Glutathione excretion in response to heterologous protein secretion in Saccharomyces cerevisiae.

Glutathione is excreted in a dose-dependent, non-stoichiometric fashion from Saccharomyces cerevisiae cells expressing and secreting Bovine Pancreatic Trypsin Inhibitor (BPTI), a small, disulfide-bonded protein. Glutathione excretion commences 40 hours following induction of BPTI synthesis. Expression of several secretory proteins with varying disulfide and cysteine contents results in glutathione excretion with no apparent requirement for protein disulfide content. Glutathione excretion is also triggered by overexpression of Kar2p/BiP, a native ER-resident protein-folding chaperone, indicating that the response is a general one not restricted to overexpression of thiol-containing heterologous proteins. Functional vesicular transport is not required at the time of glutathione excretion, and glutathione excretion requires the presence of molecular oxygen. These data are consistent with a delayed oxidative stress response potentiated by earlier heterologous secretion, but are inconsistent with secretory transport of glutathione spent as oxidizing equivalents for disulfide-bond formation in the endoplasmic reticulum.

Evaluation of reductive amperometric detection in the liquid chromatographic determination of antineoplastic platinum complexes.

The usefulness of reductive electrochemical detection at mercury drop electrodes has been determined for platinum complexes separated by solvent-generated anion-exchange high-performance liquid chromatography. Both current-sampled dropping mercury and hanging mercury drop electrodes (DME and HMDE) provide significant advantages over UV absorbance and off-line non-flame atomic absorption detection. The effects of chromatographic and polarographic parameters on analytical system performance have been investigated. By raising the detector cell temperature, the detector response to cis-dichlorodiammineplatinum(II) (DDP) can be shifted anodically to 0.0 V vs. Ag/AgCl, thereby increasing detector selectivity for this compound. The noise-limited minimum detectable quantities of DDP with DME and HMDE are 1.8 ng and 70 pg injected, respectively. DDP can be determined in untreated urine at levels below 100 ng/ml.

Automated analysis of acetaminophen and caffeine in serum using the FAST-LC system: contributions to assay imprecision in procedures based on HPLC with sample pretreatment.

Two procedures are described for the fully automated analysis of several therapeutic drugs in serum, using HPLC with on-line pretreatment (solvent extraction) of the sample. The FAST-LC system (Technicon Instruments) was used for the assay of mixtures of 1) acetaminophen, theophylline, and/or caffeine, or 2) phenylethylmalonamide, primidone, phenobarbital, carbamazepine epoxide, phenyltoin, and/or carbamazepine. The rate of sample analysis was 15/hr for the theophylline group of drugs and 12/hr for the six anticonvulsants. The precision of resulting assays was about 3% (CV), and only 75 microliter of sample was required. The precision of resulting assays, in terms of a previously reported model, is also discussed.

Liquid-chromatographic analysis for common tricyclic antidepressant drugs and their metabolites in serum or plasma with the technicon "FAST-LC" system.

We describe a single procedure for assay of seven tricyclic antidepressant drugs and metabolites in serum or plasma: protriptyline, nortriptyline, amitriptyline, desmethyldoxepin, doxepin, desipramine, and imipramine. With the Technicon "FAST-LC" system, samples are aspirated directly into the unit and pretreated via double extraction; the concentration of each drug is then determined by "high-performance" liquid chromatography. Final chromatograms are monitored at 205 nm, at analysis rates of 7.5 samples/h. Concentration and absorbance are linearly related for each drug from 0 to 1400 micrograms/L. Day-to-day CVs averaged 5 to 6% for each drug, and there is good correlation of FAST-LC values with those obtained by gas-chromatographic methods. Total sample volume is 750 microliters.

On-line liquid-chromatographic analysis for drugs in serum with the Technicon "FAST-LC" system: performance data for theophylline and for four commonly used anticonvulsants and their metabolites.

We describe a new instrument for use in assay of therpeutic drugs in serum by "high-performance" liquid chromatography, the "FAST-LC" system (Technicon). Serum samples are aspirated directly into the unit, extracted with solvent, and the evaporated and redissolved extract is injected onto a chromatographic column. We illustrate the performance of the system by assays in serum for theophylline and four anticonvulsants (primidone, phenobarbital, phenytoin, and carbamazepine) plus two of their active metabolites (phenylethylmalonamide and carbamazepine epoxide). For theophylline, final chromatograms are monitored at 270 nm, at analysis rates of 10/h. Concentration and absorbance are linearly related from 0 to 130 mg of theophylline per liter. For the anticonvulsants, chromatograms are monitored at 200 nm, at analysis rates of 7.5/h. The six individual determinations are each linear beyond the therapeutic range. For both drug panels, day-to-day CV's were 4 to 6%. Results correlate well with those by enzyme immunoassay. A total sample volume of 150 microL is required.

Pharmacokinetics of non-protein-bound platinum species following administration of cis-dichlorodiammineplatinum(II).

The pharmacokinetics of non-protein-bound platinum species derived from cis-dichlorodiammineplatinum(II) (cis-platinum) was studied under a variety of dosing conditions. Following rapid infusions (15-minute) of cis-platinum at 100 mg/m2, the unbound drug declined in a biphasic mode with a mean terminal half-life of 48 minutes. The mean beta-phase half-life after a 6-hour infusion of the same dose of cis-platinum was 26 minutes. Urinary excretion of filterable platinum was substantially greater after a 6-hour infusion than after a 15-minute injection. Concomitant administration of mannitol appeared to result in higher peak plasma concentrations and decreased urinary excretion of unbound platinum species but did not alter the terminal half-life. Renal impairment was associated with extremely high plasma levels of filterable platinum but did not affect other pharmacokinetic parameters. Preliminary data on the distribution of cis-platinum to ascitic fluid are also presented.

High-performance liquid chromatographic analysis of emetine after oxidative activation to a fluorescent product.

A clinically useful analytical method is described for monitoring plasma levels of emetine. The drug is initially extracted from plasma with dichloromethane (0.3 volumes). The extract can be analyzed directly by paired-ion reversed-phase high-performance liquid chromatography to levels of 500 ng/ml of plasma by spectrophotometric monitoring of column effluent. For analysis of emetine at lower concentrations, the dichloromethane extracts are subjected to mild mercuric acetate oxidation prior to separation, thereby converting emetine to a fluorescent product. Spectrofluorometric monitoring of the column effluent readily extends the sensitivity of the assay to 10 ng of emetine/ml of plasma. At these levels measurements can be made with a precision of +/- 4%.

Urine analysis of platinum species derived from cis-dichlorodiammineplatinum(II) by high-performance liquid chromatography following derivatization with sodium diethyldithiocarbamate.

A clinically useful method is described for the quantitative analysis of platinum species derived from cis-dichlorodiammineplatinum(II) in urine. The drug and its biodegradation products are derivatized directly in urine by reaction with sodium diethyldithiocarbamate (DDTC) to form a common product, a 2:1 DDTC-platinum adduct. This complex is stable and can be quantitatively extracted into 0.1 volumes of chloroform. An aliquot of the chloroform layer is then subjected to high-performance liquid chromatography on a muBondapak CN column and the eluent monitored spectrophotometrically at 254 nm. At this wavelength the DDTC-platinum adduct has a molar absorptivity of 43,000, and platinum levels of 25 ng/ml or urine can be detected with a precision of +/- 2.5% and an accuracy of +/- 4%.

Atomic absorption spectrophotometry of free circulating platinum species in plasma derived from cis-dichlorodiammineplatinum(II).

We describe a method of analysis for free circulating platinum species derived from cis-dichlorodiammineplatinum(II) in blood plasma. Protein-bound and free platinum species were separated from each other by centrifugal ultrafiltration. Platinum in the ultrafiltrate was converted to a cationic complex by reaction with ethylenediamine, and the product was collected on paper impregnated with cation-exchange resin, where it could be stored indefinitely without loss. The platinum was eluted from the disk with 5 mol/liter hydrochloric acid, and an aliquot of this solution was then analyzed by flameless atomic absorption spectrophotometry. The overall analytical recovery of platinum was 80 +/- 2%. The minimum quantity of cis-dichlorodiammineplatinum detectable was 35 microgram/liter of plasma at the 99% confidence level. Detector response was linearly related to drug concentration in the range from 80 microgram to 290 mg of Pt per liter of plasma. Reaction variables were made optimal, so as to yield maximum sensitivity and reproducibility (+/- 2%) consistent with minimal sample transfers and manipulations.

Gas chromatographic analysis of acetophenone oxime and its metabolites.

A gas-liquid chromatographic (GLC) method has been developed for monitoring the metabolic reduction of acetophenone oxime or oxidative metabolism of the corresponding amine, alpha-methylbenzylamine in liver homogenates. The oxime, amine, n-hydroxy-alpha-methylbenzylamine and acetophenone are quantitatively determined after GLC separation of components with temperature programming on an SP-2401-DB-coated column. The first three compounds were silylated with N,O-bis(trimethylsilyl)-acetamide prior to chromatographic analysis to enhance the stability and improve the chromatographic properties of these components. The effluent gas was monitored with flame ionization detection, and permitted quantitation of components at sub-microgram/ml levels with reproducibility between injections of +/-2%. The optimal composition of enantiomeric mixtures of (R,S)-alpha-methylbenzylamines formed during metabolic reduction of acetophenone oximes were determined by conversion to diastereomeric amides and subsequent GLC analysis.

Measurement of free-circulating cis-dichlorodiammineplatinum(II) in plasma.

Dichlorodiammineplatinum(II) is an anti-neoplastic agent that is currently undergoing clinical evaluation. We describe an analytical method for monitoring the free drug (or its breakdown products) in plasma. The method is able to distinguish between free and protein-bound drug. Plasma samples are deproteinized by centrifugal ultrafiltration. The platinum in the ultrafiltrate is converted to a cationic species by reaction with ethylenediamine and then collected on paper impregnated with cation-exchange resin. This process concentrates the samples, increases the stability of the platinum compounds (by removing the compound from solution), and places the sample in a uniform matrix of minimum thickness, which maximizes detection capabilities. Platinum was measured directly on the ion-exchange disks by X-ray fluorescence. The detection limit for free drug is 240 microgram/liter of plasma at the 3s level and fluorescence intensity is linearly related to drug concentration in the range from 570 to 5700 microgram/liter.