The LC-MS/MS-Based Measurement of Isopimaric Acid in Rat Plasma and Application of Pharmacokinetics.

Isopimaric acid (IPA) exhibits a diverse array of pharmacological activities, having been shown to function as an antihypertensive, antitumor, antibacterial, and hypocholesterolemic agent. However, few studies of the pharmacokinetics of IPA have been performed to date, and such analyses are essential to explore the in vivo mechanisms governing the biological activity of this compound. As such, we herein designed a selective LC-MS approach capable of quantifying serum IPA levels in model rats using an Agilent HC-C18 column (250 mm × 4.6 mm, 5 µm) via isocratic elution with a mobile phase composed of methanol 0.5% formic acid (91 : 9, v/v) at a 1 mL/min flow rate. Ion monitoring at m/z 301.2 [M-H]- was used to quantify IPA levels in plasma samples from these rats, while internal standard (IS) levels were assessed at m/z 455.3 [M-H]-. After validation, this approach was employed to conduct a pharmacokinetic analysis of rats administered IPA via the oral (p.o. 50, 100, or 200 mg/kg) and intravenous (i.v. 5 mg/kg) routes. Analyses of noncompartmental pharmacokinetic parameters revealed that IPA underwent secondary absorption following oral administration to these animals, with the two tested oral doses (50 and 100 mg/kg) being associated with respective absolute bioavailability values of 11.9% and 17.5%. In summary, this study may provide a foundation for future efforts to explore the mechanistic basis for the pharmacological activity of IPA, offering insights to guide its subsequent clinical utilization.

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography-electrospray tandem mass spectrometry.

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra(®) MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r(2) = 0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.

Removal of Plasmodium falciparum-infected red blood cells from whole blood by leukoreduction filters.

BACKGROUND: There has been an unexplained decrease in the incidence of transfusion-transmitted malaria in recent years. The decrease in incidence has paralleled the increasing use of leukoreduction filters. Malaria-infected red blood cells (RBCs) share surface characteristics of hemoglobin S-containing cells. Because units collected from donors with sickle trait do not filter optimally due to adherence of RBCs to the filters, the possibility that malaria-infected RBCs may also adhere to filters was investigated. STUDY DESIGN AND METHODS: Malaria-infected whole blood or calcium ionophore (A25187)-treated and control RBCs were filtered with leukoreduction filters. Quantitation of malaria-infected RBCs before and after filtration was performed by flow cytometry to determine the presence of DNA within RBCs, indicating malaria infection. Annexin V binding was also determined before and after filtration of RBCs treated with A25187. Immediately after filtration, filters were fixed and examined by scanning and transmission electron microscopy. RESULTS: There were at least three configurations of adherence of malaria-infected RBCs demonstrated within the filters. The first was direct adherence of infected RBCs to filter fibers; the second involved adherence of malaria-infected RBCs to platelets, which were adherent to filter fibers; and the third was adherence of infected RBCs to other RBCs. Filtration also resulted in preferential removal of phosphatidylserine (PS)-expressing cells as seen by the reduction of annexin V binding after filtration. This was further confirmed by electron micrographic examination of the filters in which untreated RBCs sit within the filter resting on top of filter fibers; however, calcium ionophore-treated RBCs are seen to cling tightly to the fibers. CONCLUSIONS: PS expression by RBCs leads to their adherence within leukoreduction filters. Malaria-infected RBCs are retained via more than one mechanism. The efficiency of removal requires further study.

Photometric assessment of volume changes coupled with membrane potential in valinomycin-incorporated red blood cells.

Employing photometric methods, we have attempted to derive a possible quantitative relationship between volume change and membrane potential for valinomycin-incorporated red blood cells. The cells, collected from a human, rats or bullfrogs, were suspended in test solutions composed of a mixture of NaCl and KCl in varying proportions. The osmolality of the suspending medium was appropriately fixed at different values. After the addition of valinomycin to the suspension, changes in optical density (turbidity) at 620 nm and in fluorescence from a voltage-sensitive permeant dye (diS-C3-[5]) were measured in different concentrations of external potassium ions. The changes in optical density and fluorescence intensity were converted into relative cell volume and membrane potential changes in the test cells, respectively. Cell volume increased with depolarization of the membrane. We derived an empirical equation for the volume change versus membrane potential relation curves obtained experimentally, and have also shown that the observed volume change may be plausibly represented by a hyperbolic function of transmembrane potential involving the medium osmolality as an important parameter.

Characterization of the potent in vitro and in vivo antimalarial activities of ionophore compounds.

Large-scale in vitro screening of different types of ionophores previously pinpointed nine compounds that were very active and selective in vitro against Plasmodium falciparum; their in vitro and in vivo antimalarial effects were further studied. Addition of the ionophores to synchronized P. falciparum suspensions revealed that all P. falciparum stages were sensitive to the drugs. However, the schizont stages were three- to ninefold more sensitive, and 12 h was required for complete parasite clearance. Pretreatment of healthy erythrocytes with toxic doses of ionophores for 24 to 48 h showed that the activity was not due to an irreversible effect on the host erythrocyte. No preferential ionophore adsorption in infected or uninfected erythrocytes occurred. On the other hand, ionophore molecules strongly bound to serum proteins since increasing the serum concentration from 2 to 50% led to almost a 25-fold parallel increase in the ionophore 50% inhibitory concentration. Mice infected with the malaria parasites Plasmodium vinckei petteri or Plasmodium chabaudi were successfully treated with eight ionophores in a 4-day suppressive test. The 50% effective dose after intraperitoneal administration ranged from 0.4 to 4.1 mg/kg of body weight, and the therapeutic indices were about 5 for all ionophores except monensin A methyl ether, 5-bromo lasalocid A, and gramicidin D, whose therapeutic indices were 12, 18, and 344, respectively. These three compounds were found to be curative, with no recrudescence. Gramicidin D, which presented impressive antimalarial activity, requires parenteral administration, while 5-bromo lasalocid A has the major advantage of being active after oral administration. Overall, the acceptable levels of toxicity and the good in vivo therapeutic indices in the rodent model highlight the interesting potential of these ionophores for the treatment of malaria in higher animals.

Screening of potential transport systems for methyl mercury uptake in rat erythrocytes at 5 degrees by use of inhibitors and substrates.

The current study was designed to screen the potential transport systems for methyl mercury (MeHg) uptake by isolated erythrocytes from rats at 5 degrees. Several inhibitors and substrates were used to test which potential transport system might be involved in MeHg uptake. Probenecid was used to test the organic anion transport system, valinomycin was used to test the effect of the membrane potential, D-glucose and cytochalasin B were used to test the facilitated diffusive D-glucose transport system and colchicine and vinblastine were used to test the microtubule system. The effects of Ca++, Mg++ and Na+ on MeHg uptake have been examined. Ouabain, ATP and glucose were used to test the active transport system, cysteine for the cysteine-facilitated transport system, glycine for system Gly, DL-methionine for system L, and MeHgCl and 4',4-diisothiocyano-2',2-stilbenedisulfonic acid (DIDS) for the Cl- ion transport system. The results showed that MeHg uptake might be involved in the following transport systems at 5 degrees: 1) organic anion transport system; 2) facilitated diffusive D-glucose transport system; 3) cysteine-facilitated transport system; 4) Cl- ion transport system. Moreover, the transport systems for MeHg uptake were sensitive to the membrane potential. Although the mechanisms of interaction of transport systems have not been fully clarified, evidence has been presented which support the existence of several simultaneous transport systems for MeHg uptake.

Ketorolac penetration into the cerebrospinal fluid of humans.

STUDY OBJECTIVE: To determine the cerebrospinal fluid (CSF): total plasma concentration ratio of ketorolac tromethamine following a single intramuscular (IM) dose. DESIGN: Open, single-dose, IM-administration study. SETTING: General operating theaters of a medical school hospital. PATIENTS: 29 ASA physical status I and II patients scheduled to undergo elective surgery with spinal anesthesia. INTERVENTIONS: Patients were premedicated with ketorolac 90 mg IM formulated as 3 ml of a 3% solution. Between 1 and 4 1/2 hours later, an intravenous infusion of 500 ml of compound sodium lactate was begun. Lumbar puncture was then performed, and 2 ml of CSF was collected prior to administration of the spinal anesthetic. In addition, a 5 ml sample of venous blood was taken within 5 minutes of the CSF sample. MEASUREMENTS AND MAIN RESULTS: Simultaneous plasma and CSF concentrations of ketorolac were measured between 62 and 277 minutes following IM administration in 29 patients undergoing spinal anesthesia. The CSF concentrations were on the order of 1,000 times less than the total plasma concentrations; free concentrations of ketorolac in plasma were estimated to be about 10 times more than those in CSF. There appeared to be no constant time factor relating the appearance of ketorolac in the CSF to its plasma concentration following IM administration. CONCLUSION: Although the sensitivity of central prostaglandin synthetase systems to inhibition is unknown, it is unlikely from this pharmacokinetic data that there is a major central mechanism of analgesia for ketorolac.

[Effect of ionophores A 23187 and valinomycin on acetyocholinesterase activity in rabbit erythrocytes]. / Vliianie ionofora A23187 i valinomitsina na aktivnost' atsetilkholinésterazy éritrotsitov krolika.

Activity of cholinesterase was higher in young erythrocytes when the cell populations of various age were studied; the enzymatic activity was increased after addition of valinomycine into a medium independently on the antibiotic concentration and the enzyme activation was more pronounced in the old population of erythrocytes. The same alterations were observed in presence of calcium ionophore A 23187 and nystatin. Alterations in the enzymatic activity appear to depend on changes of erythrocyte membrane properties in ageing as well as on introduction of ionophore into the membrane.

Partial purification of a calcium ionophore from human uremic plasma and normal urine.

A fraction is isolated from human uremic plasma and normal urine using a three step chromatographic separation :gel permeation on Sephadex G15, then chromatography on hydroxyapatite and finally desalting operation on Sephadex G15. The fraction thus separated is ninhydrine positive and uncouples mitochondria principally releasing the resting respiration. Calcium potentiates the uncoupling, while red ruthenium and magnesium inhibit it. These results are in good agreement with a ionophorous activity of the isolated fraction. An hypothetic physiological role of this compound is there being discussed.