(1/930) Gas-liquid chromatography of the heptafluorobutyrate derivatives of the O-methyl-glycosides on capillary columns: a method for the quantitative determination of the monosaccharide composition of glycoproteins and glycolipids.
We have developed a method involving the formation of hepta-fluorobutyrate derivatives of O-methyl-glycosides liberated from glycoproteins and glycolipids following methanolysis. The stable derivatives of the most common monosaccharides of these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc, GlcNAc, GalNAc, Neu5Ac, KDN) can be separated and quantitatively and reproducibly determined with a high degree of sensitivity level (down to 25 pmol) in the presence of lysine as an internal standard. The GlcNAc residue bound to Asn in N-glycans is quantitatively recovered as two peaks. The latter were easily distinguished from the other GlcNAc residues of N-glycans, thus allowing a considerable improvement of the data on structure of N-glycans obtained from a single carbohydrate analysis. The most common contaminants present in buffers commonly used for the isolation of soluble or membrane-bound glycoproteins (SDS, Triton X-100, DOC, TRIS, glycine, and polyacrylamide or salts, as well as monosaccharide constituents of proteoglycans or degradation products of nucleic acids) do not interfere with these determinations. A carbohydrate analysis of glycoproteins isolated from a SDS/PAGE gel or from PDVF membranes can be performed on microgram amounts without significant interferences. Since fatty acid methyl esters and sphingosine derivatives are separated from the monosaccharide peaks, the complete composition of gangliosides can be achieved in a single step starting from less than 1 microg of the initial compound purified by preparative Silicagel TLC. Using electron impact ionization mass spectrometry, reporter ions for the different classes of O-methyl-glycosides (pentoses, deoxy-hexoses, hexoses, hexosamines, uronic acids, sialic acid, and KDN) allow the identification of these compounds in very complex mixtures. The mass of each compound can be determined in the chemical ionization mode and detection of positive or negative ions. This method presents a considerable improvement compared to those using TMS derivatives. Indeed the heptafluorobutyrate derivatives are stable, and acylation of amino groups is complete. Moreover, there is no interference with contaminants and the separation between fatty acid methyl-esters and O-methyl glycosides is achieved. (+info)
(2/930) Perfluorooctanoic acid, a peroxisome-proliferating hypolipidemic agent, dissociates apolipoprotein B48 from lipoprotein particles and decreases secretion of very low density lipoproteins by cultured rat hepatocytes.
The hypolipidemic effect is evoked by various peroxisome proliferators. Modulation of gene transcription via peroxisome proliferator-activated receptor (PPAR) is generally responsible for this effect. In addition, we have found a PPAR-independent mechanism in which fibrates, known peroxisome proliferators, decrease hepatic secretion of very low density lipoproteins (VLDL) through inhibition of phosphatidylcholine synthesis via methylation of phosphatidylethanolamine (PE) (T. Nishimaki-Mogami et al., Biochim. Biophys. Acta 1304 (1996) 21-31). In the present study, we show a novel mechanism by which perfluorooctanoic acid (PFOA), a potent peroxisome proliferator and inhibitor of PE methylation, exerts its hypolipidemic effect. PFOA (100 microM) added to the medium rapidly decreased the secretion of triglyceride by cultured rat hepatocytes, which was independent of the activity of cellular PE methylation. Analysis of the density of apoB secreted into the medium showed that PFOA decreased apoB48 in VLDL, but increased apoB48 in the bottom d>1.21 fraction. This lipid-poor apoB48 was also generated by incubating medium that had been harvested from control cells with PFOA, indicating that PFOA has the ability to dissociate apoB48 from lipoprotein particles. Exposure of cells to PFOA for 2 h prior to the experiment was sufficient to generate lipid-poor apoB48, indicating that PFOA exerted its effect intracellularly. Taken together, the data suggest that a strong interaction of PFOA with apoB48 disturbs the association of apoB48 with lipids in the process of intracellular VLDL assembly, thereby inhibiting VLDL secretion. This study shows that the mechanisms of hypolipidemic effect caused by various classes of peroxisome proliferators are diverse. (+info)
(3/930) Transport of colloidal particles in lymphatics and vasculature after subcutaneous injection.
This study was designed to determine the transport of subcutaneously injected viral-size colloid particles into the lymph and the vascular system in the hind leg of the dog. Transport of two colloid particles, with average size approximately 1 and 0.41 microm, respectively, and with and without leg rotation, was tested. Leg rotation serves to enhance the lymph flow rates. The right femoral vein, lymph vessel, and left femoral artery were cannulated while the animal was under anesthesia, and samples were collected at regular intervals after subcutaneous injection of the particles at the right knee level. The number of particles in the samples were counted under fluorescence microscopy by using a hemocytometer. With and without leg rotation, both particle sets were rapidly taken up into the venous blood and into the lymph fluid. The number of particles carried away from the injection site within the first 5 min was <5% of the injected pool. Particles were also seen in arterial blood samples; this suggests reflow and a prolonged residence time in the blood. These results show that particles the size of viruses are rapidly taken up into the lymphatics and blood vessels after subcutaneous deposition. (+info)
(4/930) Dodecafluoropentane ultrasonic contrast enhancement in carotid diagnosis: preliminary results.
To assess the efficacy in carotid diagnosis of an investigational dodecafluoropentane ultrasonic contrast enhancing agent, we compared B-mode, color flow, and duplex Doppler findings in 16 patients with common carotid artery bifurcation disease after dodecafluoropentane and saline injections. Dodecafluoropentane produced enhanced backscatter in all patients for 4 to 20 min (mean, 8.4+/-4.74 min) after intravenous injection. In six patients this enhancement improved the color flow and pulsed Doppler signal detection in areas of sonographic shadowing. The enhanced color flow information changed the diagnostic impression in one case. Dodecafluoropentane produced enhanced backscatter in the carotid artery in all patients, and for a mean duration longer than that reported for other agents. It has the potential to improve the efficacy of carotid ultrasonic evaluation. (+info)
(5/930) Doppler sonographic enhancement of hepatic hemangiomas and hepatocellular carcinomas after perflenapent emulsion: preliminary study.
Ultrasonographic microbubble contrast agents improve Doppler signals by increasing blood backscatter. We retrospectively reviewed our experience with perflenapent (EchoGen), an emulsion of liquid dodecafluoropentane, in the evaluation of 13 patients with focal hepatic lesions (10 hemangiomas and six hepatocellular carcinomas). Perflenapent improved the detection of color Doppler flow signals within the lesions. The hemangiomas showed peripheral nonpulsatile signals and the hepatocellular carcinomas showed more diffuse enhancement with both arterial and venous type signals. This preliminary study suggests that perflenapent administration may aid in the sonographic differentiation of these focal lesions. (+info)
(6/930) Molecular ion fragmentation and its effects on mass isotopomer abundances of fatty acid methyl esters ionized by electron impact.
We have analyzed the isotopomer abundance ratios of an equimolar mixture of nine fatty acid methyl esters (decanoate, undecanoate, laurate, tridecanoate, myristate, pentadecanoate, palmitate, heptadecanoate, and stearate) by selected-ion monitoring gas chromatography/electron impact/mass spectrometry (GC/EI/MS). The abundance of the second lowest m/z isotopomer (IM1) increased disproportionately compared with the abundance of the lowest m/z isotopomer (IM0) as a function of: (1) increasing sample size; (2) decreasing repeller voltage; and (3) decreasing alkyl chain length. We also compared the abundance of the third lowest m/z isotopomer (IM2) and the abundance of the second lowest m/z isotopomer (IM1) of methyl palmitate and [4,4-2H2]methyl palmitate. We observed that the IM2/IM1 for methyl palmitate was significantly lower than IM2/IM1 for [4,4-2H2]methyl palmitate. From these results, as well as a consideration of basic principles of ion chemistry and ion physics, we conclude that gas-phase chemistry, specifically proton (or deuteron) transfer from fragment ions to molecules, is a major contributor to the sample size dependence observed in mass isotopomer abundance measurements of fatty acid methyl esters ionized by EI. Our results and analysis do not support hydrogen abstraction as the reaction mechanism. In addition, we calculate that rearranged molecular ions are unlikely to contribute significantly to intermolecular proton transfer because of their relatively brief lifetime. We also discuss alternative analytical techniques which might improve the precision and accuracy of isotopomer measurements by reducing molecular ion fragmentation. (+info)
(7/930) Perfluorocarbon emulsion improves oxygenation of the cat primary visual cortex.
Tissue PO2 was measured in the primary visual cortex of anesthetized, artificially ventilated, normovolemic cats to evaluate the effect of small doses [1 g perfluorocarbon (PFC)/kg] of a PFC emulsion (1 g PFC/1.1 ml emulsion; Alliance Pharmaceutical, San Diego, CA) on brain oxygenation. The change in tissue PO2 (DeltaPO2), resulting from briefly changing the respiratory gas from room air to 100% oxygen, was measured before and after intravenous infusion of the emulsion. Before emulsion, DeltaPO2 was 51.1 +/- 45.6 Torr (n = 8 cats). Increases in DeltaPO2 of 34.0 +/- 26.1 (SD) % (n = 8) and 16. 3 +/- 8.4% (n = 6) were observed after the first and second emulsion infusions, respectively. The further increase in DeltaPO2 after the third dose (7.9 +/- 10.5%; n = 7) was not statistically significant. The observed increases in tissue oxygenation as a result of the PFC infusions appear to be the result of enhanced oxygen transport to the tissue. (+info)
(8/930) Comparison of exogenous surfactant therapy, mechanical ventilation with high end-expiratory pressure and partial liquid ventilation in a model of acute lung injury.
We have compared three treatment strategies, that aim to prevent repetitive alveolar collapse, for their effect on gas exchange, lung mechanics, lung injury, protein transfer into the alveoli and surfactant system, in a model of acute lung injury. In adult rats, the lungs were ventilated mechanically with 100% oxygen and a PEEP of 6 cm H2O, and acute lung injury was induced by repeated lung lavage to obtain a PaO2 value < 13 kPa. Animals were then allocated randomly (n = 12 in each group) to receive exogenous surfactant therapy, ventilation with high PEEP (18 cm H2O), partial liquid ventilation or ventilation with low PEEP (8 cm H2O) (ventilated controls). Blood-gas values were measured hourly. At the end of the 4-h study, in six animals per group, pressure-volume curves were constructed and bronchoalveolar lavage (BAL) was performed, whereas in the remaining animals lung injury was assessed. In the ventilated control group, arterial oxygenation did not improve and protein concentration of BAL and conversion of active to non-active surfactant components increased significantly. In the three treatment groups, PaO2 increased rapidly to > 50 kPa and remained stable over the next 4 h. The protein concentration of BAL fluid increased significantly only in the partial liquid ventilation group. Conversion of active to non-active surfactant components increased significantly in the partial liquid ventilation group and in the group ventilated with high PEEP. In the surfactant group and partial liquid ventilation groups, less lung injury was found compared with the ventilated control group and the group ventilated with high PEEP. We conclude that although all three strategies improved PaO2 to > 50 kPa, the impact on protein transfer into the alveoli, surfactant system and lung injury differed markedly. (+info)