Biomedical subjects
Publications and source records attributed to C O Meese.
Pachycarpine (4), the optical antipode of the lupine alkaloid (-)-sparteine (1), has been prepared from (-)-lupanine; its metabolism was studied in rats. After isolation and chromatographic purification, streochemically homogeneous (+)-(4S)-hydroxysparteine (7) was identified as the major urinary metabolite by use of mass spectrometry and high-field NMR-spectroscopy.
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Severe forebrain ischemia was produced in rats by occluding both carotid and vertebral arteries. Following 30 min ischemia brains were recirculated for 8 or 24 h. Twelve animals subjected to 8 or 24 h recirculation (n = 6, each group) were given alpha-difluoromethylornithine (DFMO; injected intraperitoneally) immediately before recirculation. At the end of the experiments brains were frozen and samples were taken from the cerebellum, cortex, caudatoputamen and hippocampus. Samples from the left hemisphere were used for measuring ornithine decarboxylase (ODC) activity, and those from the right hemisphere for determining putrescine profiles. During recirculation ODC activity increased markedly in all brain structures, the most pronounced change being in the caudatoputamen after 8 h recirculation. Putrescine increased drastically after 8 h and even more after 24 h recirculation. DFMO-treatment significantly reduced ODC activity after 8 h recirculation and following 24 h recirculation. Putrescine, however, was significantly reduced following 24 h but not after 8 h recirculation. The discrepancy between reduction in ODC activity and putrescine levels in DFMO-treated animals was most prominent in the hippocampus after 8 h recirculation: here DFMO reduced ODC activity to control values without affecting putrescine levels. The results suggest that the observed overshoot in putrescine formation following ischemia is only partly caused by activation of ODC.
Thirty ml of an emulsion containing 3 g of trieicosapentaenoyl-glycerol (90% pure, containing 5% arachidonic acid (AA)) was infused intravenously in 2 male healthy volunteers. Urine samples were collected for 24 h before and 48 h after the infusion in 5 periods. Urinary metabolites of prostaglandin (PG) I2/3 and thromboxane (TX) A2/3 (PGI2/3-M and TXB2/3-M, respectively) were extracted from the urinary samples and measured by GC-MS. Excretion of PGI3-M was markedly enhanced right after the infusion. Because PGI3 was produced without involvement of intestinal absorption of eicosapentaenoic acid (EPA), enhanced PGI3 formation was strongly suggested to take place in the vasculature. From the marked increment in TXB2/3-M after the infusion it was calculated that conversion rate of EPA to TXA3 was 8% of that of AA to TXA2 in this in vivo condition.
Electron impact mass spectra of methyl ester/trimethylsilyl ether derivatives of prostaglandin F2 alpha and methyl ester/methoxime/trimethylsilyl ether derivatives of prostaglandin E2, prostaglandin D2, 6-oxo-prostaglandin F1 alpha and 2,3-dinor-6-oxo-prostaglandin F1 alpha are presented. Most of the prostaglandins studied have additionally been 2H-labelled at different sites in order to assign the corresponding fragment ions. Collisionally activated decomposition mass spectra of the most intense parent ions in the high-mass region were taken. High-intensity, prostaglandin-characteristic daughter fragments will allow a reliable quantification of prostaglandins in biological fluids and a reduction of sample clean-up.
Electron impact mass spectra of methyl ester/methoxime/trimethylsilyl ether derivatives of thromboxane B2 (TxB2) and 2,3-dinor-TxB2 and methyl ester/trimethylsilyl ether derivative of 11-dehydro-TxB2 are presented. Additionally, the derivatives of (2H4)-thromboxanes and methyl ester (2H3)-methoxime/trimethylsilyl ether derivatives of TxB2 and 2,3-dinor-TxB2 and (2H3)-methyl ester/trimethylsilyl ether derivative of 11-dehydro-TxB2 were investigated. Collision-induced dissociation mass spectra of the most intense parent ion in the high-mass region were taken. Collisionally activated decomposition mass spectra of the [C(12)-C(20)]+ ion of TxB2 and 2,3-dinor-TxB2 show an intense but not specific daughter ion, whereas the collision-induced dissociation mass spectrum of the [M-(C(16)-C(20)]+ ion of 11-dehydro-TxB2 results in the formation of numerous daughter ions.
Negative ion chemical ionization mass spectra of prostaglandin F2 alpha, prostaglandin E2, prostaglandin D2, 6-oxo-prostaglandin F1 alpha, 2,3-dinor-6-oxo-prostaglandin F1 alpha, thromboxane B2, 2,3-dinor-thromboxane B2, and 11-dehydro-thromboxane B2 pentafluorobenzyl ester (PFB)/methoxime/trimethylsilyl ether derivatives are presented. Collisionally activated decomposition mass spectra of the [M-PFB]- ions at collision energies of 8-24 eV and argon collision gas pressures of 1-2m Torr almost show only fragmentation of trimethylsilanol, (CH3)2Si = CHOH, (CH3)2Si = CH2 and methanol whereas, except for carbon dioxide loss, only few low-intensity fragments from the carbon skeleton of the prostanoids are observed.
A stable isotope assay for the quantification of debrisoquine (1) and its major urinary metabolite 4-hydroxydebrisoquine (2) is described. The method consists of extractive derivatization of 1 and 2 by use of 1,3-diketones, chiral derivatization of the 4-hydroxy group of 2, and gas chromatography/negative ion chemical ionization mass spectrometry in the presence of deuterated analogues of 1 and 2. In comparison with synthetic R-(-)-2 and S-(+)-2 it is shown that in vivo benzylic 4-hydroxylation of 1 is highly stereoselective, leading predominantly to S-(+)-4-hydroxydebrisoquine (enantiomeric excess greater than or equal to 90%).
Debrisoquine (DQ) has no chiral centre, but hydroxylation in position 4 leads to formation of an asymmetric carbon centre with two possible enantiomers, their absolute configuration being R(-) and S(+)-4-hydroxydebrisoquine (4-OHDQ). Since the absolute stereochemistry of the 4-hydroxylation of DQ in man is unknown, the enantioselectivity of this process was studied in panels of extensive (EM) and poor metabolizers (PM) of DQ. In EM subjects 4-hydroxylation of DQ leads almost exclusively to the formation of S(+)-4-OHDQ. In contrast, PM subjects were not only characterized by a decreased total 4-OHDQ formation but also a marked loss of enantioselectivity in product formation. Between 5 to 36% of total 4-OHDQ was excreted as R(-)-4-OHDQ.
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The metabolism of [1-14C]arachidonic acid by microsomal and purified prostaglandin (PG) H synthase was investigated. HPLC analysis confirmed that arachidonic acid (20:4) was extensively converted into prostaglandin G2 (PGG2) and/or prostaglandin H2 (PGH2) but several minor labelled products were formed in addition. Their formation, mediated by PGH synthase was established by inhibition with aspirin and indomethacin [Hecker, M., Hatzelmann, A. & Ullrich, V. (1987) Biochem. Pharmacol. 36, 851-855]. Upon comparison with authentic reference material these unknown PGH synthase metabolites were identified with respect to chromatographic properties, ultraviolet spectroscopy and mass spectrometry as 11 (R)-hydroperoxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11-OOH-20:4), 12(S)-hydroperoxy-5Z,8E,10E-heptadecatrienoic acid (OOH-17:3), 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (OH-17:3), 15(RS)-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoi c acid (15-OOH-20:4), 15(RS)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-OH-20:4), 13-hydroxy-5Z,14Z-prostaglandin H2, 15(S)-hydroxy-8-iso-5Z,13E-prostaglandin H2 and 15-oxo-prostaglandin H2. Unlike PGG2 and PGH2, 8-iso-PGH2, 13-hydroxy-PGH2 and 15-oxo-PGH2 failed to induce aggregation of washed human platelets and to form thromboxane upon incubation with homogeneous human platelet thromboxane synthase. In contrast to the formation of OOH-17:3, 15-oxo-PGH2 and OH-17:3 which can be attributed to the heme-catalyzed decomposition of PGG2 and PGH2, 11-OOH-20:4,15-(O)OH-20:4-,8 iso-PGH2 and 13-hydroxy-PGH2 represent potential side products of arachidonic acid conversion into PG endoperoxides. Their formation allows to conclude on PGH synthase mechanism and its intermediates for which an extended reaction scheme is proposed.
11-Dehydrothromboxane B2 is one of the major enzymatic metabolites of thromboxane B2 (TXB2), a biologically inactive product of thromboxane A2. The short half-life of thromboxane A2 and ex vivo production of thromboxane B2 by platelet activation make these prostanoid metabolites inappropriate as indices of systemic thromboxane biosynthesis, whereas 11-dehydro-TXB2 has been shown to reflect the release of thromboxane A2 in the human blood circulation. Analysis of 11-dehydro-TXB2 in plasma and urine was performed by gas chromatography-mass spectrometry-mass spectrometry using the chemically synthesized tetradeuterated compound as an internal standard. The high selectivity of triple-stage quadrupole mass spectrometry (tandem mass spectrometry) considerably facilitates sample purification as compared to single quadrupole mass spectrometric determination. Plasma concentrations in five healthy male volunteers were in the range 0.8-2.5 pg/ml. Urinary excretion of 11-dehydro-TXB2 was higher than that of 2,3-dinor-TXB2: 1.2 +/- 0.36 micrograms/24 h vs 0.53 +/- 0.33 micrograms/24 h (n = 5). Thus 11-dehydro-TXB2 appears at present to be the best index metabolite of systemic TXA2 activity in plasma as well as in urine.
The ex vivo stability of leukotrienes (LT) C4 and D4 in human urine was tested in an experimental model. The various LTs were identified/measured by HPLC, liquid scintillation counting, RIA, UV-absorbance and GC-MS. If fresh urine was incubated under physiological conditions added LTC4 was converted in a time-dependent manner to LTD4 (t1/2 about 0.5 h) and to LTE4 (t1/2 about 6 h), respectively. At the end of the 22 h incubation period less than 10% of the initial LTC4 was detectable. LTB4 was stable under these conditions. Therefore the enzymatic urinary metabolism of LTs should be considered in experimental and clinical studies when urine is collected under different conditions.
The present study was designed to investigate the effects of salicylate on the antiplatelet action of acetylsalicylic acid as well as on in vivo prostanoid formation and platelet function in healthy volunteers. In the first study six female volunteers received 350 mg acetylsalicylic acid intravenously, with and without previous oral administration of sodium salicylate (1200 mg daily for 3 days). Urinary prostanoid excretion as well as platelet aggregation and thromboxane formation were measured before and during salicylate and after acetylsalicylic acid. In the second study seven female volunteers received sodium salicylate (52.6 mg kg-1) or acetylsalicylic acid (60.7 mg kg-1) for 8 days in a randomized cross-over protocol. Urinary prostanoid excretion, platelet aggregation and thromboxane formation as well as salicylate plasma concentrations were determined before, during and after administration of each drug. Sodium salicylate did not impair the complete suppression of arachidonic acid-induced platelet thromboxane formation and aggregation obtained by the single intravenous dose of acetylsalicylic acid in the first study. Sodium salicylate in the second study did not affect urinary excretion of prostaglandin E2, its major urinary metabolite (7 alpha-hydroxy-5,11-diketo-tetranor-prostane-1,16-dioic acid), and 2,3-dinor-6-keto-prostaglandin F1 alpha, the main urinary metabolite of epoprostenol (prostacyclin, PGI2). In contrast, acetylsalicylic acid significantly decreased excretion rates of these prostanoids by 64, 59 and 61%, respectively. In both studies platelet aggregation and thromboxane formation induced by collagen, thrombin or arachidonic acid were not significantly affected by salicylate administration, whereas acetylsalicylic acid inhibited platelet aggregation induced by all three agents as well as thrombin- and arachidonic acid induced thromboxane formation.(ABSTRACT TRUNCATED AT 250 WORDS)
A stable isotope dilution assay for the simultaneous determination of two metabolites of prostacyclin (1), 6-oxo-prostaglandin F1 alpha (2a) and 2,3-dinor-6-oxo-prostaglandin F1 alpha (3a), in human seminal fluid and human urine is described. A new chemical total synthesis of deuterated internal standard, 18,18,19,19-(2H4)-2,3-dinor-6-oxo-PGF1 alpha (3b), is presented and enables specific and sensitive quantification based on negative ion chemical ionization mass spectrometry. 2a and 3a were analysed as their methoxime pentafluorobenzyl ester tris(trimethylsilyl) ether derivatives in the selected ion monitoring mode registrating the [M-181]- fragments with a detection limit for both prostanoids of 10 pg per injection. The two metabolites occur in human seminal fluid in very low concentrations (2a: 2.8 ng ml-1; 3a: 1.7 ng ml-1) and cannot contribute significantly to the urinary metabolite levels which are in the range of 108-265 ng/24 h for 3a and 124-574 ng/24 h for 2a.
A highly sensitive and specific assay for the quantification of thromboxane B2 (TXB2)(1) in human urine is described. The method is based on the use of low-blank (1H less than or equal to 0.2%) tetradeuterated internal standard 2 (18, 18, 19, 19-2H4-thromboxane B2), whose chemical synthesis is reported. After purification and high-performance liquid chromatography (HPLC) samples are derivatized to give an open-chain derivative of thromboxane B2, the methoxime pentafluorobenzyl ester tris(trimethylsilyl) ether (TXB2-MO-PFB-TMS3), most suitable for negative ion chemical ionization mass spectrometry. In the selected ion monitoring mode limits of detection per injection for pure standards and biological samples of 10 pg and 30 pg, respectively, are established. Normal urinary excretion of 1 in humans is 37-112 ng/24 h (n = 12).
The synthesis of a number of N-acyl-5-aminosalicylic acids and their derivatives is described. These compounds allow the sensitive and specific mass spectrometric determination of unlabelled or deuterium-labelled N-acetyl-5-aminosalicylic acid in biological samples. An in vivo study shows that the labelled compound, administered to rats, is excreted isotopically unchanged to at least 97%, and that no significant deacetylation/acetylation mechanism exists for this metabolite N-acetyl-5-aminosalicylic acid of salicylazosulphapyridine.
In two healthy male subjects the disposition of deuterated N-(2H3) acetyl-5-aminosalicylic acid (d3-ac-5-AS) was investigated after a single rectal dose of 500 mg d3-ac-5-AS. Urine and plasma were analysed by h.p.l.c. and gas chromatography mass-spectrometry. Peak concentrations of around 0.5 microgram/ml occurred within 6 h and plasma concentrations declined thereafter with a half-life (t1/2) of about 6 h which was confirmed by urinary excretion data. Renal clearance of d3-ac-5-AS ranged between 200 and 300 ml/min and only 4.4-11.2% of the dose could be recovered in the 48 h urine. Since no undeuterated ac-5-AS could be detected in any of the plasma and urine samples an irreversible acetylation of 5-AS is assumed in man.