Automatic determination of hydroperoxides of phosphatidylcholine and phosphatidylethanolamine in human plasma

An automatic method for the determination of hydroperoxides of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is reported. Sample plasma was deproteinized with a fourfold volume of methanol. After centrifugation, the supernatant was injected directly into an HPLC system without further treatment. The hydroperoxides of PC and PE were concentrated and washed on an ODS column followed by introduction into two analytical columns, a silica gel and an aminopropylsilica gel column, which were connected in series, by column switching. After the separation, they were detected by postcolumn detection with diphenyl-1-pyrenylphosphine. The compounds were determined at picomole levels within 30 min with good reproducibilities. By using only a silica gel column as an analytical column, PC hydroperoxides were determined within 20 min, and samples could be injected into it at 15-min intervals. Those methods made it possible to inject a sample of up to 2 ml at one time and up to 8 ml by repeated injections and to determine phospholipid hydroperoxides in human plasma at picomole levels.     

The preparative isolation of lecithin

Lecithin can be prepared on a relatively large scale, free of colored impurities, by a simple two-column procedure. Commercial crude egg lecithin is partially purified by a single-step passage through an alumina column. It is then purified by a two-step passage through a prepacked, commercial silica gel column. The lecithin is prepared in solvent-free form for weighing by lyophilization from cyclohexane. Toxic solvents (chloroform and methanol) are avoided by the use of ethanol, isopropanol, hexane, and water. The elutions are easily monitored by a flow cell in an ordinary spectrophotometer set at 215 nm. Study of the column parameters has made it possible to use heavy loads with a relatively small column and minimal solvent.     

After-potential of spinal axons in vivo

A method is evolved whereby the after-potential sequence intrinsic to longitudinal dorsal column myelinated fibers may be studied in isolation from those events occurring in intact spinal cord subjected to an afferent volley. The recovery sequence intrinsic to dorsal column fibers, after refractoriness is over, is characterized by supernormality approximately four to five times greater than that seen in peripheral nerve. This supemormality averages 15.7 ± 4 per cent (current calibration) at peak and decays exponentially with a half-time of 7.5 msecs. It is not followed by subnormality unless conditioning is repeated more than three times at frequencies greater than 100/sec. Characterization of the recovery curve of dorsal column fibers permits by exclusion, the allocation of the origin of DRV to structures more centrally located. DCV (the dorsal column counterpart of DRV) is seen to exist equally developed in active and passive dorsal column fibers.     

Control of Gaseous Environment of Organisms using a Diffusion Column Technique

A technique based on the theory of steady-state diffusion ofgases in a long column provides a simple means of controllingthe gas concentration in biological experiments. The diffusioncolumn consists of a sand-filled tube with side-arms along itslength. Selected gas concentrations are applied at both endsof the column and maintain a predictable concentration gradientalong the column. The gas composition at each side-arm is thusknown and can be adjusted by alteration of the controlling concentrationsat the ends of the column. The response to gas concentrationof small bodies such as seeds and micro-organisms can be testedby placing them in small cuvettes attached to the side-arms.The technique is well suited for experiments needing a closelygraded series of gaseous concentrations. The design and useof an electrode to monitor oxygen concentration is also described.     

A double-vented tetraphasic continuous column approach to MuDPIT analysis on long capillary columns demonstrates superior proteomic coverage

A double-vented serial tetraphasic capillary column approach is applied to proteomic MuDPIT-type analysis using extended length capillary reverse-phase columns. The heart of the tetraphasic device consists of a triphasic MuDPIT trap located upstream of a venting tee. The trap is followed by a 60 cm high-resolution capillary column. A conventional high-flow HPLC is used to develop gradients at standard flow rates and pressures. The double-vented triphasic MuDPIT trapping device relieves the capillary separation column from the salt burden during the on-line cation-exchange portion of the analysis. Two configurations are presented, a double-vented continuous column model and a discontinuous model in which the triphasic MuDPIT trap is installed on a six-port valve; both configurations were tested with 60 and 10 cm capillary columns. All four systems were challenged with a trypsin digest of undepleted human serum, and a matrix of proteomic results for the different models and column lengths are compared.     

Simplified in-line sample preparation for amino acid analysis in carbohydrate containing samples

This report describes a new, automated chromatographic procedure eliminating carbohydrates from amino acid samples prior to their analysis by anion-exchange chromatography and integrated amperometric detection. In the first step, a sample is brought onto a short cation-exchange column (trap column) in hydrogen form. Carbohydrates are passing through this column, while only amino acids are retained. Subsequently, the cation-exchange column, holding the amino acid fraction, is switched in-line with the gradient pump and separator column. The mobile phase used at the beginning of the separation (NaOH; pH 12.7) transfers amino acids from the trap column onto the anion-exchange column and the amino acid separation is completed without any interference by carbohydrates. All common amino acids are recovered following the carbohydrate removal step. The average value of their recovery is 88.1%. The calibration plots were tested between 12.5 and 500 pmol (amounts injected). The mean value of correlation coefficients of calibration plots was calculated as 0.99. The mean value of relative standard deviations from five replicates was 3.9%. The usefulness of the method is illustrated with two chromatograms of a carrot juice sample obtained before and after the in-line removal of carbohydrates.     

Tissue dynamics of steady state growth in Hydra littoralis. II. Patterns of tissue movement

In the column of hydra, tissues continually move away from a region located just underthe whorl of tentacles. Above this subtentacular region, tissues proceed into the hypostome and tentacles; below it tissues pass into the buds or continue down the stalk. These movements represent a steady state pattern of tissue renewal in which column growth is balanced by tissue loss at the body extremities. But the existence of a subtentacular zone in which tissue appears stationary does not necessarily indicate that growth is restrictedto this region, as is commonly stated. The body column of hydra can be viewed as an expanding cylinder whose elongation is balanced by tissue loss at the two ends. In such a body there must be one region from whichtissue appears to emanate, regardless of how growth is distributed along the cylinder. Only the rates at which tissues move will be characteristic of the underlying growthpattern. In Hydra littoralis, the measured rates of tissue movement down the gastric column are consistent with the distributions of mitotic figures, which indicate that growth is spread out along the column.     

New system for preparative electrochromatography of proteins

Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate proteins and other molecules based on differences in electrophoretic mobility. Because the separation is electrically driven, the need for additional chemical reagents is reduced. Two major impediments to scale-up of electrochromatography columns, removal of heat and electrolysis gases, have historically limited the diameter of packed columns to 2.5 cm ID with volumes of approximately 55 mL. We report a novel electrochromatography column that effectively removes electrolysis gases and minimizes heating. A vital component of this system is a new electrode design that couples a platinum gauze with an ultrafiltration membrane across both ends of the column. Use of a methacrylate base stationary phase enabled axial voltage gradients of 10 to 20 V/cm. Thermocouples inserted radially in the column at four axial positions showed that the flow of a 4 degrees C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 degrees C. The new column design, with dimensions of 3.81 cm ID x 38.1 cm long and bed volume of 400 mL, was demonstrated by separating mixtures of BSA and myoglobin. The column was operated in a horizontal position with radial sample injection and withdrawal at the ends of the packed bed. These experiments are a first step in demonstrating that scale-up of electrochromatography columns can be achieved by choosing appropriate flow rates, voltage gradients, and stationary phase.     

Efficacy of a pentaiodide resin disinfectant on Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) oocysts in vitro

The resin-I5 column developed at Kansas State University was tested for efficacy against oocysts of Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae). Cesium chloride gradient-purified oocysts were passed through 1.0-cm-diameter columns with lengths of 2.5, 5.0, and 10.0 cm at 23 C. Following column passage, oocyst viability was determined both in vitro by excystation and in vivo by the ability to establish infections in suckling mice. Oocysts were found to be retained by the pentaiodide resin in a linear fashion, probably by electrostatic interactions. Linear regression analysis revealed 100% of the oocysts should be removed in such a manner using a column length of greater than or equal to 25.7 cm. When compared to untreated control oocysts, less than 12% of the oocysts that passed through the columns appeared to be affected by the resin, as assessed by excystation. Inoculation of suckling mice with these column-treated oocysts supported the excystation data and revealed the coccidian to be viable. These results indicate that oocysts of C. parvum are retained on the pentaiodide column in a 1-hit manner and that, although killing of parasites may occur within the column, the greatest effect that the column may have on the parasite is as an electrostatic retention device.     

Effect of resin disinfectants-I3 and -I5 on Giardia muris and Giardia lamblia.

The resin-I5 column developed in our laboratories rendered aqueous suspensions containing up to 5 X 10(4) cysts of Giardia muris or Giardia lamblia per ml incapable of excystation. The inhibition of excystation was effective at both 4 and 25 degrees C. The addition of Na2S2O3 to column eluates containing cysts appeared to partially reverse the disinfectant action, and the reversal was more pronounced at 4 degrees C than at 25 degrees C. In contrast, the rapid removal of cysts from the column eluates by centrifugation and filtration or the use of other reductants, notably cysteine and glutathione, did not similarly reverse the disinfectant properties of the column. Based on these data, we suggest that the disinfecting agent is acquired by the cyst in its passage through the resin column and that either the disinfecting agent or its reaction can be partially and specifically neutralized by Na2S2O3. We hypothesize that the time between disinfectant acquisition and activity is a function of the thickness of the Giardia cyst wall and consequently takes longer at the lower temperature. Nevertheless, resin-I5 appears to inactivate a larger number of cysts in a shorter period of time with lower residual halogen levels than do agents of other published methods.     

Identification and Characterization of Activating and Conjugating Enzymes of the Ubiquitin System from the Unicellular Alga Chiamydomonas reinhardtii

Abstract: Using a biochemical approach we identified families of ubiquitin-activating and ubiquitin-conjugating enzymes in Chiamydomonas reinhardtii . The family of ubiquitin-activating enzymes, characterized by their ability to form thioesters with ubiquitin and eluting off a ubiquitin affinity column by ATP-depletion probably consists of at least four members. Whereas one of these enzymes is active under a broad range of pH values, thioesterof the other UBAs with ubiquitin is restricted to pH 7.5. Two ubiquitin-activating enzymes are metabolically phosphory-lated which is assumed to be an activity control mechanism. Most of the 7 ubiquitin-conjugating enzymes detected in this study were found to bind rather tightly to an anion exchange column, and eluted off the column at specific salt concentra tions. Two of the ubiquitin-conjugating enzymes described here did, however, not bind to this column. These enzymes can, as all other C. reinhardtii ubiquitin-conjugating enzymes, perform thioester formation with ubiquitin regardless of the source (plant/animal) of the ubiquitin-activating enzyme.     

Dynamic model of a nitrifying fixed bed column: Simulation of the biomass distribution of Nitrosomonas and Nitrobacter and of transient behaviour of the column

A dynamic model for a fixed bed nitrifying column with recirculation of the liquid and gas phases was developed. Liquid RTD experiments demonstrated that the liquid phase was perfectly mixed inside the column. Hete- rogeneity of biomass distribution on the solid phase (beads) was represented by an N-tanks in series model, and a back-mixing term was set to account for the well-mixed liquid phase throughout the column. In autotrophic conditions, competition for oxygen is the cause of the spatial segregation of the two species. Nitrosomonas is concentrated on beads at the bottom of the bed whereas Nitrobacter is more widely distributed. This is consistent with biomass distribution results reported by Cox et al. [17] in a nitrifying fixed bed column. Nitrification takes place at the bottom of the column, always in oxygen gas-liquid mass transfer limiting conditions. Nevertheless, considering the whole process, nitrification is complete (>98% of NH₃ oxidised) and there is no oxygen limitation (the outlet dissolved oxygen concentration is not limiting). The dynamic behaviour of the column, in conditions set up to avoid biofilm diffusion limitation, was simulated for different NH₃-load variations and oxygen shutdowns. The simulated behaviour of the column can be compared to results reported by Bazin et al. [16]. This confirms that the output transient nitrite peaks are higher when changes in the process conditions produce a rearrangement of biomass distribution in the fixed bed.     

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.     

Optimization of the determination of 4-aminopyridine in human serum and urine by column liquid chromatography

Two convenient reversed-phase column liquid chromatographic procedures are described for the determination of 4-aminopyridine in human serum and urine. A 0.5-ml aliquot of serum after the addition of a 0.5-ml solution of 4-(aminomethyl)pyridine in 0.1M Na₂HPO₄ as the internal standard is passed through a 1-ml BondElut C₁₈ silica extraction column. The column is selectively washed to remove acidic, neutral and weakly basic compounds. The desired compounds are eluted with a 0.3-ml aliquot of 35% perchloric acid-methanol (1:100, v/v). A 10-μl aliquot of the eluate is infected onto a 150 × 4.6 mm I.D. column packed with 5-μm C₁₈ silica particles that is eluted at ambient temperature with a mobile phase containing octanesulfonic acid as the ion-pairing agent. The peaks are monitored at 263 nm. A 0.25-ml aliquot of urine or 0.5 ml of serum is mixed with N-propionylprocainamide as the internal standard and subjected to benzoylation by Schotten Baumann reaction. The reaction mixture is adjusted to pH 5.5–6 and extracted with a BondElut C₁₈ extraction column. An aliquot of the eluate is chromatographed at ambient temperature with a mobile phase containing tetramethylammonium perchlorate. The peaks are monitored at 278 nm.     

Enantiomeric analysis of amino acids by using comprehensive two-dimensional gas chromatography

The chiral separation of amino acids (AA) derivatised with ethyl chloroformate by using comprehensive two-dimensional gas chromatography is reported. A commercially available enantioselective capillary column (Chirasil-l-Val) has been tested as first-dimension column. Two nonenantioselective stationary phases (BPX50 and BP1) with different column lengths were combined with the enantioselective column, which represent chiral/polar and chiral/low-polarity column sets, respectively. These column sets were evaluated to determine the most useful column combination to provide improved separation efficiency of enantioselective AA analysis. Separations of AA mixtures derivatised either as their N-trifluoroacetyl methyl esters or with methyl chloroformate, performed on a chiral/low-polarity column set, are also shown. The method was demonstrated for chiral analysis of AAs in different beer samples. The major AA in the beer samples was proline with amounts ranging from around 65-95% with minor contents of glycine and the l-enantiomers of alanine, valine, leucine, and isoleucine. Small amounts of d-alanine, at about 1, 1.5, and 15% were detected in the three samples.     

A highly sensitive high-performance liquid chromatography method for the estimation of ascorbic and dehydroascorbic acid in tissues, biological fluids, and foods

A highly sensitive procedure for determining ascorbic acid (AA) and dehydroascorbic acid (DHAA) by high-performance liquid chromatography with electrochemical detection in biological fluids, tissues, and foods is described. AA is separated in a C18 reverse-phase column after extraction from the sample with metaphosphoric acid. An aliquot of 20 microliter of diluted extract is injected into the column for the estimation of AA. DHAA is indirectly estimated by converting it to AA after reduction with DL-homocysteine at pH 7.0-7.2 for 30 min at 25 degrees C. After dilution, a 20-microliter aliquot is injected into the column to obtain total vitamin C (AA + DHAA). The concentration of DHAA is calculated by subtraction. AA can be reproducibly quantified at concentrations as low as 50 pg/20 microliter of sample extract. The method described here used a specially designed mobile phase, gave greater stability and a noiseless baseline, and increased substantially the sensitivity and precision. The procedure is rapid, analysis being completed within 10 min after sample preparation, and has been successfully applied to biological fluids, tissues, and foods.     

Direct determination of tamoxifen and its four major metabolites in plasma using coupled column high-performance liquid chromatography

A rapid, rugged and fully automated method has been developed for the determination of tamoxifen and its major metabolites in plasma. The system is based upon an in-line extraction process combined with column switching to a coupled analytical column. The plasma sample is deproteinated by the addition of acetonitrile before injection onto a semi-permeable surface (SPS) cyano guard column (1.0 × 0.46 cm I.D.). After washing the guard column briefly with water, the sample is eluted with a mobile phase composed of 35% acetonitrile in 20 mM potassium phosphate buffer (pH 3). The eluent is directed through a cyano analytical column (25 × 0.46 cm I.D.) and a photochemical reactor where the analytes are converted to highly fluorescent phenanthrene derivatives. Tamoxifen, 4-hydroxytamoxifen, N-desdimethyltamoxifen, N-desmethyltamoxifen and tamoxifen-ol are eluted in that order at a flow-rate of 1.0 ml/min. The method has been validated for use in a clinical study utilizing tamoxifen in the treatment of recurrent cerebral astrocytomas.     

Direct enantioseparation of mandelic acid by high-performance liquid chromatography using a phenyl column precoated with a small amount of cyclodextrin additive in a mobile phase

Direct enantioseparation of mandelic acid by high-performance liquid chromatography (HPLC) with a reversed phase column and a mobile phase containing a small amount of hydroxylpropyl-β-cyclodextrin (HP-β-CD) was studied as an efficient method for saving consumption of the CD additive. As a result, it was proposed that racemic mandelic acid can be analyzed with a phenyl column by using a mobile phase composed of 10 mM ammonium acetate buffer (pH 4.2) and 0.02% (w/v) HP-β-CD at a flow rate of 1.0 mL/min at 40°C after the passage of 10 mM ammonium acetate buffer (pH 4.2) containing 0.1% (w/v) HP-β-CD as a precoating mobile phase for 60 min. It is suggested that HP-β-CD is bound with a phenyl group on the surface of the stationary phase to allow a phenyl column to act as a transient chiral column, and injected mandelic acid can form the ternary complex with the adsorbed HP-β-CD. The longer retention time of D-mandelic acid than the L-isomer for HPLC can be explained from the higher stability of the HP-β-CD complex with D-mandelic acid, which was confirmed by CE experiment with HP-β-CD as a selector. The efficiency of a phenyl column compared with other stationary phases was also discussed.     

Enhanced sequence coverage in tryptic fragment analysis by two-dimensional HPLC/MS using a monolithic silica capillary column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Enhanced Sequence Coverage in Tryptic Fragment Analysis by Two-Dimensional HPLC/MS Using a Monolithic Silica Capillary Column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.