On the relation between toxin production and protein synthesis byCorynebacterium diphtheriae and the iron content of the culture medium

Summary An investigation of the quantitative relationship between toxin production and protein synthesis byC. diphtheriae and the iron content of the medium has been reported on. Both functions can be expressed quantitatively,viz., Lf/ml and Lf/mg P.N.Both functions appeared to be narrowly related to the iron concentration of the medium. The optimal Fe-concentration for the greatest purity of the toxin (Lf/mg P.N.) lies, however, lower than that for the maximal yield of toxin (Lf/ml).In connection with the fact that for the subsequent purification of the toxoid to be obtained from the diphtheria toxin, starting from as pure as possible raw material is essential, a somewhat lower production of toxin than an optimal iron concentration might yield, has to be accepted.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography

A sensitive and automated method for the separation and individual determination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and phosphate buffer, pH 7.4 A 1.0-ml volume of plasma was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) were added to the extract before injection into the LC system. The enantiomeric separation of tramadol was achieved using a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phase was a mixture of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaClO₄ concentration were optimized with respect to enantiomeric resolution. The method developed was validated. Recoveries for both enantiomers of tramadol were about 100%. The method was found to be linear in the 2.5–150 ng/ml concentration range [r²=0.999 for (+)- and (−)-tramadol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (−)-tramadol, respectively.     

Simple procedure for purification of diphtheria toxin and separation of its fragments by hydrophobic chromatography

Diphtheria toxin and fragment B bind to hydrocarbon-coated agaroses. Fragment A of the toxin is not adsorbed to such resins. Using Seph-C₄, the toxin and fragment B can be eluted from the column after adsorption by increasing the ionic strength of the eluent. The toxin is also eluted from the Seph-C₆ column, but fragment B is eluted only in the denatured form. Purification of the toxin can be achieved simply by passing the growth medium supernatant through a small size Seph-C₆ column and eluting the toxin by 0.1 m NaCl. The fragments of diphtheria toxin obtained after mild trypsin treatment can be separated purely on a Seph-C₄ column. The hydrophobic chromatography system may thus serve as a tool for purification of the toxin and its fragments: it may also be useful in large-scale preparations.     

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution–acetonitrile (80:20). The fluorescence of the column effluent was monitored at λ_ex=338 and λ_em=425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r²=0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.     

Determination of the angiotensin-converting enzyme inhibitor lisinopril in urine using solid-phase extraction and reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method is described for assaying lisinopril in human urine. Urine (1 ml) containing lisinopril and enalaprilat (internal standard) was acidified with 10 μl of 6 M nitric acid, passed through a Sep-Pak C₁₈ cartridge and eluted with 3 ml of 10% acetonitrile, followed by 6 ml of distilled water. The separations were carried out using a μBondapak C₁₈ column with a mobile phase comprising acetonitrile (60 ml), methanol (10 ml) and tetrahydrofuran (10 ml) in 15 mM phosphate buffer (920 ml) at pH 2.90. Separations were performed at 40°C and detection was at 206 nm. Standard calibration plots of lisinopril in urine were linear (r> 0.998) and recovery was greater than 64%. The lowest quantifiable concentration was 0.5 μg/ml. Within-day and between-day imprecision (coefficient of variation) ranged from 2.51% to 9.26%, and inaccuracy was less than 8.3%.     

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and toxoids

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG₁ monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse antitoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and toxoids. The detection sensitivity of diphtheria toxin/toxoid equaled 0.0005 Lf/ml; tetanus toxin and toxoid were detected with sensitivities of 20 LD₅₀/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum toxoids (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E toxoid (0.001 UI/ml).     

Optimization of diphtheria toxin production by <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> using a casein-based medium in a fermenter

The Td-based combined vaccine contains only small amounts of the diphtheria toxoid antigen. However, a high level of purity is necessary for this antigen. The diphtheria toxin is produced by growing Corynebacterium diphtheriae in a semisynthetic, casein-based medium in a fermenter. In order to obtain a highly pure diphtheria toxoid, the optimal conditions to express the toxin at 300 Lf/mL in a fermenter culture were determined. When C. diphtheriae was cultivated in a fermenter and a high concentration of toxin was obtained, specific patterns for the pH and dissolved oxygen levels identified. Overall, the fermenter cultivation process was divided into four stages according to variations in the pH. A specific range of K _La in the fermenter (0.0092 ~ 0.0093/sec) was required to produce high level expression of diphtheria toxin. The amount of toxin expression varied significantly according to culture conditions. Agitation and aeration in the fermenter affected toxin expression, even when the optimal K _La value for toxin production was maintained. A previous study has reported that the amounts of agitation and aeration are important factors when cultivating fungus in the fermenter to produce chitinolytic enzyme. A mass production of diphtheria toxoid with a purity level greater than 2,500 Lf/ mgPN was obtained through purification and detoxification from this optimized toxin production.     

Reversed-phase high-performance liquid chromatography of nicotinic acid mononucleotide for measurement of quinolinate phosphoribosyltransferase

A system has been developed for the determination of quinolinate phosphoribosyltransferase (QPRT) activity in liver and kidney homogenates using HPLC. A product, nicotinic acid mononucleotide (NaMN), is separated by reversed-phase chromatography (a Tosoh ODS 80TS was used as an analytical column) using a mixture of 10 mM KH₂PO₄–K₂HPO₄ buffer (pH 7.0) containing 1.48 g/l tetra-n-butylammonium bromide–acetonitrile (9:1, v/v) as a mobile phase. The flow-rate was 1.0 ml/min, the detection wavelength was 265 nm. The column temperature was maintained at 40°C. Under these conditions, NaMN was eluted at about 8.1 min. Sample preparation was very straightforward. The reaction mixture of QPRT assay was stopped by immersing the tube into a boiling water bath, the resulting supernatant was filtered, and the filtrate was directly injected into a HPLC system. The total HPLC analysis time was approximately 20 min.     

Development of a routine analysis method for liposome encapsulated recombinant interleukin-2

This paper describes the development of an isocratic reversed-phase high-performance liquid chromatographic method for the routine analysis of recombinant interleukin-2 (rIL-2) in liposome samples. The chromatographic system employed a C₄ column maintained at 30°C eluted with 52.5% (w/w) acetonitrile in water, containing 100 mM NaClO₄ and 10 mM HClO₄. To remove phospholipid interference the chromatographic method was combined with a lipid-extraction procedure. No significant loss of rIL-2 was noted upon inclusion of this extraction step. The protein eluted from the column with a capacity factor (k′) of 5.8. The method was validated for robustness, linearity, precision and reproducibility. It was shown that the method was linear over a sample concentration range of 1–100 μg/ml. Upon assessment of the intra-day and inter-day precision, the relative standard deviations (RSD) were within the range of the methodical error (approximately 5%), except at the lower concentration of 10 μg/ml, where the intra-day RSD was relatively high (17.8%). The recovery of rIL-2 upon liposome preparation and subsequent analysis of the samples was in the range 94±9%. The results indicate that the method is suitable for routine quantitation of rIL-2 in liposomal samples.     

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 μm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5±2.8% with a linear range of 0.1–100 μg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA–2-thiobarbituric acid (TBA) complex. The separation of MDA–TBA complex was performed using a 250×4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 μl (composed of 100 μl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 μl of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95°C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA–TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.     

Simultaneous determination of estriol and estriol 3-sulfate in serum by column-switching semi-micro high-performance liquid chromatography with ultraviolet and electrochemical detection

A column-switching HPLC with semi-microcolumn enabled us a direct and simultaneous analysis of estriol (E3) and estriol 3-sulfate (E3 S) in human serum in combination with ultraviolet (for E3 S) and electrochemical (for E3) detectors. The mobile phases (phosphate buffer pH 7.0) contained 5 mM tetra-n-butylammonium ion (TBA) as a counter ion for E3 S. Serum samples were diluted with 200 mM phosphate buffer (pH 7.0) containing 100 mM TBA, then injected to the pre-column. After serum proteins had flowed out from the pre-column, E3 and E3 S were transferred to the enrichment column. Subsequently the analytes were eluted to the analytical column. Detection limits of E3 and E3 S in human serum were 2.5 ng/ml and 295 ng/ml. Serum E3 and E3 S levels (mean±SD) of umbilical artery from 18 full-term healthy neonates were 33±23 ng/ml and 1.26±0.69 μg/ml, respectively.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

The coagglutination reaction for detecting diphtheria toxin

High-titer antidiphtheria antitoxic rabbit serum has been obtained, and on the basis of this serum a coagglutinating diagnosticum has been developed. The sensitivity of the test has been found to depend on the content of antitoxic antibodies in the serum and on its purity. Diagnostica prepared from native serum containing 500 I. U./ml (a titer of 1:51, 200 in the passive hemagglutination test) permit the detection of 0.02-0.03 Lf/ml of diphtheria toxin. A decrease in antibody titer to 5-25 I. U./ml leads to a drop in sensitivity to 0.2-2 Lf/ml. The use of LgG fraction and pure antibodies increases the sensitivity of the test to 0.002-0.003 Lf/ml. The possibility of detecting toxin in Corynebacterium diphtheriae strains is shown.     

Determination of cocaine and its metabolites in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 μl). The separation used a 2.1-mm I.D. reversed-phase Brownlee C₁₈ column with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.2, containing 1.29·10⁻⁴M tetrabutylammonium phosphate (12.5:10:77.5, v/v/v). The detection limit was 2.5 ng/ml for all the compounds using an ultraviolet detector operated at 235 nm. The method was used to study the pharmacokinetics of cocaine after an intravenous (i.v.) bolus dose (4 mg/kg).     

Purification and hydrophobic properties of the δ-endotoxin in the parasporal crystal produced by Bacillus thuringiensis var. entomocidus

Parasporal crystals of Bacillus thuringiensis var. entomocidus were separated from spores and other cell debris by the water-chloroform biphase procedure. The solubilization and fractionation were carried out under mild conditions at 4°C. Crystals were solubilized in 0.01 M dithiothreitol and 0.2 M glycine NaOH buffer at pH 10.0. The solution was treated overnight with 0.01 M Tris-HCl buffer, pH 5.5, containing 0.1% Triton N-101 and 0.1% sodium cholate, and then placed on a Sepharose 6B column, equilibrated, and later developed with the same buffer. Under these conditions, four fractions were obtained, one of which had a molecular weight ranging from 60,000 to 70,000, and demonstrated a high insecticidal activity on second instar larvae of Spodoptera litioralis. The LC₅₀ value of this fraction was a half of that of the solubilized crystals. The other three fractions had a lower activity. The active fraction was further fractionated on an octyl-Sepharose 4B resin. Elution of this column with the same buffer separated the proteins into two fractions. The first eluted fraction was highly active, while the second demonstrated a very low activity. The active fraction was further purified by loading on a short column of octyl-Sepharose 4B and eluted with a linear gradient of the same detergents. Under these conditions, the highly active fraction gave a sharp and symmetrical peak that revealed five close bands at the pH range of 6.1–6.5 on isoelectric focusing gel electrophoresis.     

Simplified procedure for the analysis of 3- and 4-hydroxyproline

A column chromatographic analysis of 3-hydroxyproline (3-Hyp), 4-hydroxyproline (4-Hyp), and γ-carboxyglutamic acid (Gla) is described. The analyses of urine and plasma were performed with a JLC-6AH amino acid analyzer. A 0.15 M sodium citrate buffer, pH 2.1, was used for elution. Urinary Gla, 3-Hyp, and 4-Hyp were among the seventeen peaks eluted before asparti acid. Hyp, Gla, glutamine, and asparagine in plasma were separated by elution with 0.2 M sodium citrate buffer, pH 3.25, containing 10% methanol. This single-column procedure achieves the sequential separation and quantitation of Gla, 3-Hyp, and 4-Hyp in urine as well as plasma, and is applicable to the diagnosis of collager, metabolism disorders.     

Determination of fumagillin in muscle tissue of rainbow trout using automated ion-pairing liquid chromatography

A high-performance liquid chromatographic assay is described as a routine analytical method for the determination of fumagillin in rainbow trout muscle tissue. Muscle tissue samples (1 g) containing fumagillin were deproteinized with 8 ml of an acetonitrile-water mixture (2:6, v/v). The extracts were purified with a Bond Elut Octyl C₈ cartridge column, washed with a water-methanol mixture (95:5, v/v; 4 ml) and fumagillin was eluted with acetonitrile (1 ml). Analytical separations were performed by reversed-phase HPLC with UV detection at 351 nm under gradient conditions. The mobile phase was acetonitrile-0.005 M tetrabutyl ammonium phosphate in water (pH 7.8). The assay is specific and reproducible within the fumagillin range of 20–1000 ng/g and recovery at 20 ng/g was 69.2%. Sample preparation involves the use of a robotic sample preparation system. Gravimetric validation of all operations enabled Good Laboratory Practices to be observed.     

Introduction of Additional Charges as an Aid in Protein Purification: Isolation of Elongation Factor 2 from Sulfolobus acidocaldarius by Preparative Isoelectric Focusing Before and After ADP-Ribosylation

Diphtheria toxin catalyzes the ADP-ribosylation of elongation factor 2 (EF-2) in eukaryotes and archaebacteria. As the reaction is strictly EF-2 specific and introduces two negative charges into the molecule, the resulting shift in the isoelectric point (pI) by 0.2 pH units was used to establish a new purification method for EF-2 from Sulfolobus acidocaldarius. The cells were lysed with dithiothreitol at pH 9 and EF-2 was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-200, and three isoelectric focusing steps. The EF-2-containing fractions from the first isoelectric focusing step at pH 4-9 were refocused in a more narrow pH-gradient (pH 5-7). The EF-2 peak from the second step was eluted, collecting only the fractions above the pH region where ADP-ribosylated EF-2 would focus. The EF-2 was then ADP-ribosylated with diphtheria toxin and NAD and subjected to further isoelectric focusing (pH 5-7). The EF-2 was almost homogeneous since ADP-ribosylation had shifted it into a region of the pH gradient free of contaminating proteins. Diphtheria toxin was immobilized on CNBr-activated Sepharose to prevent a possible contamination by proteins from the diphtheria toxin preparation which might have the same pI as ADP-ribosylated EF-2. Finally, the ADP-ribosyl group was removed by equilibrium dialysis using diphtheria toxin and nicotinamide at pH 6.3. The obtained EF-2 was active in protein synthesis.