Inactivation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by a plasma acetylhydrolase: Higher activities in hypertensive rats

We have partially characterized the properties of a specific acetylhydrolase in plasma from spontaneous hypertensive rats. This enzyme inactivates 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine (a lipid involved in platelet aggregating, hypotensive, and allergic responses) by removal of the acetate group. The extent of acetate hydrolysis was linear with both time and protein concentration, and the enzyme had an apparent K_m of 2.5 μM and a V_max of 2.6 nmol/min/mg protein. As with an intracellular acetylhydrolase previously characterized by us, the plasma activity was not affected by addition of phosphatidylcholine, EDTA, or Ca²⁺. However, in contrast to the acetylhydrolase activity in the rat kidney soluble fraction, the plasma activity was associated with a higher molecular weight protein resolved on a Sepharose 6B column and the plasma acetylhydrolase was not inhibited by treatment with trypsin, pronase, or subtilisin. We also compared the acetylhydrolase activity in plasma of age-matched spontaneous hypertensive rats and their normotensive controls, and found approximately 20% higher levels of activity in plasma from the hypertensive animals ($\text{P}$ <0.01).     

Adjustment of digestion enzyme composition improves islet isolation outcome from marginal grade human donor pancreata

Despite improvements and recent attempts to standardize techniques to isolate islets from human donor pancreata, there still exists the problem of consistently recovering sufficient quantities of high quality islets. Moreover, achieving consistent recoveries of high numbers of good quality islets becomes even more challenging from marginal grade human donor pancreata with prolonged cold ischemic times. In this study, we investigate whether addition of Pefabloc SC, a serine protease inhibitor, in combination with Pulmozyme, a recombinant human DNase I, to Liberase HI improves islet isolation outcome from marginal grade human donor pancreata (cold ischemic time > 12 h). Twenty-three marginal grade human donor pancreata were randomly digested using four different enzyme preparations: (1) Liberase alone (n = 6), (2) +Pefabloc (n = 7), (3) +Pefabloc/Pulmozyme (n = 5), and (4) +Pulmozyme (n = 5). Overall, there were no significant differences in donor age, body mass index (BMI), pancreas weight, and cold ischemic time. After purification, significantly higher islet yields (3,281 ± 590 IE/g) were obtained with the Pefabloc/Pulmozyme group as compared to the Liberase alone (1,615 ± 305 IE/g) or the Pefabloc group (1,255 ± 261 IE/g) (P < 0.05). Significant improvements in islet viability were also noted from the Pefabloc/Pulmozyme group (87.3 ± 4.4%) as opposed to islets isolated from the Pefabloc group (75.2 ± 3.9%) (P < 0.05). No significant differences in insulin secretory response to glucose stimulation among the four groups were observed, which indicates that the addition of Pefabloc and/or Pulmozyme does not have a detrimental effect on the functionality of islets. It is concluded that the addition of Pefabloc in combination with Pulmozyme to the Liberse HI significantly improves islet isolation outcome and potentially impacts the viability and morphology of the islets obtained from marginal grade human donor pancreata with prolonged cold ischemic times. This study was presented in a poster session at the American Transplant Congress 2005 in Seattle, Washington, USA (May 2005).     

Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (Δsse ^MGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. K_M and k _cat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of Δsse ^MGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.     

Characterization of the enzymatic hydrolysis of acetate from alkylacetylglycerols in the de novo pathway of PAF biosynthesis

This report describes the partial characterization of the enzymatic activity responsible for the hydrolysis of acetate from 1-alkyl-2-acetyl-sn-glycerol, the immediate precursor in the de novo synthesis of PAF (platelet-activating factor or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by Ehrlich ascites cells. The highest acetylhydrolase activity for this neutral lipid was associated with the membrane fractions from Ehrlich ascites cells (> 90% of total activity); only a minimal level of activity (< 10%) was observed in the cytosol which contrasts with the cytosolic site of PAF acetylhydrolase in normal cells. Hydrolysis of 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction at pH 7.5 and 37°C gave apparent values for K_m and V_max of 45 μM and 179 nmol/min per mg protein, respectively. Hydrolysis of acetate from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction was not affected by 5 mM concentrations of Ca⁺², Mg⁺² or EDTA, but was significantly inhibited (80% reduction) by 10 mM NaF. Based on differences in both the subcellular distribution and response to inhibition by NaF, the neutral lipid acetylhydrolase does not appear to be the same enzyme that hydrolyzes acetate from platelet-activating factor. In contrast to inhibition of diacylglycerol lipase by p-chloromercuribenzoate and N-ethylmaleimide, we found no significant inhibition of acetate hydrolysis from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by either of these compounds. Also, p-nitrophenyl acetate (a nonspecific esterase substrate) failed to inhibit acetate hydrolysis of 1-[³H])hexadecyl-2-acetyl-sn-glycerol. Our studies of this enzyme would indicate that it may play an important role in regulating the levels of platelet-activating factor synthesized by the de novo pathway via hydrolysis of the immediate precursor of PAF.     

Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis

Stimulated inflammatory cells synthesize platelet-activating factor (PAF), but lysates of these cells show little enhancement in PAF synthase activity. We show that human neutrophils contain intracellular plasma PAF acetylhydrolase (PLA2G7), an enzyme normally secreted by monocytes. The esterase inhibitors methyl arachidonoylfluorophosphonate (MAFP), its linoleoyl homolog, and Pefabloc inhibit plasma PAF acetylhydrolase. All of these inhibitors induced PAF accumulation by quiescent neutrophils and monocytes that was equivalent to agonist stimulation. Agonist stimulation after esterase inhibition did not further increase PAF accumulation. PAF acetylhydrolase activity in intact neutrophils was reduced, but not abolished, by agonist stimulation. Erythrocytes, which do not participate in the acute inflammatory response, inexplicably express the type I PAF acetylhydrolase, whose only known substrate is PAF. Inhibition of this enzyme by MAFP caused PAF accumulation by erythrocytes, which was hemolytic in the absence of PAF acetylhydrolase activity. We propose that PAF is continuously synthesized by a nonselective acyltransferase activity(ies) found even in noninflammatory cells as a component of membrane remodeling, which is then selectively and continually degraded by intracellular PAF acetylhydrolase activity to modulate PAF production.     

Differences in the regulation of RyR2 from human,sheep, and rat by Ca2+ and Mg2+ in the cytoplasm and in the lumen of the sarcoplasmic reticulum

Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca²⁺ and Mg²⁺ plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca²⁺- and Mg²⁺-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca²⁺ activation (K_a = 4 µM) and inhibition by cytoplasmic Mg²⁺ (K_i = 10 µM at 100 nM Ca²⁺) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca²⁺, RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg²⁺ inhibition than those from sheep and rat. The K_a values for luminal Ca²⁺ activation were similar in the three species (35 µM for human, 12 µM for sheep, and 10 µM for rat). From the relationship between open probability and luminal [Ca²⁺], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ∼10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg²⁺ as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca²⁺ and Mg²⁺ only occurred when cytoplasmic [Ca²⁺] was <3 µM. The activation response of RyR2 to luminal and cytoplasmic Ca²⁺ was strongly dependent on the Mg²⁺ concentration. Addition of physiological levels (1 mM) of Mg²⁺ raised the K_a for cytoplasmic Ca²⁺ to 30 µM (human and sheep) or 90 µM (rat) and raised the K_a for luminal Ca²⁺ to ∼1 mM in all species. This is the first report of the regulation by Ca²⁺ and Mg²⁺ of native RyR2 receptor activity from healthy human hearts.     

The cloning,expression, and comparative analysis of peroxiredoxin 6 from various sources

Human, rat, Xenopus, and Drosophila (DPx2540 and DPx6005) peroxiredoxin cDNAs were cloned and expressed in Escherichia coli. The recombinant enzymes were compared with respect to enzymatic activity toward various substrates and protection of plasmid DNA from the Fenton reaction products. The activity toward H₂O₂ decreased in the following order: DPx2540 > human Prx6 > Xenopus Prx6 > rat Prx6 > DPx6005. The activity toward tret-butyl hydroperoxide decreased in the following order: DPx2540 = DPx6005 > rat Prx6 > Xenopus Prx6 > human Prx6. The efficiency of plasmid DNA protection from oxidative damage mediated by the Fenton reaction decreased in the order of DPx2540 > DPx6005 = rat Prx6 = human Prx6 > Xenopus Prx6. The optimal temperature for activity of all enzymes was 37°C. Peroxiredoxins from rat, Xenopus, and Drosophila (DPx6005) retained no less than 50% of their activity in a wider temperature range (10–50°C) as compared with the human and Drosophila (DPx2540) enzymes (25–45°C). The thermostability of the enzymes decreased in the following order: DPx6005 = rat > human > Xenopus > DPx2540. The results confirmed a negative correlation between the activity and stability of peroxiredoxin 6, especially in the case of the Xenopus and Drosophila enzymes.     

In Vitro Metabolism of 20(R)-25-Methoxyl-Dammarane-3, 12, 20-Triol from Panax notoginseng in Human,Monkey, Dog,Rat, and Mouse Liver Microsomes

The present study characterized in vitro metabolites of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol (20(R)-25-OCH₃-PPD) in mouse, rat, dog, monkey and human liver microsomes. 20(R)-25-OCH₃-PPD was incubated with liver microsomes in the presence of NADPH. The reaction mixtures and the metabolites were identified on the basis of their mass profiles using LC-Q/TOF and were quantified using triple quadrupole instrument by multiple reaction monitoring. A total of 7 metabolites (M1–M7) of the phase I metabolites were detected in all species. 25(R)-OCH₃-PPD was metabolized by hydroxylation, dehydrogenation, and O-demethylation. Enzyme kinetic of 20(R)-25-OCH₃-PPD metabolism was evaluated in rat and human hepatic microsomes. Incubations studies with selective chemical inhibitors demonstrated that the metabolism of 20(R)-25-OCH₃-PPD was primarily mediated by CYP3A4. We conclude that 20(R)-25-OCH₃-PPD was metabolized extensively in mammalian species of mouse, rat, dog, monkey, and human. CYP3A4-catalyzed oxygenation metabolism played an important role in the disposition of 25(R)-OCH₃-PPD, especially at the C-20 hydroxyl group.     

Isoform selective PLD inhibition by novel,chiral 2,8-diazaspiro[4.5]decan-1-one derivatives

This letter describes the on-going SAR efforts to develop PLD1, PLD2 and dual PLD1/2 inhibitors with improved physiochemical and disposition properties as well as securing intellectual property position. Previous PLD inhibitors, based on a triazaspiro[4.5]decanone core proved to be highly selective PLD2 inhibitors, but with low plasma free fraction (rat, human f_u?<?0.03), high predicted hepatic clearance (rat CL_hep?>?65?mL/min/kg) and very short half-lives in vivo (t_1/2?<?0.15?h). Removal of a nitrogen atom from this core generated a 2,8-diazaspiro[4.5]decanone core, harboring a new chiral center, as well as increased sp³ character. This new core demonstrated enantioselective inhibition of the individual PLD isoforms, enhanced free fraction (rat, human f_u?<?0.13), engendered moderate predicted hepatic clearance (rat CL_hep?～?43?mL/min/kg), improved half-lives in vivo (t_1/2?>?3?h), and led to the first issued US patent claiming composition of matter for small molecule PLD inhibitors.     

Inhibition of lipoprotein-associated phospholipase A2 diminishes the death-inducing effects of oxidised LDL on human monocyte-macrophages

The death of macrophages contributes to atheroma formation. Oxidation renders low-density lipoprotein (LDL) cytotoxic to human monocyte-macrophages. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also termed platelet-activating factor acetylhydrolase, hydrolyses oxidised phospholipids. Inhibition of Lp-PLA2 by diisopropyl fluorophosphate or Pefabloc (broad-spectrum serine esterase/protease inhibitors), or SB222657 (a specific inhibitor of Lp-PLA2) did not prevent LDL oxidation, but diminished the ensuing toxicity and apoptosis induction when the LDL was oxidised, and inhibited the rise in lysophosphatidylcholine levels that occurred in the inhibitors' absence. Hydrolysis products of oxidised phospholipids thus account for over a third of the cytotoxic and apoptosis-inducing effects of oxidised LDL on macrophages.     

Polysaccharide peptides from Coriolus versicolor competitively inhibit tolbutamide 4-hydroxylation in specific human CYP2C9 isoform and pooled human liver microsomes

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited CYP2C11-mediated tolbutamide 4-hydroxylation in the rat both in vitro and in vivo. In this study, the effects of water extractable fraction of PSP on tolbutamide 4-hydroxylation was investigated in pooled human liver microsomes and in specific human CYP2C9 isoform. PSP (2.5-20 μM) dose-dependently decreased the biotransformation of tolbutamide to 4-hydroxy-tolbutamide. Enzyme kinetics studies showed inhibition of tolbutamide 4-hydroxylase activity was competitive and concentration-dependent. In pooled human liver microsomes, PSP had a K_i value of 14.2 μM compared to sulfaphenazole, a human CYP2C9 inhibitor, showed a K_i value of 0.32 μM. In human CYP2C9 isoform, the K_i value of PSP was 29.5 μM and the K_i value of sulfaphenazole was 0.04 μM. This study demonstrated that PSP can competitively inhibit tolbutamide 4-hydroxylation in both pooled human liver microsomes and specific human CYP2C9 in vitro. This study compliments previous findings in the rat that PSP can inhibit human tolbutamide 4-hydroxylase, but the relatively high K_i values in human CYP2C9 would suggest a low potential for PSP to cause herb-drug interaction.     

Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification

In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; M_r 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to M_r 52000 (PLAT Y₁) and M_r 45000 (PLAT Y₂) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y₂ had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y₁- and PLAT Y₂-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y₂ accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y₁ and PLAT Y₂ fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y₁ and PLAT Y₂ for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y₁ and PLAT Y₂. The major glutathione S-transferase from the PLAT Y₂ fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y₂-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.     

Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue

The initiation or progression of periodontitis might involve a local renin-angiotensin system (RAS) in periodontal tissue. The aim of this study was to further characterize the local RAS in human and rat periodontal tissues between healthy and periodontally-affected tissue. Components of the RAS were investigated using in vitro, ex vivo and in vivo experiments involving both human and Wistar rat periodontium. Although not upregulated when challenged with P. gingivalis-lipopolysaccharide, human gingival and periodontal ligament fibroblasts expressed RAS components. Likewise, healthy and inflamed human gingiva expressed RAS components, some of which were shown to be functional, yet no differences in expression were found between healthy and diseased gingiva. However, in inflamed tissue the immunoreactivity was greater for the AT₁R compared to AT₂R in fibroblasts. When compared to healthy tissue, ACE activity was increased in human gingiva from volunteers with gingivitis. Human-gingiva homogenates generated Ang II, Ang 1-9 and Ang 1-7 when incubated with precursors. In gingiva homogenates, Ang II formation from Ang I was nearly abolished only when captopril and chymostatin were combined. Ang 1-7 formation was significantly greater when human gingiva homogenates were incubated with chymostatin alone compared to incubation without any inhibitor, only captopril, or captopril and chymostatin. In rat gingiva, RAS components were also found; their expression was not different between healthy and experimentally induced periodontitis (EP) groups. However, renin inhibition (aliskiren) and an AT₁R antagonist (losartan) significantly blocked EP-alveolar-bone loss in rats. Collectively, these data are consistent with the hypothesis that a local RAS system is not only present but is also functional in both human and rat periodontal tissue. Furthermore, blocking AT₁R and renin can significantly prevent periodontal bone loss induced by EP in rats.     

Synthesis and in vitro activity of N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine P2X(7) antagonists

Synthesis and biological evaluation of a novel class of substituted N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine derivatives resulted in the identification of potent P2X₇ antagonists. These compounds were assayed for activity at both the human and rat P2X₇ receptors. On the benzyl moiety, a variety of functional groups were tolerated, including both electron-withdrawing and electron-donating substituents. Ortho-substitution on the benzyl group provided the greatest potency. The ortho-substituted analogs showed approximately 2.5-fold greater potency at human compared to rat P2X₇ receptors. Compounds 12 and 38 displayed hP2X₇pIC₅₀s >7.8 with less than 2-fold difference in potency at the rP2X₇.     

Human umbilical blood vessel converts all cis-5, 8, 11, 14, 17 eicosapentaenoic acid to prostaglandin I3

All cis-5, 8, 11, 14, 17 eicosapentaenoic acid (EPA) is presented being evaluated for dietary prophylactic use in thrombo-embolic disorders. EPA inhibits the production of TXA₂ and platelet aggregation. We here present results demonstrating that human umbilical arteries convert ¹⁴C- EPA to a substance that in aqueous solutions decomposes to 14C-δ¹⁷-6-keto-PGF_1α. The conversion rate in rat aortic tissue was found substantially lower. These results in combination with earlier data indicating that EPA does not influence the conversion of arachidonic acid (AA) into PGI₂ in human vascular tissue, encourage further research along the lines initiated by the findings of high EPA/AA ratio and low incidence of myocardial infarction in Greenland Eskimos.     

Pharmacological characterization of DPTN and other selective A3 adenosine receptor antagonists

The A₃ adenosine receptor (AR) is emerging as an attractive drug target. Antagonists are proposed for the potential treatment of glaucoma and asthma. However, currently available A₃AR antagonists are potent in human and some large animals, but weak or inactive in mouse and rat. In this study, we re-synthesized a previously reported A₃AR antagonist, DPTN, and evaluated its affinity and selectivity at human, mouse, and rat ARs. We showed that DPTN, indeed, is a potent A₃AR antagonist for all three species tested, albeit a little less selective for mouse and rat A₃AR in comparison to the human A₃AR. DPTN’s K_i values at respective A₁, A_2A, A_2B, and A₃ receptors were (nM) 162, 121, 230, and 1.65 (human); 411, 830, 189, and 9.61 (mouse); and 333, 1147, 163, and 8.53 (rat). Its antagonist activity at both human and mouse A₃ARs was confirmed in a cyclic AMP functional assay. Considering controversial use of currently commercially available A₃AR antagonists in rats and mice, we also re-examined other commonly used and selective A₃AR antagonists under the same experimental conditions. The K_i values of MRS1523 were shown to be 43.9, 349, and 216 nM at human, mouse, and rat A₃ARs, respectively. MRS1191 and MRS1334 showed incomplete inhibition of [¹²⁵I]I-AB-MECA binding to mouse and rat A₃ARs, while potent human A₃AR antagonists, MRS1220, MRE3008F20, PSB10, PSB-11, and VUF5574 were largely inactive. Thus, we demonstrated that DPTN and MRS1523 are among the only validated A₃AR antagonists that can be possibly used (at an appropriate concentration) in mouse or rat to confirm an A₃AR-related mechanism or function.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09823-5.     

Gas chromatographic–mass spectrometric analysis of perillyl alcohol and metabolites in plasma

Perillyl alcohol (POH), a metabolite of d-limonene and a component of the lavender oil, is currently in Phase I clinical trials both as a chemopreventative and chemotherapeutic agent. In vivo, POH is metabolized to less active perillic acid (PA) and cis- and trans-dihydroperillic acids [DHPA, 4-(1′-methylethenyl)-cyclohexane-1-carboxylic acid]. Previous pharmacokinetic studies using a GC–MS method detected POH metabolites but not POH itself; thus these studies lacked information on the parent drug. The present report describes a sensitive GC–MS method for the quantitation of POH and metabolites using stable-isotopically labeled internal standards. The residue obtained from CH₂Cl₂ extraction of a plasma sample was silylated. The products were separated on a capillary column and analyzed by an ion-trap GC–MS using NH₃ chemical ionization. POH-d₃ was used as the internal standard for POH while ¹³C-PA-d₂ was used as the internal standards for the metabolites. The quantitation limits for POH, PA, cis- and trans-DPA were <10 ng/ml using 1–2 ml plasma. The assay was validated in rat and human plasma. The assay was linear from 2 to 2000 ng/ml for POH, 10 to 1000 ng/ml for PA and trans-DHPA, and 20 to 1000 ng/ml for cis-DHPA monitored. The within-run and between-run coefficients of variation were all <8%. Preliminary pharmacokinetic data from a rat following i.v. administration of POH at 23 mg/kg and from a patient receiving POH at 500 mg/m² p.o. was also provided. Intact POH, PA, cis- and trans-DHPA were all detected in plasma in both cases. Two new major metabolites were found in human and one in the rat plasma.