Characterization of the thromboxane synthase pathway product 12-oxoheptadeca-5(Z)-8(E)-10(E)-trienoic acid as a thromboxane A2 receptor antagonist with minimal intrinsic activity.

Thromboxane synthase forms thromboxane (TX) A2 and 12(S)-hydroxyheptadeca-5(Z)-8(E)-10(E)-trienoic acid (HHT) at equimolar amounts. Twelve-oxoheptadeca-5(Z)-8(E)-10(E)-trienoic acid (Oxo-HT) is the primary metabolite of HHT and has been described to be an inhibitor of platelet aggregation. Functional studies, Schild analysis and competitive binding studies were performed to clarify its mode of action. Oxo-HT was prepared biosynthetically as well as chemosynthetically, purified and characterized by gas chromatography and mass spectrometry. Platelet activation was assessed by determination of shape change, aggregation, fibrinogen binding and P-selectin expression using optical aggregometry and flow cytometry. Oxo-HT 0.1 nM to 50 microM did not induce platelet activation. Furthermore, it had no effect on platelet activation induced by thrombin, ADP or PAF. In contrast, Oxo-HT inhibited platelet aggregation, fibrinogen binding and P-selectin expression induced by U46619 in a competitive manner. Schild analysis for U146619-induced fibrinogen binding and P-selectin expression revealed pA2 values of 6.1 and 6.6, respectively, which correspond to Kd values of approximately 0.8 microM and 0.3 microM, respectively. Oxo-HT also inhibited U46619 induced shape change (IC50 is approximately equal to 10 microM). However, Oxo-HT over a concentration range of 0.1-1 microM enhanced the partial shape change induced by low concentrations of U46619. Thus Oxo-HT seems to possess a minimal agonistic potential, which alone is not sufficient to trigger a platelet activation but can enhance low levels of platelet activation. Oxo-HT blocked the binding of [3H]SQ 29548 in a concentration-dependent manner, whereas HHT did not displace [3H]SQ 29548. The Kd of Oxo-HT determined from competition binding studies was 7.7 microM, about 10-25-fold higher than the apparent Kd determined by Schild analysis. This discrepancy might be due to a desensitization of the TXA2 receptor triggered by the minimal intrinsic activity of Oxo-HT. We conclude that Oxo-HT is a naturally occurring specific TXA2 receptor antagonist with minimal intrinsic activity. Oxo-HT may contribute to the regulation of TXA2-induced platelet activation in vivo.

Mechanism of cytochrome P450 2D6-catalyzed sparteine metabolism in humans.

Two different reaction mechanisms for the formation of the two human enamine-structured sparteine metabolites by cytochrome P450 2D6 have been discussed in the literature. These mechanisms are either initial one-electron oxidation of N1 of sparteine followed by deprotonation of the aminium radical cation, resulting in the formation of different carbon radicals and oxygen rebound of the carbon radicals, or oxidation of the carbon atoms adjacent to N1 by the enzyme, directly producing the respective carbon radicals. With a spectrum of deuterium-labeled isotopomers of sparteine, stereoselectivity and kinetic isotope effects of human sparteine metabolism were investigated by in vitro and in vivo experiments and were compared with chemical oxidation of 17-oxosparteine. These experiments revealed that the major human sparteine metabolite 2,3-didehydrosparteine is formed via highly stereoselective abstraction of the 2 beta-hydrogen atom; the deuterium label was completely retained during metabolism when 2R-[2H]sparteine was used as substrate. Chemical oxidation of 17-oxosparteine by Ce4+, as a model for one-electron oxidation of N1 of a sparteine-like structure, resulted in the sole formation of the 5,6-unsaturated enamine, and no 2,3-unsaturated enamine, structurally equivalent to the human major metabolite, was found. An unequivocal discrimination between the two possible reaction mechanisms was not possible by simple interpretation of the magnitude of the kinetic deuterium isotope effects. However, results of competitive and noncompetitive experiments revealed the presence of a nondissociative enzymatic mechanism for the formation of the two sparteine metabolites, i.e., the sparteine molecule that is bound to the substrate binding site of cytochrome P450 2D6 performs orientational changes without dissociating from the activated enzyme/substrate complex before the product-determining first irreversible reaction step. These results agree with the hypothesis that sparteine metabolism proceeds by direct carbon oxidation. Because electron transfer from amines to P450 may occur over some distance, the possibility of a sequential electron-proton transfer reaction during sparteine metabolism cannot be ruled out completely as an alternative reaction mechanism for sparteine metabolism.

Determination of prostaglandin E1 and its main plasma metabolites 15-keto-prostaglandin E0 and prostaglandin E0 by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry.

Prostaglandin E1 (PGE1), 15-keto-PGE0 and PGE0 in plasma were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of deuterated internal standards, the prostaglandins were extracted by a solid-phase cartridge and derivatized to the pentafluorobenzyl ester methoxime. The samples were purified by thin-layer chromatography, converted to the trimethylsilyl ethers and quantified by gas chromatography/triple-stage quadrupole mass spectrometry. The parent ions in the negative ion chemical ionization mode were [M-pentafluorobenzyl]- ([P]-), the daughter ions used for quantification were [P-(CH3)3SiOH]- (PGE0 and 15-keto-PGE0) and [P-2(CH3)3SiOH]- (PGE1), respectively. Plasma concentrations in healthy subjects were at about 1-3 pg ml-1 for PGE1 and PGE0 and 2-15 pg ml-1 for 15-keto-PGE0. After infusion of 60 micrograms PGE1 in 2 h, the concentrations in plasma were 3-10 pg ml-1 for PGE1, 8-17 pg ml-1 for PGE0 and 115-205 pg ml-1 for 15-keto-PGE0.

Rapid determination of CYP2D6 phenotype during propafenone therapy by analysing urinary excretion of propafenone glucuronides.

Metabolism of the antiarrhythmic, propafenone, cosegregates with the sparteine/debrisoquine polymorphism. Patients devoid of CYP2D6 activity have a higher incidence of adverse effects than those with normal enzyme function. In this paper we present a method for rapid assignment of CYP2D6 phenotype using urinary excretion of intact glucuronides of propafenone (PPFG). After establishing an HPLC assay, urinary excretion of PPFG was quantified during one dosage interval and related to individual CYP2D6 activity as determined by phenotyping. We observed a close correlation of urinary excretion of PPFG with individual CYP2D6 activity (r = 0.84, P < 0.01) and conclude that this method is suitable for rapid assignment of phenotype during propafenone therapy.

Re-evaluation of the metabolism of carbocisteine in a British white population.

It has been claimed that the amino acid derivative carbocisteine is predominantly metabolized by sulfoxidation and that this pathway exhibits a genetic polymorphism. Moreover, those subjects with a 'poor metabolizer' phenotype have been thought to have a genetic predisposition to developing certain diseases. We have confirmed the observations of others that this marker drug does not undergo significant S-oxidation. Furthermore, a novel urinary metabolite, S-(carboxymethylthio)-L-cysteine (CMTC) has recently been identified. To determine if a genetic polymorphism for this biotransformation pathway exists, metabolic ratios (% urinary excretion carbocisteine/% urinary excretion CMTC) for 120 healthy volunteers were assessed using high-performance thin-layer chromatography. Urinary excretion of the parent drug ranged from 6% of the dose administered to 56% (mean +/- SD, 23.4 +/- 0.8%). No cysteinyl sulfoxide metabolites were identified in the urine samples. The amount excreted as CMTC exhibited a 12-fold variation but only accounted for mean of 4.4% (1-12%) of the dose given. Two individuals initially had high metabolic ratios (> 30), however, on rechallenge both their MRs were less than 5. Therefore, carbocisteine is not an appropriate probe drug for sulfoxidation. The formation of the novel metabolite CMTC appears to exhibit polymorphism, although the considerable intra-subject variation for its formation does not allow assignment of a phenotype.

Evaluation of deuterated cholesterol and deuterated sitostanol for measurement of cholesterol absorption in humans.

The continuous isotope feeding method of Crouse and Grundy (1978. J. Lipid Res. 19: 967-971) for measurement of dietary cholesterol absorption has been modified by using markers labeled with stable isotopes ([2,2,4,4,6-2H5]cholesterol or [25,26,26,26,27,27,27-2H4]cholesterol or [26,26,26,27,27,27-2H6] cholesterol and [5,6,22,23-2H4]sitostanol) quantified by gas-liquid chromatography-selected ion monitoring. Tracing of the isotope distribution of the authentic markers and after their intestinal passage, including the microbiological products (coprostanol and coprostanone) revealed stability of the labels. The new method was evaluated in six monkeys on two occasions by comparison with the original method using radioactively labeled cholesterol and sitosterol. The results obtained by the two different methods were in excellent agreement, and absorption ranged from 49% to 73% (mean 60%) for the stable isotope method and from 51% to 69% (mean 62%) for the radioactive method. The coefficient of variation of cholesterol absorption in animals ranged from 3.9% to 15.1% (mean 7.1%) for stable isotopes and 1.9% to 13.6% (mean 5.7%) for radioactive isotopes. In twelve subjects cholesterol absorption was measured by the new method from total fecal samples frozen immediately and compared to results obtained from small fecal aliquots (approximately 1 g) sent by ordinary mail to the laboratory. A significant correlation of cholesterol absorption between the two different sample handlings was obtained (r = 0.981, P < 0.001). In addition, measurement of cholesterol absorption twice in seven volunteers 2 weeks apart revealed identical results. Thus, the new method is extremely safe and reproducible without radioactive exposure to the subjects and labortory staff and can be used on women of child-bearing age.

Mass determination of 15-hydroxyprostaglandin dehydrogenase from human placenta and kinetic studies with (5Z, 8E, 10E, 12S)-12-hydroxy-5,8,10-heptadecatrienoic acid as substrate.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the first step in the metabolism of prostaglandins which is usually associated with physiological inactivation. A highly purified homogenous enzyme preparation from human placenta was used to determine the molecular mass and lack of quaternary structure of the enzyme. Furthermore we have examined enzyme kinetics of the purified enzyme with (5Z,8E,10E,12S)-12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) an equimolar coproduct of thromboxane biosynthesis. Using gel electrophoresis and gel filtration on FPLC, we could estimate a molecular mass of 28 +/- 1 kDa, indicating that the enzyme consists of one single protein chain. The exact molecular mass of the monomer was calculated by matrix-assisted laser desorption/ionization mass spectrometry to 28740 +/- 30 Da. (5Z,8E,10E)-12-oxo-5,8,10-heptadecatrienoic acid (oxo-HT) could be identified as the only product obtained from the enzymatic reaction with HHT. Quantification of this metabolite was achieved by gas chromatography/tandem mass spectrometry. The calculated enzyme kinetic constants for the formation of the metabolic product [Km (HHT) = 9.68 microM, Vi = 12.78 mU/micrograms] were in agreement with those determined for NADH formation (Km = 7.65 microM, Vi = 11.79 mU/micrograms). This demonstrates that HHT shows high affinity to the enzyme which is comparable to prostaglandin E2 (PGE2). As the product oxo-HT is a potent inhibitor of platelet aggregation, dehydrogenation of HHT might represent a biological activation step.

Quinidine inhibition of debrisoquine S(+)-4- and 7-hydroxylations in Chinese of different CYP2D6 genotypes.

Pronounced differences in the CYP2D6 gene between Chinese and Caucasians have previously been described. There was a low frequency of detrimental mutations in the Chinese CYP2D6 gene causing the poor metabolizer (PM) phenotype. In contrast to Caucasians where the Xba I 44 kb allele is almost always associated with the PM phenotype, Chinese with the 44/44 kb RFLP pattern are extensive metabolizers (EM). In order to evaluate whether the debrisoquine hydroxylation seen in subjects with this haplotype is catalysed by a functionally similar enzyme to CYP2D6 or is catalysed by another type of P450 isozyme, product selectivity of the 4-hydroxylation was studied in 27 Chinese. The inhibition of CYP2D6 by quinidine was also investigated. In the 26 Chinese EM the S(+)-4-hydroxy enantiomer was found to be the major urinary metabolite of debrisoquine with an enantiomeric excess of 96.8-100%, which is similar to that in Caucasians. A correlation between the amount of S(+)-4-hydroxy and the minor 7-hydroxy metabolites excreted in urine (r = 0.72; p < 0.001) was seen. The amount of these two metabolites excreted was less in Chinese EM of debrisoquine with the 44/44 kb RFLP pattern, than in those with the wild type 29/29 kb pattern (p < 0.01). The stereoselectivity was very high in both groups. All Chinese homozygous for the 44 kb fragment (n = 5) were transformed to apparent PM after a single 100 mg dose of quinidine similarly to five Caucasian EM. Both the S(+)-4- and 7-hydroxylations of debrisoquine were inhibited by quinidine in both populations. This study shows that the cytochrome P450 catalysing the 4- and 7-hydroxylations of debrisoquine in Chinese EM has the same properties (product stereoselectivity and inhibition by quinidine) as the CYP2D6 in Caucasian EM.

A nuclear magnetic resonance study of sparteine delta metabolite structure.

Originally, the enamines 2,3- and 5,6-didehydrosparteine (2a and 3a, respectively) had been characterized by GC/MS as metabolites after administration of (-)-sparteine sulfate (1a.H2SO4). Since the existence of free enamines in aqueous medium seemed rather doubtful, the metabolism of (-)-sparteine was reinvestigated by high-resolution 1H-, 2H-, and 13C-NMR spectroscopy. When synthetic 1,2-didehydrosparteinium monoperchlorate (4a.ClO4) is dissolved in aqueous medium at pH 4-9, sterically uniform (2S)-hydroxysparteine (6a) is formed, as is proven unequivocally by 1H- and 13C-NMR. However, at pH less than or equal to 2, this carbinolamine eliminates water under reconstitution of the iminium structure 4a. No carbinolamine is formed, in contrast, from synthetic 1,6-didehydrosparteinium monoperchlorate (5a.ClO4). Upon oral application of [2R-2H]sparteine (1b), as the sulfate, the respective carbinolamine and iminium structures 6b and 5b are identified in the urines by 2H-NMR spectroscopy. Reversed-phase TLC likewise confirms the different structural principle of the two main sparteine metabolites. The free enamine bases, characterized by GC/MS, thus must be regarded as artifacts, formed in the work-up requisite for GC analysis.

Enantiomer/enantiomer interaction of (S)- and (R)-propafenone for cytochrome P450IID6-catalyzed 5-hydroxylation: in vitro evaluation of the mechanism.

Many drugs are used as racemates, and the enantiomers may differ in terms of pharmacological properties and disposition. Stereoselective disposition of the enantiomers can arise from metabolism of the enantiomers via different routes catalyzed by different enzymes. In contrast, the enantiomers may be metabolized by the same enzyme at different rates. In the latter case, the enantiomers can compete for this metabolic step, giving rise to the possibility of an enantiomer/enantiomer interaction. We have chosen the antiarrhythmic propafenone, for which in vivo data indicated an interaction between (S)- and (R)-propafenone, as a model substance to study the mechanism underlying that interaction in human liver microsomes. We used the cytochrome P450IID6-mediated 5-hydroxylation of propafenone as a model pathway, because this metabolic step constitutes the major route of biotransformation of propafenone. The Michaelis-Menten kinetics for 5-hydroxylation were determined after incubation of (R)- and (S)-propafenone and a pseudoracemate consisting of (S)-[2H4]propafenone and (R)-propafenone. Inhibition experiments were performed using (S)-[2H4]propafenone as an inhibitor of the 5-hydroxylation of (R)-propafenone, and vice versa. The kinetic model of mixed alternative substrates was used to simulate inhibition experiments. Experimental data were compared with those predicted by this model. We observed a substantial stereoselectivity after incubation of the individual enantiomers [(S)-propafenone: Vmax, 10.2 pmol/micrograms/hr, and Km, 5.3 microM; (R)-propafenone: Vmax, 5.5 pmol/micrograms/hr, and Km, 3.0 microM]. In contrast, no substrate stereoselectivity was observed after incubation of the pseudoracemate [3.1 pmol/micrograms/hr for (S)-[2H4]propafenone and 3.3 pmol/micrograms/hr for (R)-propafenone]. Application of the model revealed Ki values of 2.9 and 5.2 microM for the inhibition of 5-hydroxylation of (S)-[2H4]-propafenone by (R)-propafenone and for inhibition of 5-hydroxylation of (R)-propafenone by (S)-[2H4]-propafenone, respectively. The predicted and the experimental data were in good agreement, and both indicated the mode of inhibition to be competitive. In conclusion, the enantiomers of propafenone interact with respect to 5-hydroxylation, with (R)-propafenone being a more potent inhibitor than the S-enantiomer with respect to cytochrome P450IID6-mediated 5-hydroxylation. Because beta-blocking properties of propafenone reside in the S-enantiomer, inhibition of metabolism of this enantiomer by (R)-propafenone may have therapeutic consequences.

Regioselectivity and stereoselectivity of the metabolism of the chiral quinolizidine alkaloids sparteine and pachycarpine in the rat.

1. The metabolism of (-)-sparteine and (+)-sparteine (pachycarpine) was investigated in male Sprague-Dawley rats by g.l.c.-mass spectrometry, and 13C- and 2H-n.m.r. spectroscopy. The structure of the major metabolite of (-)-sparteine was confirmed to be 2,3-didehydrosparteine by g.l.c.-mass spectrometry after alkaline sample work-up. 2H-n.m.r. spectroscopy showed that this metabolite exhibits the structure of the carbinolamine (2S)-hydroxysparteine in aqueous solution of neutral pH. No other metabolites with an enamine structure were observed by g.l.c.-mass spectrometry and 13C-n.m.r. spectroscopy. 2. Pachycarpine is metabolized in vivo and in vitro stereoselectively to the aliphatic alcohol (4S)-hydroxypachycarpine as the main metabolite. 3. The formation of the 2,3-didehydrosparteine proceeds via stereospecific abstraction of the axial 2 beta hydrogen atom. Inhibition in vitro studied with purified rat liver microsomes demonstrated that both sparteine enantiomers are metabolized by the same cytochrome P450 isozyme. Therefore this enzyme exhibits marked substrate and product stereoselectivity for the metabolism of the two enantiomeric quinolizidine alkaloids.

Smoking alters thromboxane metabolism in man.

Chronic smoking is a major risk factor of atherosclerosis and coronary heart disease. The measurement of three major thromboxane A2 metabolites, 11-dehydrothromboxane B2, 2,3-dinorthromboxane B2 and thromboxane B2, in the urines of 13 apparently healthy smokers (average 39 years, range 27-56 years) showed significantly elevated excretion rates for all thromboxane A2 metabolites as compared to 10 apparently healthy age-matched non-smokers (average 37 years, range 26-56 years). Importantly, characteristic alterations in the thromboxane A2 metabolite pattern were found in the urines of smokers. The contribution of 2,3-dinorthromboxane B2 to total measured excretion of thromboxane A2 metabolites was 59.2% in smokers (404.0 +/- 53.0 pg/mg creatinine) versus 19.4% in non-smokers (85.2 +/- 8.3 pg/mg creatinine), that of 11-dehydrothromboxane B2 35.7% in smokers (673.2 +/- 88.9 pg/mg creatinine) as compared to 75.5% in non-smokers (332.6 +/- 30.9 pg/mg creatinine). The contribution of thromboxane B2 (57.5 +/- 7.7 pg/mg creatinine in smokers versus 21.9 +/- 1.5 pg/mg creatinine in non-smokers) was similar at 5.1%. The excretion of cotinine, the major urinary metabolite of nicotine that correlates well with the reported daily cigarette consumption (r = 0.97, P less than 0.0001), showed a good correlation to thromboxane A2 metabolite excretion (2,3-dinorthromboxane B2: r = 0.92, P less than 0.0001; 11-dehydrothromboxane B2; r = 0.87, P less than 0.0001).

Identification of thiodiglycolic acid, thiodiglycolic acid sulfoxide, and (3-carboxymethylthio)lactic acid as major human biotransformation products of S-carboxymethyl-L-cysteine.

S-Carboxymethyl-L-cysteine (CMC) is used both as an orally administered mucolytic agent and as a probe drug for uncovering polymorphic sulfoxidation of other sulfur-containing drugs in humans. However, several recent studies could not confirm the formation of significant amounts of urinary sulfoxides of CMC or its decarboxylation product S-methyl-L-cysteine. The metabolism of CMC and a 13C-labeled isotopomer was therefore reinvestigated in 11 and 14 humans, respectively, and emphasis was laid on monitoring of potential alternative metabolic pathways. Combined capillary gas chromatography/electron impact or negative-ion chemical ionization mass spectrometry employing stable isotope-labeled analogues as internal standards were used for identification and quantification of CMC metabolites in human urine. Three nitrogen-free metabolites that were identified as thiodiglycolic acid (TDGA, mean: 19.8% of the dose/24 hr), thiodiglycolic acid sulfoxide (TDGA-SO, mean: 13.3% of the dose/24 hr), and (3-carboxymethylthio)lactic acid (TLA, mean: 2.1% of the dose/8 hr), cumulatively account for about one-third of the dose during a urinary collection period of 24 hr. In addition, trace amounts of both TDGA and TLA exist as endogenous components in urine from persons not administered exogenous CMC at levels of about 5 and 1 nmol/ml, respectively. Both major metabolites TDGA and TDGA-SO, that were not considered in previous sulfoxidation phenotyping, are predominantly excreted after 8 hr. These results demonstrate the existence of a pyruvate-like metabolic pathway and suggest the necessity of a revision of the hitherto accepted biotransformation route of CMC in humans.

Syntheses of metabolites of S-carboxymethyl-L-cysteine and S-methyl-L-cysteine and of some isotopically labelled (2H, 13C) analogues.

The chemical syntheses of human metabolites of S-carboxymethyl-L-cysteine (3) and S-methyl-L-cysteine (12) are described. The additional preparation of some 2H- and 13C-labelled isotopomers enabled the direct evaluation of the stabilities of 3 and 12 under physiological conditions and also facilitated the unambiguous assignments of the signals in the 13C-NMR spectra of all compounds mentioned.

Tracing the human metabolism of stable isotope-labelled drugs by ex vivo NMR spectroscopy. A revision of S-carboxymethyl-L-cysteine biotransformation.

A direct structural identification, and quantitative assessment below the 50 nmol/ml level, of the full pattern of renally excreted metabolites is made possible by 13C NMR measurements of untreated urine samples when stable isotope-labelled (13C) drug analogues are administered to humans. The full potential of the new ex vivo NMR approach is exemplified by a study, for a group of volunteers, of S-carboxymethyl-L-cysteine metabolism. The metabolic sulphoxidation pathway of S-carboxymethyl-L-cysteine in man, accepted so far, needs to be profoundly revised on the basis of the 13C NMR results.

Measurement of 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid and its metabolite 12-oxo-5Z,8E,10E-heptadecatrienoic acid in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry.

Thromboxane A2, the predominant product of arachidonic acid metabolism in the blood platelet, is a potent vasoconstrictor and platelet agonist. During its biosynthesis from cyclic endoperoxide, 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (HHT) is formed in equal amounts. The further metabolism of HHT, catalyzed by 15-hydroxyprostaglandin dehydrogenase, leads to 12-oxo-5Z,8E,10E-heptadecatrienoic acid (Oxo-HT). Sample workup procedures are described which allow for the sensitive and reproducible determination of these two arachidonic acid metabolites in human plasma by gas chromatography-mass spectrometry in the presence of deuterated analogues as internal standards. HHT is derivatized to the pentafluorobenzyl ester tert-butyldimethylsilyl ether. In order to enable quantification of low concentrations of about 10 pg/ml in nonstimulated human plasma, the samples have to be purified by HPLC. Oxo-HT is derivatized to the pentafluorobenzyl ester, which is purified by HPLC, and then derivatized to the trimethylsilyloxime. The method allows quantification of Oxo-HT in concentrations down to 10 pg/ml plasma. The reported methods have been used to measure HHT and Oxo-HT in stimulated platelet rich plasma and to quantify HHT in nonstimulated plasma. Determination of endogenous levels of these two arachidonic acid metabolites may give new insights into the overall biosynthesis of thromboxane A2 in man.

Determination of 11 alpha-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostan oic acid and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoi c acid by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry.

11 alpha-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostano ic acid (PGE-M) and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGF-M) in urine were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of the 2H7-labeled internal standard, O-methylhydroxylamine hydrochloride in acetate buffer was added either directly (PGE-M) or after standing overnight at pH 10 (PGF-M) to form the methoxime. The sample was acidified to pH 2.5 and PGE-M and PGF-M were extracted with ethyl acetate/hexane. Then the prostanoids were derivatized to the pentafluorobenzyl ester and purified by thin-layer chromatography and the trimethylsilyl ether was formed. The products were quantified by gas chromatography/triple-stage quadrupole mass spectrometry. For PGE-M, the fragment ions m/z 349 and m/z 356 (2H7 standard) (daughter ions of m/z 637 and m/z 644 (2H7 standard] were used. The results of the PGE-M assay were compared with those of an assay using the [2H3]methoxime as the internal standard. For determination of PGF-M, the daughter ions m/z 484 and m/z 491 (2H7 standard) with the parent ions m/z 682 and m/z 689 (2H7 standard) were chosen.

Unusual metabolism of prostacyclin in infants with persistent septic pulmonary hypertension.

In urine of healthy man, the major metabolite of prostacyclin is 2,3-dinor-6-oxo-prostaglandin F1 alpha. The excretion rates of this compound as well as of 2,3-dinor-thromboxane B2, a major metabolite of thromboxane A2, in two newborns with septic persistent pulmonary hypertension were about 30- to 50-fold higher than the normal range (2,3-dinor-6-oxo-prostaglandin F1 alpha: 3-15 ng/h/1.73 m2; 2,3-dinor-thromboxane B2: 8-25 ng/h/1.73 m2). The ratios of 2,3-dinor-6-oxo-13,14-dihydro-prostaglandin F1 alpha/2,3-dinor-6-oxo-prostaglandin F1 alpha in these two infants were about 100% and 800%, respectively whereas in controls the excretion of the 13,14-dihydro metabolite was found to be about 10-25% of 2,3-dinor-6-oxo-prostaglandin F1 alpha. Thus in patients with septic persistent pulmonary hypertension and extremely high excretion rates of prostacyclin and thromboxane A2 metabolites, the pattern of metabolites differs from those of healthy man.