 6% (v/v) exogenous ethanol concentration. This strain appears to be a good yeast for industrial production of ethanol from inulin substrate.

Culture Media↗