Spin label studies on rat liver and heart plasma membranes: effects of temperature, calcium, and lanthanum on membrane fluidity.

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(12,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(12,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19 degrees and 28 degrees C) and heart (between 21 degrees and 32 degrees C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl2 to I(12,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37 degrees C. Similarly, titrating I(12,3)-labeled heart plasma membranes with either CaCl2 or LaCl3 decreased the lipid fluidity at 37 degrees C, although the magnitude of the La3+ effect was larger and occurred at lower concentrations than that induced by Ca2+; addition of 0.2 mM La3+ or 3.2 mM Ca2+ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe--probe interactions. Ca2+ and La3+ at these concentrations decrease the activities of such plasma membrane enzymes as Na+, K+-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.     

Gas chromatographic assay for the new antitumor agent pyrazine-2-diazohydroxide (diazohydroxide) and its stability in buffer, blood and plasma

Diazohydroxide is a new antitumor agent being considered for clinical trial. A sensitive and specific assay for diazohydroxide in physiological media, plasma and blood has been developed based on conversion of diazohydroxide to 2-chloropyrazine in the presence of strong hydrochloric acid. The 2-chloropyrazine is extracted into the ethyl acetate and separated by capillary gas chromatography with nitrogen-phosphorus detection. Using 0.2 ml plasma the assay was linear up to 100 micrograms/ml diazohydroxide and had a lower limit of detectability for diazohydroxide of 50 ng/ml. The coefficient of variation of the assay at 1 micrograms/ml was 6.7%. Breakdown of diazohydroxide was rapid under mild acid conditions but slower under alkaline conditions,. The half-life of diazohydroxide in 0.1 M sodium phosphate buffer, pH 6.0, at room temperature was 5 min and at pH 8.0, 480 min. Breakdown of diazohydroxide in plasma was biphasic. In fresh mouse plasma diazohydroxide had a terminal half-life at 37 degrees C of 72 min while in fresh human plasma the terminal half-life was 23 min and in fresh blood 21 min. Diazohydroxide accumulated in red blood cells at 37 degrees C to a concentration 68% above the concentration in plasma. Diazohydroxide was 49% bound to human plasma proteins at room temperature.     

A quantitative LC/MSMS method for determination of edoxaban,a Xa inhibitor and its pharmacokinetic application in patients after total knee arthroplasty

Edoxaban was extracted from human plasma by simple protein precipitation with acetonitrile, followed by quantitative determination using a liquid chromatography–mass spectrometry method. The recoveries of edoxaban and the internal standard (ticlopidine) from human plasma were >85%, and the within‐ and between‐day coefficients of variation were within 15%. The limit of quantification in human plasma was 1 ng/mL. The concentration of edoxaban in blood decreased at room temperature, but remained unchanged for 1 week at 4°C. On the other hand, the concentration in plasma at both −20 and −80°C remained unchanged for 5 months. These results indicated that blood samples should be centrifuged immediately or stored at 4°C, and that plasma samples should be stored below −20°C until analysis. This method was applied to human plasma obtained from four patients after total knee arthroplasty. Analysis of edoxaban pharmacokinetics demonstrated an absorption time lag of 4h, a maximum concentration of 110 ± 26 ng/mL and an oral clearance of 37 ± 16 L/h. The analytical methods established in this study will be suitable for determining the concentrations of edoxaban in human plasma.     

Stability of penicillin antibiotic residues in meat during storage. Ampicillin

Incurred samples from a pig treated with ampicillin, one of the most important penicillin antibiotic drugs used in food-producing animal treatments, were analyzed at the residue level of the drug in muscle tissue (approximately 100 microg kg(-1)) during their freezing storage and using three different techniques: quantitative microbiological assay, HPLC-UV and LC-MS. Two parameters have been specifically monitored: storage temperature (-20 and -75 degrees C) and storage packaging (ground meat or bulk meat). No significant decrease was observed during the first 3 months of storage monitoring at -20 and -75 degrees C. On the contrary, the sample preparation significantly affected the drug concentration in muscle from the very beginning of the storage. Grinding the meat before storage allowed to keep the drug near the higher level of concentration (approximately 100 microg kg(-1)) when bulk meat stored frozen systematically led to a decreased value (approximately 75 microg kg(-1)). After 8 months of storage at -20 degrees C, a significant decrease arose and was never observed at -75 degrees C. All the results were similarly obtained with the three different techniques used simultaneously, which allows to indicate a good correlation between the techniques.     

Liquid chromatographic assay for the cyclic depsipeptide aplidine, a new marine antitumor drug, in whole blood using derivatization with trans-4'-hydrazino-2-stilbazole

A sensitive bio-analytical assay for the depsipeptide aplidine in plasma has been modified and tested for human whole blood samples. The adapted method is based on reversed-phase liquid chromatography and fluorescence detection of the trans-4'-hydrazino-2-stilbazole derivative of the analyte. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, the derivatization with the hydrazino reagent is performed in a water-acetonitrile mixture at pH = 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2-100 ng/mL range, with 2 ng/mL being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The stability of aplidine in whole blood at ambient temperature and at 37 degrees C is limited; recoveries in the range 60-85% were observed after 7 h. Further, adequate stability of aplidine in plasma at -80 and -20 degrees C for 35 months could now be demonstrated.     

Driving in vitro selection of anti-HIV-1 TAR aptamers by magnesium concentration and temperature

In vitro selection with either DNA or RNA libraries was performed against the TAR RNA element of HIV-1. The role of the selection conditions on the outcome of the selection was evaluated by varying the magnesium concentration and the temperature. The selection stringency was demonstrated to determine i) the affinity of the best identified aptamers for the TAR target, and ii) the type of interaction between the two partners. Selections performed with a DNA library under low (4 degrees C, 10 mM magnesium) and high stringency (23 degrees C, 3 mM magnesium) led to the emergence of "kissing aptamers"; but even if the motif interacting directly with the TAR loop were identical in the two kinds of aptamers, the consensus was extended from eight to thirteen nucleotides when the Mg(2+) concentration was decreased from 10 to 3 mM. Similar kissing aptamers were selected at 23 degrees C and 37 degrees C starting with two different RNA libraries under identical ionic conditions. In addition, selection performed at 37 degrees C yielded a significant proportion of antisense sequences. Only antisense RNAs complementary to the TAR loop competitively inhibited the association of a Tat peptide with TAR.     

Stability studies of testosterone and epitestosterone glucuronides in urine

The stability of testosterone glucuronide (TG), epitestosterone glucuronide (EG) and the T/E ratio in urine has been studied. Samples were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Urine samples were submitted to a solid-liquid cleanup followed by extraction of unconjugated testosterone (T) and epitestosterone (E) with tert-butyl methyl ether (free fraction). The remaining aqueous phase was hydrolyzed with beta-glucuronidase and extracted at alkaline pH with n-pentane. Analytes were analyzed by GC/MS as their enol-trimethylsilyl (TMS) derivatives. The urine for stability testing was obtained from an excretion study after the administration of T to healthy volunteers. The homogeneity of the sample was verified before starting the stability study. The stability of TG and EG was evaluated at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 degrees C and -20 degrees C was determined at different time intervals for 22 months. For short-term stability testing, analyte concentration was evaluated in urine stored at 37 degrees C for 3 and 7 days. The effect of repeated freezing (at -20 degrees C) and thawing (at room temperature) was studied for up to three cycles. Data obtained in this work demonstrated the stability of TG, EG and the T/E ratio in sterilized urine samples stored at 4 and -20 degrees C for 22 months and after going through repeated freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 37 degrees C due to the partial cleavage of the glucuronide conjugates; however, the T/E ratio was not affected. These results show the feasibility of preparing reference materials containing TG and EG to be used for quality control purposes.     

Determination of the endocannabinoid anandamide in human plasma by high-performance liquid chromatography

Anandamide (N-arachidonylethanolamine) is an endogenous cannabinoid receptor ligand that has been implicated in various physiological and pathophysiological functions. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with fluorometric detection was validated and applied for the analysis of anandamide in human plasma. Following derivatization with the fluorogenic reagent 4-(N,N-dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methyl-amino)-2,1,3-benzoxadiazole (DBD-COCl), the analyte was separated using an acetonitrile-water gradient at a flow rate of 0.8 mL/min, and spectrophotometric detection at 560 nm with an excitation wavelength of 450 nm. The retention times for anandamide and R+-methanandamide (internal standard) were 27.1 and 30.7 min, respectively. The validated quantification range was 1-15 ng/mL. The developed procedure was applied to determine anandamide levels in human plasma following a 24 h incubation of human whole blood at 37 degrees C in the presence or absence of phenylmethylsulfonyl fluoride, an inhibitor of the anandamide-degrading enzyme fatty acid amide hydrolase. Anandamide levels determined under both conditions were within the validated concentration range with anandamide levels being 2.3-fold higher in plasma from PMSF-treated blood.     

Theoretical analysis of localized heating in human skin subjected to high voltage pulses

Electroporation, the increase in the permeability of bilayer lipid membranes by the application of high voltage pulses, has the potential to serve as a mechanism for transdermal drug delivery. However, the associated current flow through the skin will increase the skin temperature and might affect nearby epidermal cells, lipid structure or even transported therapeutic molecules. Here, thermal conduction and thermal convection models are used to provide upper and lower bounds on the local temperature rise, as well as the thermal damage, during electroporation from exponential voltage pulses (70 V maximum) with a 1 ms and a 10 ms pulse time constant. The peak temperature rise predicted by the conduction model ranges from 19 degrees C for a 1 ms time constant pulse to 70 degrees C for the 10 ms time constant pulse. The convection (mass transport) model predicts a 18 degrees C peak rise for 1 ms time constant pulses and a 51 degrees C peak rise for a 10 ms time constant pulse. The convection model compares more favorably with previous experimental studies and companion observations of the local temperature rise during electroporation. Therefore, it is expected that skin electroporation can be employed at a level which is able to transport molecules transdermally without causing significant thermal damage to the tissue.     

Protein synthesis in Escherichia coli at 4 degrees C

Two-dimensional electrophoresis technology was used to investigate protein synthesis by the mesophilic bacterium Escherichia coli at low temperature. It was confirmed that protein synthesis in E. coli decreased strongly after a temperature downshift from 37 to 4 degrees C. After incubation for 150 min at 4 degrees C, however, the number of synthesized proteins represented 60% of the overall polypeptide number observed at 37 degrees C. Furthermore, the analysis of autoradiograms revealed the overexpression of 69 proteins by shocked bacteria, showing that the number of cold-induced proteins has been significantly underestimated so far.     

Examination of the in vitro degradation of [14C] pentaerythritol tetranitrate in rat and human blood with an improved thin-layer radiochromatographic procedure

Improvements were made on a reported thin-layer radiochromatographic assay for the determination of [14C]pentaerythritol tetranitrate (PETN) and its metabolites in whole blood, using methanol instead of dioxane as the extracting solvent. Recovery of total radioactivity for the entire work-up procedure was greater than 90%, and the distribution of PETN and its metabolites in degraded blood samples was found to be reproducible. This modified method appeared simpler and yielded better recovery of radioactivity than the literature method. In vitro metabolism of [14C]PETN in rat and human blood was examined by incubation of the drug with fresh blood at 37 degrees C for 60 min. In rat blood, the half-life of PETN degradation was about 15 min producing the trinitrate, dinitrate and mononitrate metabolites. Human blood was also capable of degrading PETN in vitro, but at a lower rate than rat blood, yielding only the trinitrate metabolite in quantifiable amounts during the incubation period. Equilibrium of PETN between plasma and red blood cells was observed within 1 min after PETN addition to both rat and human blood. The apparent plasma/red blood cells partition ratios of PETN were 1.1 and 1.7 for rat and human blood, respectively. PETN degradation was approximately ten times slower in rat plasma than in rat blood, suggesting that enzymes in erythrocytes are important for PETN metabolism in rat whole blood.     

The influence of blood sample storage time on the propofol concentration in plasma and solid blood elements

In order to describe the changes of propofol concentration in whole blood and in its components during the blood storage we examined venous blood samples collected from patients anaesthetized either with or without propofol. Blood samples from patients anaesthetized without propofol were spike with propofol 45 min before analysis. Propofol concentration was examined in whole blood, plasma, rinsed formed elements and rinsed and lysed formed blood elements by means of HPLC after 1, 4, 7, 13, 21, 25 and 28 days of storage. There was significant decrease in plasma concentration of propofol during the first few days of sample storage followed by its increase during subsequent days. The opposite phenomenon was observed for formed blood elements. The findings support the hypothesis that propofol distribution between blood components changes in time.     

Stability of inorganic mercury and methylmercury on yeast-silica gel microcolumns: field sampling capabilities

The stability of methylmercury and inorganic mercury retained on yeast-silica gel microcolumns was established and compared with the stability of these species in solution. Yeast-silica gel columns with the retained analytes were stored for two months at three different temperatures: -20 degrees C, 4 degrees C and room temperature. At regular time intervals, both mercury species were eluted and quantified by cold vapor atomic absorption spectrometry (CVAAS). Methylmercury was found stable in the columns over the two-month period at the three different temperatures tested while the concentration of inorganic mercury decreased after one week's storage even at -20 degrees C. These results are of great interest since the use of these microcolumns allows the preconcentration and storage of mercury species until analysis, thus saving laboratory space and avoiding the problems associated with maintaining species integrity in aqueous solution.     

Analysis of the interaction between human plasma fibronectin and gelatin by affinity electrophoresis

The interaction between human plasma fibronectin and gelatin was analyzed by affinity electrophoresis, in which the fibronectin was subjected to electrophoresis in a 4% polyacrylamide gel in the presence and absence of gelatin, as an affinity ligand, and the fibronectin band was stained by an immunoblotting method. The apparent dissociation constants (Kd) of fibronectin for gelatin were calculated from affinity plots based on the original affinity equation at different pHs, urea concentrations, and temperatures. The fibronectin exhibited much lower affinity in the presence of urea. The Kds at 37 degrees C were 1.49 X 10(-7) M, 2.50 X 10(-6) M, and 3.58 X 10(-6) M with 2 M, 3 M, and 4 M urea, respectively. The van't Hoff plots of Kd values against absolute temperature (T) showed that the value of log Kd decreased in proportion to the increase in the value of 1/T within the range of 15-50 degrees C. The standard enthalpy, the standard free energy change at 37 degrees C, and the entropy change at 37 degrees C for association were calculated to be -124.7 kJ/mol, -33.23 kJ/mol, and -295.1 J/mol/deg, respectively. These results suggest that a hydrophilic interaction, such as hydrogen bond or van der Waals interaction, plays an important role in the binding of plasma fibronectin to gelatin.     

A novel liquid chromatography/tandem mass spectrometry based depletion method for measuring red blood cell partitioning of pharmaceutical compounds in drug discovery

A novel liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based depletion method for measuring compound partitioning between human plasma and red blood cells (RBC) in a drug discovery environment is presented. Conventionally, RBC partitioning is determined by separate measurements of drug concentrations in equilibrating plasma and whole blood or RBC using separate standards prepared in their respective matrices, i.e., in plasma and whole blood or RBC lysates. The process is very tedious, labor-intensive, and difficult to automate. In addition, interferences from the heme and other highly abundant cellular composites make the measurement of the drug concentration in whole blood or RBC inevitably variable even with a highly specific LC/MS/MS method. Therefore, there is an imminent need to develop a straightforward and fast method to assess the partitioning of drug-like compounds in RBC.This work describes an LC/MS/MS-based depletion assay that measures the compound concentration in plasma that has been equilibrating with RBC. Compounds were spiked into fresh human whole blood and plasma respectively to a final concentration of 500 nM. Both the spiked whole blood and plasma control were incubated at 37 degrees C for up to 60 min. During the time course, aliquots of plasma and whole blood from both incubation mixtures were sampled at 10 and 60 min. The whole blood samples were centrifuged to yield the plasma. The plasma samples from both incubations were extracted using a protein precipitation method, and analyzed using LC/MS/MS under the multiple-reaction monitoring (MRM) mode. The RBC partitioning ratio was calculated using the analyte peak area responses of the plasma samples through an equation deduced in this work.The method was first tested using two commercial compounds, phenoprobamate and acetazolamide, to determine the optimal incubation conditions and the concentration dependency of the assay. The assay reproducibility was also assessed by three inter-day assays for phenoprobamate. This method was further evaluated using 20 commercial compounds of different classes with a wide range of RBC partitioning coefficients and the results were compared with those reported in the literature. Excellent correlation (R2=0.9396) was found between the measured and literature values. In addition, several proprietary compounds were assayed using both the new and traditional methods and the measured partitioning ratios from the two methods are equivalent. The experiments in this work demonstrate that the LC/MS/MS-based depletion method can provide direct and accurate measurement of RBC partitioning for compounds in drug discovery.     

Influence of parenteral fat emulsion Intralipos and citric acid on blood viscosity and erythrocyte morphology in vitro

In most studies fat emulsion had been administered by enteral route. Recently a parenteral soybean-oil emulsion has been developed. In this study we assessed the effects of a parenteral soybean-oil emulsion (Intralipos((R))) and citric acid on blood rheology and erythrocyte morphology in vitro. Porcine blood was incubated in vitro with increasing concentrations of fat emulsion Intralipos((R)) and citric acid at 37 degrees C for 1h. Viscosity of plasma and whole blood was measured using a FASCO-2050 digital viscometer. Red blood cell morphology was examined by light microscopy. The viscosity of whole blood represented an ascending dose-dependence for different Intralipos((R)) concentrations at high shear rate of 90 and 225s(-1), however, it decreased with citric acid concentrations. On the other hand, the whole blood viscosity also declined with citric acid concentrations in presence of 30% Intralipos((R)) (v/v), and there is a minimal viscosity at 0.67% citric acid (v/v). There are some thorns on the blood membrane at 40% Intralipos((R)) as compared with control (no Intralipos((R)) addition), which indicates the Intralipos((R)) compound may affect blood cell membranes, and resulted in whole blood viscosity increase. We concluded that the intravenous soybean-oil preparation Intralipos((R)) interacts with the erythrocyte membrane, and citric acid could alleviate the whole blood viscosity.     

Covalently Bound Antibody on Polystyrene Latex Beads: Formation, Stability, and Use in Analyses of White Blood Cell Populations

CD4 or CD8 antibodies were covalently bound to latex beads by reaction of activated CD4 or CD8 monoclonal antibodies with 2-μm-diameter, 1,3-diaminopropane (DAP) coupled, polystyrene aldehyde/sulfate latex beads. Spectrophotometric analyses of the filtrates of the antibody-bead conjugation mixtures for unreacted antibody allowed construction of binding curves of antibody for the polystyrene bead surface and evaluation of binding constants for association of antibody with bead, ranging from 1.5x10(7) to 1.6x10(7) M(-1) for CD4 and CD8 antibodies. The reaction of the antibody thiol group with the activated maleimide group on the bead at pH 7.2-7.3 was complete within 10-15 min. The kinetics of CD4 or CD8 monoclonal antibody displacement from the surface of covalently conjugated antibody-polystyrene latex beads was followed as a function of temperature (5, 22, and 37 degrees C) and the nature of the final diluent for the antibody-coated beads by measuring the concentration of antibody in the filtrates of conjugated beads by an ELISA (enzyme-linked immunosorbent assay). The displacement reaction showed a pseudo-zero-order dependence of the rate, with constants, k(1), ranging from 0.65x10(-17) to 270x10(-17) M s(-1). The functionality of antibody-coated beads suspended in various media was also monitored in a biological cell assay with whole blood. The cell assay depends on forming a layer of beads around targeted lymphocytes to distinguish them from nontargeted lymphocytes by differences in dc or rf conductivity or median angle light scatter. Covalently bound CD4 and CD8 antibody beads stored in one set of media at 5, 22, and 37 degrees C over a period of 16 weeks showed excellent results in the STKS assay with various blood donors, which correlated well (correlation coefficients of 0.99 for CD4 data and 0.93 for CD8 data) with reference results obtained with fluorescent markers by flow cytometry. Covalently bound CD4/CD8 beads stored for 2 weeks in BSA buffer at 5-37 degrees C performed equally well in providing accurate values of the percentage of CD4- or CD8-positive cells in the total white blood cell population, whereas the same beads stored in the 47-50 degrees C range showed some failures in performance. Comparison with antibody concentrations in filtrates of adsorbed antibody-bead suspensions showed 2- to 10-fold greater amounts of free antibody at comparable elapsed time, media, and temperature conditions. A threshold of 1-2 μg/mL of free antibody was necessary before adverse effects on the biological cell assay were noticeable. Copyright 2001 Academic Press.     

The stability of doxorubicin and ldarubicin in plasma and whole blood

Summary The stability of doxorubicin and idarubicin was studied in spiked human EDTA-, heparin-plasma and whole blood at room temperature (RT) and at +4°C. The analysis was performed by capillary electrophoresis (CE). Under each sel of conditions, doxorubicin and idarubicin concentrations decreased rapidly with time; the decrease was insignificantly more rapid with doxorubicin. Neither doxorubicinol nor idarubicinol were detectable. In order to obtain comparable plasma levels, doxorubicin and idarubicin containing blood samples should be centrifuged and stored at −20°C immediately.     

Study of the stability of selenium compounds in human urine and determination by mixed ion-pair reversed-phase chromatography with ICP-MS detection

The stability of five selenium compounds, selenate, Se(VI), selenourea, SeUr, trimethylselenonium ion, TMSe(+), selenomethionine, SeMet, and selenoethionine, SeEt, at concentrations from 30-60 micro g L(-1) in a pooled human urine, stored in dark at -20 degrees C, 4 degrees C, or ambient temperature (ca. 25 degrees C), without addition of any stabilizing reagent was evaluated. The investigated Se species were determined independently by mixed ion-pair reversed-phase liquid chromatography with inductively coupled plasma mass spectrometric (ICP-MS) detection. The general trend is the lower the temperature used for storage, the higher the stability of Se species, when other conditions such as light, acidity, and container material are kept constant. On the basis of these results it is considered that the storage of urine samples at -20 degrees C for a short-term (within one month) is safe for Se speciation analysis. Long-term storage of urine samples for speciation analysis should, however, be undertaken with caution.     

High-performance liquid chromatographic assay for the antitumor glycoside phyllanthoside and its stability in plasma of several species

Phyllanthoside is a glycoside isolated from the roots of the Central American tree Phyllanthus acuminatus Vahl with antitumor activity against murine B-16 melanoma and P-388 leukemia. We report a reversed-phase high-performance liquid chromatographic assay for phyllanthoside in plasma using a 25-cm RP-18, 5-micron column with a linear 10-min gradient of 50% to 100% methanol in 0.3 M sodium acetate, pH 4.0, at a flow-rate of 1.5 ml/min. Eluting peaks were detected at 270 nm. The lower limit of sensitivity of the assay for phyllanthoside in 0.5 ml plasma following ethyl acetate extraction at pH 7.0 was 0.25 micrograms/ml and the coefficient of variation at 1 microgram/ml was +/- 7.4%. Phyllanthoside was very rapidly broken down by mouse and rat plasma in vitro to an unidentified less polar metabolite. Formation of this metabolite was completely inhibited by preheating mouse plasma to 100 degrees C for 10 min. When mouse plasma was diluted 1:50 with water the half-life of phyllanthoside disappearance at 37 degrees C was 2.0 min. Breakdown of phyllanthoside in plasma from other species was slower than in mouse and the initial half-life at 37 degrees C in dog plasma was 30 min, in monkey plasma 33 min and in human plasma 38 min. The same less polar metabolite as in mouse plasma was formed slowly by plasma of monkey and dog. Phyllanthoside did not accumulate in human red blood cells. Binding of phyllanthoside to human plasma protein determined by ultrafiltration at 4 degrees C was 70%.