Microwaves and the cell membrane. III. Protein shedding is oxygen and temperature dependent: evidence for cation bridge involvement

Microwaves (2450 MHz, 60 mW/g) are shown to result in the release or shedding of at least 11 low-molecular-weight proteins (less than or equal to 31,000 Da) from rabbit erythrocytes maintained in physiological buffer. Protein release was detected by gel electrophoresis of cell-free supernatants using sensitive silver staining. This release is oxygen dependent and occurs in 30 min for exposures conducted within the special temperature region of 17-21 degrees C, which is linked to a structural or conformational transition in the cell membrane. Shedding of 26,000 and 24,000 Da proteins is unique to microwave treatment, with enhanced release of 28,000 and less than or equal to 15,000 Da species during microwave compared to sham exposures. Two-dimensional isoelectric focusing further reveals that proteins of less than or equal to 14,000 Da shed during microwave treatment exhibit a pI of 6.8-7.3 not seen in sham-treated cells. Treatment of erythrocytes with a serine-directed protease inhibitor does not prevent release of proteins. However, when erythrocytes are maintained at 17-21 degrees C by conventional heating in the absence of divalent cations, release of 28,000-31,000 and less than or equal to 14,000 Da components is detected. This indicates that cation-bridge stability may be important for release of these proteins. The above results provide evidence that microwaves alter erythrocyte protein composition at temperatures linked to a transition in the cell membrane and that destabilization of salt bridges may play a role in an interaction mechanism for protein release.     

Chlorophyll analysis by high-performance liquid chromatography

The separation and determination of chlorophylls by high-performance liquid chromatography (HPLC) is described. Chlorophylls and their derivatives were separated by reversed-phase HPLC based on hydrophobic interaction between solute and support, using an octadecyl silica column and elution with 100% methanol. Separated pigments were detected fluorometrically with a sensitivity in the picomole range: the fluorescence response was linear over a wide pigment concentration range. Resolution of five chlorophylls a and four protochlorophyll species esterified with different alcohols was achieved within 22 min in a single experiment. This method can be used for the determination of chlorophyll b, bacteriochlorophyll a esters and products synthesized from chlorophyll, but not for nonesterified pigments, i.e., chlorophyllide, protochlorophyllide and chlorophyll c. The chromatographic mobility of chlorophyll a esterified with different alcohols increases with increasing number of carbon atoms in the esterifying alcohols. The plots obtained from the logarithm of the capacity factor (k′) of these pigments versus the numbers of carbon atoms of the alcohol molecule gave a straight line, thus permitting the estimation of the chain length of unknown pigment esterifying alcohols. This HPLC separation technique did not cause the formation of artifacts. The deviation of the individual retention time for each pigment is less than ±0.5%, thus making this method suitable for the rapid identification and quantification of unknown pigments.     

Stereoselective analysis of ketorolac in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) analytical method is described for the quantification of the (R)- and (S)-enantiomers of ketorolac when present together in human plasma. The method involves derivatization with thionyl chloride/(S)-1-phenylethylamine and subsequent reversed-phase chromatography of the diastereomeric (S)-1-phenylethylamides of (R)- and (S)-ketorolac. The method is suitable for the analysis of large numbers of plasma samples and has been applied in this report to a pharmacokinetic study of ketorolac enantiomers upon intramuscular administration of racemic drug to a human subject. The limit of quantification for each enantiomer of ketorolac is 50 ng/ml (signal-to-noise ratio > 10). © 1993 Wiley-Liss, Inc.     

The analysis of aflatoxins by high-performance liquid chromatography.

The potential of high-performance liquid chromatography as a technique for separating aflatoxins B₁ B₂, G₁, G₂, B_2a, Q₁, M₁, P₁, aflatoxicol, and a degradation product of aflatoxin B₁, 2,3-dihydrodiol, has been assessed. A microparticulate silica adsorption column used with a 1:1 chloroform -dichloromethane eluant provided good resolution of aflatoxins B₁, B₂, G₁, and G₂ but the addition of 1% propan-2-ol was necessary for the elution of aflatoxins M₁ and Q₁. By selecting appropriate solvent mixtures, good resolution of all of the aflatoxins studied was obtained using columns containing an octadecyl (C₁₈) reversed-phase bonded to a microparticulate support. Details are given for resolving: (1) aflatoxins B₁, B₂, G₁, and G₂ using a 5% tetrahydrofuran-15% dimethylformamide in water eluant and (2) aflatoxins B₁ B_2a, Q₁ M₁ P₁ aflatoxicol, and a product of aflatoxin B₁ 2,3-dihydrodiol treated with Tris-buffer, using either 15% dimethylformamide in water or 10% tetrahydrofuran in water as eluant.     

Simultaneous quantitative determination of cilostazol and its metabolites in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cilostazol, a quinolinone derivative, and its known metabolites OPC-13015, OPC-13213, OPC-13217, OPC-13366, OPC-13269, OPC-13326 and OPC-13388 in human plasma was developed and validated. Cilostazol, its metabolites and two internal standards, OPC-3930 and OPC-13112, were extracted from human plasma by a combination of liquid–liquid and liquid–solid phase extractions, with combined organic solvents of n-butanol, methanol, chloroform, methyl-tert.-butyl ether, and a Sep-Pak silica column. The combined extract was then evaporated and the residue was reconstituted in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto a HPLC system and was subjected to reversed-phase HPLC on a 5 μm ODS-80TM column to obtain quality chromatograph and good peak resolution. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the two internal standards were well separated at baseline from each other with resolution factor being 74 and 138. This HPLC method was demonstrated to be specific for all analytes of interest with no significant interference from the endogenous substances of human plasma. The lower limit of quantitation was 20 ng/ml for cilostazol and all metabolites. The method was validated initially for an extended linear range of 20–600 ng/ml for all metabolites and cilostazol, and has been revised later for a linear range of 20–1200 ng/ml for cilostazol and two major and active metabolites OPC-13015 and OPC-13213. The overall accuracy (relative recovery) of this method was established to be 98.5% to 104.9% for analytes with overall precision (CV) being 1.5% to 9.0%. The long-term stability of clinical plasma samples was established for at least one year at −20°C. Two internal standards of OPC-3930 and OPC-13112 were evaluated and validated. However, the data indicated that there was no significant difference for all accuracy and precision obtained by using either OPC-3930 or OPC-13112. OPC-3930 was chosen as the internal standard for the analysis of plasma samples from clinical studies due to its shorter retention time. During the validation standard curves had correlation coefficients greater than or equal to 0.998 for cilostazol and the seven metabolites. These data clearly demonstrate the reliability and reproducibility of the method.     

Chlorpheniramine : I. Rapid quantitative analysis of chlorpheniramine in plasma,saliva and urine by high-performance liquid chromatography

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.     

Trace analysis of SN-38 in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method with fluorescence detection was developed and validated for the quantitation of SN-38, the active metabolite of irinotecan (CPT-11), a new anticancer drug. This method uses solid-phase extraction with a C₁₈ column for sample clean-up and concentration following acidification of human plasma with two volumes of 0.1 M HCl. Using blank plasma spiked with SN-38, we found the assay to be linear over the concentration range of 10–500 pM (3.9–195 pg/ml) with acceptable total and within-day imprecision. The recovery of SN-38 ranged from 48.3% (10 pM) to 91.5% (500 pM) whereas that of the internal standard, 20-(S)-camptothecin, was 96.9% (500 pM). This method represents a sizeable increase in sensitivity over other published methods and is shown to be suitable for the measurement of ‘trough' concentrations of SN-38 during the treatment of patients with a weekly regimen of irinotecan.     

Direct separation and quantitative analysis of thyroxine and triiodothyronine enantiomers in pharmaceuticals by high-performance liquid chromatography

A rapid reversed-phase type HPLC method for the simultaneous separation and analysis of D- and L-thyroxine (D- and L-T(4)) and triiodothyronine (T(3)) was developed using a quinine-derived chiral stationary phase and applied for a quantitative assay of the enantiomeric impurity of the drugs in pharmaceutical formulations of levothyroxine. The influence of operating parameters has been studied for the optimization of the separation and also in order to gain an insight into the retention mechanism. Validation of the method included linearity, precision and accuracy which revealed R.S.D. values of <3.3% for intra-assay precision and percent error ranging from -6 to +2.1% for various defined validation samples, proving satisfactory accuracy. Quantitation was performed over the range of 0.5-500 microg ml(-1) with limits of detection and quantitation lower than 0.1 and 0.5 microg ml(-1), respectively, for both analytes. Further, the determination of 0.1% impurity, of D-T(4) as well as L- and D-T(3) in levothyroxine sodium tablets proved to be feasible.     

Separation and quantitative analysis of alkyl sulfobetaine-type detergents by high-performance liquid chromatography and light-scattering detection

An improved high-performance liquid chromatographic (HPLC) method for the separation of zwitterionic detergents is described. It is based on a reversed-phase liquid chromatography with evaporative light-scattering detection (ELSD). The method was shown to be highly specific, allowing the separation of three detergents of the alkyl sulfobetaine family: 3-(N-dodecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB12), 3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB14) and 3-(N-hexadecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB16). It was further used to develop a quantitation method for SB14, which was validated for linearity, precision, robustness, limits of detection and quantitation, specificity and accuracy. Linearity was found in the range of 50-500 microg/ml with a correlation coefficient of 0.9938+/-0.0029. The mean value of slope and intercept were 1.567+/-0.06 and 0.1541+/-0.0271, respectively. The limits of detection (LOD) and quantitation (LOQ) were 2 and 10 microg/ml, respectively. The validated method was used to determine the concentration of SB14 in different biological samples, specially in bulks of a recombinant membrane protein, the Klebsiella pneumoniae outer membrane protein A, which is produced at the pilot scale for human clinical studies.     

Microwaves and the cell membrane. II. Temperature, plasma, and oxygen mediate microwave-induced membrane permeability in the erythrocyte

Microwaves (2450 MHz) are shown to increase 22Na permeability of rabbit erythrocytes for exposures only within the narrow temperature range of 17.7 to 19.5 degrees C (Tc) which coincides with a nonlinearity in the Arrhenius plot reflecting an apparent membrane phase transition. Significantly, this response is not observed for cholesterol-loaded erythrocyte membranes which exhibit a linear Arrhenius plot and no apparent phase transition at Tc. The permeability increase at Tc is a nonlinear function of absorbed power but is a linear function of the internal electric field strength of the sample and saturates at approximately 400 mW/g and 600 V/m, respectively. The permeability increase was found to be reversible and transient in that immediately following termination of exposure sodium influx is significantly reduced but returns to normal within 60 min. Extracellular factors exert a significant influence on the microwave effect. The presence of plasma markedly potentiates the increase in 22Na permeability at Tc. Oxygen also modulates the microwave effect with relative hypoxia (5 mm Hg) and hyperoxia (760 mm Hg) enhancing the permeability increase. In contrast, the presence of two antioxidants, ascorbic acid or mercaptoethanol, inhibits the effect. These findings raise important questions about the physical and chemical nature of microwave interactions with cell membranes and also shed light on earlier studies reporting either positive or negative effects on membrane permeability.     

A sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography

A simple and sensitive method was developed for quantitative analysis of phospholipid molecular species. Diacylglycerols were prepared from phospholipids by phospholipase C treatment and converted to the corresponding dinitrobenzoyl derivatives, which could be sensitively detected at 254 nm. The derivatives of 21 molecular species were resolved by high-performance liquid chromatography with an octadecylsilyl reversed-phase column. All the derivatives had the same peak area per mol, and peak areas were proportional to the amounts of the derivatives. Quantification was carried out at the picomole level.     

Mutational analysis of the human dihydropyrimidine dehydrogenase gene by denaturing high-performance liquid chromatography

Mutations in the DPYD gene, which encodes dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in the catabolism of pyrimidines, are responsible for an inborn error of metabolism associated with thymine-uraciluria and neurological symptoms. Because the antimetabolite 5-fluorouracil (5-FU) is metabolized by the same enzyme, deficient DPYD alleles may also constitute a risk factor for severe toxicity following treatment with this anticancer drug. The aim of this study was to develop a comprehensive and rapid method to detect sequence variations within the DPYD gene. Using polymerase chain reaction (PCR) amplification and denaturing high-performance liquid chromatography (DHPLC), we established a protocol that makes it possible to screen all 23 exons of the DPYD gene and their exon-intron boundaries for both known and unknown mutations under identical conditions. A novel one-step PCR mutagenesis procedure was developed to generate heterozygous mutant amplicons as positive controls to optimize DHPLC detection of any sequence variation. DHPLC analysis was shown to result in mutation-specific elution profiles and to be able to distinguish different base changes within the same exon or different heterozygous combinations of mutations within the same exon. By analyzing the DPYD gene in 16 affected individuals, a total of 47 base changes were detected, representing eight known mutations and three novel intronic base changes. Sequence analysis confirmed all base changes detected. This method will be useful in identifying patients at risk for toxicity prior to 5-FU treatment, as well as in the analysis of individual patients with thymine-uraciluria.     

Monooxygenase and epoxide hydratase analysis by high-performance liquid chromatography

A simple HPLC method for the estimation of monooxygenase and epoxide hydratase activity is described using trans-stilbene and trans-stilbene oxide as substrates. The analysis method also allows the determination of benzoin, benzil, and benzoic acid, which have been proposed as other metabolites of trans-stilbene outside the traditional epoxide-diol pathway. We report the application of this method in an investigation of the biochemical potential of cell suspension cultures of Phaseolus vulgaris.     

Corticosteroid analysis in biological fluids by high-performance liquid chromatography

A sensitive, specific, and reproducible high-performance liquid chromatographic assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples are extracted with methylene chloride, washed with sodium hydroxide and then water, and chromatographed on a microparticulate silica gel column with UV detection at 254 nm. Sensitivity was greater than 15 ng for all four steroids. Specificity was supported by use of dual wavelength UV detection and/or radioimmunoassay. The assay has been applied in pharmacokinetic studies and a typical plasma concentration—time profile for the three steroids is presented for one subject who received 50 mg of prednisone.     

Purification of liver and hepatoma membrane proteins by high-performance liquid chromatography

This paper documents the recovery of selected proteins from hepatic plasma membranes. Initial purification, achieved by a series of stepwise extractions, facilitates the subsequent purification by HPLC. Examples are provided to illustrate the recovery of specific proteins from two Morris hepatoma lines and the liver.     

A highly sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography

A highly sensitive method was developed for quantitative analysis of phospholipid molecular species. Diradylglycerols prepared from phospholipids with phospholipase C were converted to the anthroyl-diradylglycerol derivatives, which could be separated into molecular species and sensitively quantified by reverse-phase HPLC using a fluorescence detector. All the molecular species of the derivatives had the same peak area per mole, and the peak areas were proportional to the amounts of the derivatives. Quantification could be carried out at the femtomole level.     

Sensitive analysis of ethanolamine- and serine-containing phosphoglycerides by high-performance liquid chromatography.

A highly sensitive method for the separation and quantitative measurement of phospholipids containing primary amino groups, such as phosphatidylethanolamine, phosphatidylserine and lysophosphatidylethanolamine, is described. The method involves a simple and quantitative derivative formation of the phospholipids containing amino groups to their u.v.-absorbing biphenylcarbonyl derivatives. These have molar extinction coefficients of about 23,000 at 268nm. The phospholipid derivatives are then separated and non-destructively determined by high-performance liquid chromatography. The amino phospholipids containing vinyl ether bonds (plasmalogens) can be determined separately from the diacyl- and alkylacyl-amino phospholipids. The lower limit of detection by high-performance liquid-chromatographic analysis of the phospholipid derivatives is about 10-13pmol or 0.3-0.4ng of phospholipid P. The quantitative range of derivative formation and analysis by high-performance liquid chromatography of the phospholipids containing amino groups was shown to be 10-500nmol. The method was shown to be applicable to the analysis of phospholipids containing amino groups in tissue samples.