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Biomedical subjects

J F Newton

Publications and source records attributed to J F Newton.

At least 19 recordsLinked to original sources
PubMed2001

Simultaneous determination of delta9-tetrahydrocannabinol and 11-nor-9-carboxy-delta9-tetrahydrocannabinol in human plasma by solid-phase extraction and gas chromatography-negative ion chemical ionization-mass spectrometry.

Delta9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCA) in human plasma can be simultaneously detected using solid-phase extraction with gas chromatography and negative ion chemical ionization mass spectrometry. THC-d3 and THCA-d3 are added as internal standards; protein is precipitated with acetonitrile and the resulting supernatants diluted with 0.1 M sodium acetate (pH 7.0) prior to application to the solid-phase extraction columns. THC and THCA were eluted separately and then pooled, dried under air, and derivatized with trifluoroacetic anhydride and hexafluoroisopropanol. The derivatized THC-d0 gives abundant molecular anions (m/z 410), and the derivatized THCA-d0 gives abundant fragment ions (m/z 422) formed by loss of (CF3)2CHOH from its molecular anion. The recoveries of THC and THCA were 74% and 17%, respectively. The lower and upper limits of quantitation were 0.5 and 100 ng/mL for THC and 2.5 ng/mL and 100 ng/mL for THCA. The within-run accuracy and precision for THC (measured at 0.5, 1, 10 and 75 ng/mL) ranged from 98 to 106% (% target) and 4.1 to 9.5 (%CV), respectively. For THCA, the within-run accuracy and precision (measured at 2.5, 5, 10, and 75 ng/mL) ranged from 89 to 101% and 4.3 to 7.5%, respectively. The between-run accuracy and precision for THC ranged from 92 to 110% and 0.4 to 12.4%, respectively. The between-run accuracy and precision for THCA ranged from 97 to 103% and 6.5 to 12.3%, respectively. In processed samples stored in reconstituted form at -20 degrees C, THC and THCA were stable for at least three days. THC and THCA stored in plasma were stable following three freeze/thaw cycles. THC and THCA in whole blood at room temperature for 6 h, or in plasma stored at room temperature for 24 h, did not show significant change. Storage in polypropylene containers for 7 days at -20 degrees C and the presence of 1% sodium fluoride or the cannabinoid receptor antagonist, SR141716, at 1 microg/mL did not interfere with the quantitation of THC and THCA. In three individuals who smoked marijuana under controlled dosing conditions, peak THC concentrations of 151, 266, and 99 ng/mL were seen in the first plasma samples drawn immediately after the end of smoking, and corresponding peak THCA concentrations of 41, 52, and 17 ng/mL occurred at 0.33 to 1 h after cessation of smoking.

Adult↗
PubMed1997

Drug quantitation on a benchtop liquid chromatography-tandem mass spectrometry system.

The specificity and selectivity of LC-MS-MS is illustrated to explain why LC-MS-MS has become the method of choice for quantitation within the pharmaceutical industry. Two assays are described that demonstrate the facility with which new ion trap technology can utilize the selectivity and sensitivity of LC-MS-MS to quantitate trace level components within complex matrices, in particular human plasma. One assay undergoes a validation procedure and demonstrates the utility of this new technology for drug quantitation within a regulated environment.

Calibration↗
PubMed1996

Bicyclic N-hydroxyurea inhibitors of 5-lipoxygenase: pharmacodynamic, pharmacokinetic, and in vitro metabolic studies characterizing N-hydroxy-N-(2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl)urea.

A series of N-hydroxyurea derivatives have been prepared and examined as inhibitors of 5-lipoxygenase. Oral activity was established by examining the inhibition of LTB4 biosynthesis in an ex vivo assay in the mouse. The pharmacodynamic performance in the mouse of selected compounds was assessed using an ex vivo LTB4 assay and an adoptive peritoneal anaphylaxis assay at extended pretreat times. Compounds with an extended duration of action were re-examined as the individual enantiomers in the ex vivo assay, and the (S) enantiomer of N-hydroxy-N-[2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl]urea, (+)-1a (SB 202235), was selected as the compound with the best overall profile. Higher plasma concentrations and longer plasma half-lives were found for (+)-1a relative to its enantiomer in the mouse, monkey, and dog. In vitro metabolic studies in mouse liver microsomes established enantiospecific glucuronidation as a likely mechanism for the observed differences between the enantiomers of 1a. Enantioselective glucuronidation favoring (-)-1a was also found in human liver microsomes.

Animals↗
PubMed1991

Comparative biochemical and morphometric changes associated with induction of the hepatic mixed function oxidase system in the rat.

This study characterized the induction of the rat hepatic cytochrome P-450-dependent mixed function oxidase system by SK&F 86002 [6-(4'-fluorophenyl)-5-(4'-pyridyl)-2,3-dihydroimidazo-(2,1-b)thia zole], an inhibitor of both the cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism. The induction characteristics of SK&F 86002 were compared to those of the classical inducer, phenobarbital, and morphological features of both SK&F 86002 and phenobarbital induced hepatocellular hypertrophy were quantitated. Rats were administered either SK&F 86002 (6, 18, or 60 mg/kg/day, po) or phenobarbital (8, 24, 80 mg/kg/day, ip) for 3 or 14 consecutive days. Liver to body weight ratio, total hepatic microsomal protein and cytochrome P-450 content, ethoxycoumarin-O-deethylase (ECOD) and leukotriene B4(LTB4) omega- and omega-1 hydroxylase were measured. Ultrastructural morphometry of the liver from control, and high dose SK&F 86002 (60 mg/kg/day) and phenobarbital (80 mg/kg/day) treated rats was completed. On day 3, phenobarbital increased liver to body weight ratio but only at the 80 mg/kg/day dosage; microsomal protein content was unchanged. ECOD activity increased in a dose-dependent fashion. LTB4 omega- and omega-1 hydroxylase activities were unaffected. Administration of SK&F 86002 for 3 days increased the liver to body weight ratio at both the 18 and 60 mg/kg/day dosage; microsomal protein content was unchanged. ECOD activity was significantly increased by the 60 mg/kg/day dosages of SK&F 86002. On day 14, phenobarbital increased the liver to body weight ratio and microsomal protein content but again only at the 80 mg/kg/day dosage. Cytochrome P-450 content was increased by all dosages.(ABSTRACT TRUNCATED AT 250 WORDS)

7-Alkoxycoumarin O-Dealkylase↗
PubMed1990

SK&F S-106203 inhibits leukotriene C4, leukotriene D4 and leukotriene E4 vasopressor responses in the conscious rat.

1. The purpose of these experiments was to investigate the effects of the selective peptidoleukotriene receptor antagonist, SK&F S-106203, on leukotriene C4 (LTC4), LTD4 and LTE4 vasopressor responses in the conscious, normotensive rat. SK&F S-106203 was administered as a bolus followed by a continuous infusion in order to provide information on the relationship between antagonism of leukotriene responses and steady-state plasma concentrations. 2. Infusion of SK&F S-106203 at doses of 0.2 mgkg-1 + 1 mgkg-1 h-1, 1 mgkg-1 + 3 mgkg- h-1 or 2 mgkg-1 + 10 mgkg-1 h-1 produced dose-dependent steady-state plasma drug concentrations of 1.0, 3.2 and 23.8 micrograms ml-1, respectively. Plasma SK&F S-106203 concentrations appeared to increase in a linear fashion at doses of 1 and 3 mgkg-1 h-1; at the highest dose the increment in plasma drug concentrations (i.e., 7-8 fold) was greater than the increment in dose (i.e., 3 fold), suggesting saturation of the primary clearance mechanism(s) at this dose. 3. SK&F S-106203 (2 mgkg-1 + 10 mgkg-1 h-1) had no effect on noradrenaline-, vasopressin-, isoprenaline-, or U 46619-induced responses. 4. SK&F S-106203 produced dose-dependent rightward shifts in the LTC4 and LTE4 dose-response curves. Administration of SK&F S-106203 at doses of 0.2mg kg1 + 1 mg kg1 h-, mg kg' + 3mgkg-'h-1, or 2mgkg-' + lOmgkg-1h'- produced dose-ratios of 1.0, 3.1 and 19.9, respectively, against LTC4 responses, and dose-ratios of 1.6, 3.8 and 9.1, respectively, against LTE4 responses. 5. Against LTD4 responses, SK&F S-106203 at doses of 0.2mgkg- + mgkg-1 h-, mg kg' + 3 mg kg- 1h - ', or 2 mg kg- + 10 mg kg- h- produced dose-ratios of 2.5, 2.8, and 11.4, respectively. Administration of D-penicillamine, a non-competitive LTD4 dipeptidase inhibitor, had no effect on LTD4 responses. 6. The similarity in the LTD4 dose-ratios at the two lower infusion rates, despite increases in the plasma drug concentrations, suggests the existence of pharmacologically heterogeneous LTD4 receptors. These results indicate that SK&F S-106203 is a potent, selective and apparently competitive antagonist of LTC4, LTD4 and LTE4 vascular responses in the intact rat.

Animals↗
PubMed1989

Selective antagonism of peptidoleukotriene responses does not reduce myocardial damage or neutrophil accumulation following coronary artery occlusion with reperfusion.

This study was designed to assess the effect of a peptidoleukotriene receptor antagonist, SK&F 104353, for limiting myocardial damage and neutrophil accumulation in rats subjected to myocardial reperfusion injury (MI/R). In conscious rats, SK&F 10,4353 (25 mg/kg, i.v.) antagonized LTD4-induced vasopressor responses by 90% and 60% at 1 and 4 hr, respectively, indicating effective blockade of peptido-leukotriene responses. In another group of animals subjected to 30 min of coronary artery occlusion with reperfusion for 24 hr, myocardial injury and neutrophil infiltration were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) activity, respectively, in the left ventricular free wall (LVFW). Myocardial CPK levels were 8.1 +/- 0.2 U/mg protein in Sham-MI/R vehicle-treated animals, and were significantly decreased to 6.4 +/- 0.6 U/mg protein in MI/R-vehicle animals. Myocardial MPO values were 1.5 +/- 0.5 U/g LVFW in Sham-MI/R vehicle-treated animals, and significantly increased to 4.3 +/- 0.6 U/g LVFW in MI/R-vehicle animals. Administration of SK&F 10,4353 (25 mg/kg, i.v.) 1 min prior to coronary occlusion and 3.5 hr post reperfusion had no effect on the loss of myocardial CPK specific activity or the increase in MPO levels (p greater than 0.05, compared to the MI/R-vehicle group). Thus, at a dose that antagonized LTD4-induced vasopressor responses, SK&F 104353 did not attenuate either the extent of myocardial injury or inflammatory cell accumulation associated with myocardial ischemia/reperfusion. These results suggest that peptidoleukotrienes do not contribute to the progression of myocardial ischemic/reperfusion injury.

Animals↗
PubMed1989

The bronchopulmonary pharmacology of SK&F 104353 in anesthetized guinea pigs: demonstration of potent and selective antagonism of responses to peptidoleukotrienes.

The bronchopulmonary pharmacology of SK&F 104353 [2(S)-hydroxy-3(R)-[2(2-carboxyethyl)thio]-3[2-(8- phenyloctyl)phenyl]-propanoic acid], a potent and selective leukotriene (LT) receptor antagonist in vitro, was assessed in anesthetized, spontaneously breathing guinea pigs. Aerosol administration of SK&F 104353 (5-2000 micrograms/ml x 100 breaths) reduced in a concentration-dependent manner the response to a standard LTD4 challenge (4.33 micrograms/ml x 5 breaths) given 30 min later. Inhalation of a 2000 micrograms/ml solution abolished LTD4-induced bronchoconstriction for at least 2 hr. The i.v. administration of SK&F 104353 10 min before challenge antagonized LTD4-induced bronchoconstriction with an ID50 of 0.55 mumol/kg (0.25 mg/kg). Substantial antagonism of LTD4-induced bronchospasm was observed for at least 60 min after i.v. administration of 5 mumol/kg of SK&F 104353. Infusion of SK&F 104353 at various rates revealed that a steady-state plasma concentration of 0.125 microM (0.06 micrograms/ml) reduced LTD4-induced bronchoconstriction by 60%. In addition to preventing the response to LTD4, i.v. administered SK&F 104353 (10 mumol/kg) rapidly and completely reversed ongoing LTD4-induced bronchoconstriction. SK&F 104353 also was effective when given intraduodenally 1 hr before LTD4 challenge, although the ID50 (32 mumol/kg) was 60-fold greater than the i.v. ID50. Given intragastrically, 100 mumol/kg of SK&F 104353 abolished the response to LTD4 for 1 hr, and reduced the response for 6 hr. SK&F 104353 (20 mumol/kg i.v.) had no effect on the bronchoconstriction induced by aerosolized acetylcholine, histamine or U-44069, but did antagonize the response to LTC4. SK&F 104353 alone did not produce bronchoconstriction when administered by any route or dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerosols↗
PubMed1989

Antagonism of leukotriene C4, leukotriene D4 and leukotriene E4 vasoconstrictor responses in the conscious rat with the peptidoleukotriene receptor antagonist SK&F 104353: evidence for leukotriene D4 receptor heterogeneity.

The purpose of these experiments was to investigate the effects of the selective peptidoleukotriene receptor antagonist, SK&F 104353, on leukotriene (LT)C4, LTD4 and LTE4 vasopressor responses in conscious, normotensive rats. Steady-state plasma concentrations of SK&F 104353 at infusion rates of 0.2 mg/kg + 1 mg/kg/hr, 1 mg/kg + 3 mg/kg/hr or 2 mg/kg + 10 mg/kg/hr were 0.5, 1.6 and 9.4 micrograms/ml, respectively, indicating that the plasma concentrations of SK&F 104353 were related directly to the infusion rate. LTC4, LTD4 and LTE4 (0.17-170 nmol/kg i.v.) produced dose-dependent increases in mean blood pressure. The ED20 dose (i.e., dose required to increase blood pressure 20 mm Hg) of LTC4, LTD4 or LTE4 was 2.7 +/- 0.4, 2.2 +/- 0.3 and 109 +/- 17 nmol/kg, respectively. SK&F 104353 produced dose-dependent, parallel shifts to the right in the LTC4 dose-response curve. Administration of SK&F 104353 at doses of 0.2 mg/kg + 1 mg/kg/hr, 1 mg/kg + 3 mg/kg/hr or 2 mg/kg + 10 mg/kg/hr produced dose ratios (i.e., ratio of ED20 in presence of SK&F 104353 to that of the vehicle group) of 6, 12 and 26, respectively. Against LTD4 responses, SK&F 104353 at doses of 0.1 mg/kg + 0.3 mg/kg/hr or 0.2 mg/kg + 1 mg/kg/hr produced dose ratios of 3 and 9, respectively. At a dose of 1 mg/kg + 3 mg/kg/hr, there was no further increase in the dose ratio, whereas a dose of 2 mg/kg + 10 mg/kg/hr resulted in a dose ratio of greater than 100.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Concentration-dependent, stereoselective inhibition of the endotoxin-induced hemoconcentration in conscious rats with the peptidoleukotriene receptor antagonist SK & F 104353.

Endotoxemia is associated with increases in a number of humoral mediators including vasopressin, thromboxane and leukotrienes (LT), all of which may participate in the pathophysiologic responses to endotoxemia. Previous studies from our laboratory demonstrated that endotoxin-induced hemoconcentration was attenuated with a peptidoleukotriene receptor antagonist, SK & F 104353. The purpose of this study was to investigate further the mechanism of endotoxin-induced hemoconcentration. Injection of LTD4 (51 nmol/kg, i.v.) produced an increase in the hematocrit of conscious male Sprague-Dawley rats: administration of SK & F 104353 (2 mg/kg, i.v. + 10 mg/kg/h, i.v. infusion) blocked completely this response to exogenous LTD4. Injection of Salmonella enteritidis endotoxin (30 mg/kg, i.v.) increased the hematocrit from 41 +/- 1 vol% to 55 +/- 1 vol%. Following pretreatment with SK & F 104353 (2 mg/kg, i.v. + 10 mg/kg/h, i.v.), the hemoconcentration was attenuated to 46 +/- 1 vol% (p less than 0.01). Simultaneous determination of plasma drug concentrations over a range of doses indicated that inhibition of the hemoconcentration produced by SK & F 104353 was concentration-dependent (IC30 = 0.5 microgram/ml). The IC30 for the stereoisomer, SK & F 104373, was 50 micrograms/ml. The 5-lipoxygenase/cyclooxygenase inhibitors, SK & F 86002 and BW 775C, also attenuated the endotoxin-induced increase in hematocrit, whereas indomethacin, heparin, daltroban, or the selective V1 vasopressin receptor antagonist [d(CH2)5Tyr(Me)]AVP did not significantly affect the endotoxin-induced hemoconcentration. The endotoxin-induced hemoconcentration was inhibited in a concentration-dependent, stereoselective manner with a peptidoleukotriene receptor antagonist, and by 5-lipoxygenase inhibitors, indicating that this response is mediated by peptidoleukotrienes.

Animals↗
PubMed1988

Ovarian effects of SK&F 86002-A2 in the rat: site of action.

In a preliminary 30-day study, oral administration of SK&F 86002-A2, an inhibitor of prostaglandin and leukotriene synthesis, blocked ovulation and altered ovarian structure and hormone production in rats. The purpose of the present study was to determine if the locus of action of SK&F 86002-A2 for these effects was the ovary or some other site in the female reproductive system, using a number of experimental approaches. A single sc or intraovarian injection of SK&F 86002-A2 did not block spontaneous or gonadotropin-induced ovulation in proestrous rats, whereas indomethacin, a positive control, acutely disrupted the ovulatory process. Since neither route of administration blocked ovulation, integrated pituitary and ovarian events were not negatively affected by a single injection of SK&F 86002-A2 at doses which caused ovarian dysfunction when administered repeatedly for 30 days. In contrast to a single dose, oral administration of SK&F 86002-A2 to hypophysectomized rats for 2 weeks suppressed follicular growth and estradiol production in response to sc administration of pregnant mare serum gonadotropin. Although ovarian function was suppressed in hypophysectomized rats, LH surges induced by estradiol in ovariectomized rats were not affected by administration of SK&F 86002-A2 for 2 weeks. Thus, hypothalamic/pituitary dysfunction did not contribute to the ovarian effects of SK&F 86002 that occurred after repeated dosing. In conclusion, these results indicate that disruption of ovarian cycles by SK&F 86002-A2 is related to a direct effect on the ovary, and not to altered hypothalamic/pituitary function and LH release. Specifically, SK&F 86002-A2 may suppress the ovarian response to gonadotrophin, retarding follicular growth and estrogen production. The ovarian effects are consistent with a pharmacological expression of the inhibitory action of SK&F 86002-A2 on prostaglandin and leukotriene synthesis.

Administration, Oral↗
PubMed1988

Determination of microsomal lauric acid hydroxylase activity by HPLC with flow-through radiochemical quantitation.

An assay for the microsomal hydroxylation of lauric acid (LA), based on HPLC with flow-through radiochemical detection, has been developed. Conditions were optimized for resolution and quantitation of three microsomal metabolites of LA, one of which has not been reported previously as a metabolite of LA in mammalian microsomal incubations. These products, 12-(omega)-hydroxy-LA, 11-(omega-1)-hydroxy-LA, and a novel metabolite, 10-(omega-2)-hydroxy-LA, were isolated by HPLC and identified by gas chromatography/mass spectrometry. In the presence of NADPH, the formation of all three metabolites was linear with time and microsomal protein concentration. Hydrogen peroxide also supported the microsomal metabolism of LA, although the ratio of metabolites was substantially different than that produced by NADPH-supported microsomes. Several biochemical probes (metyrapone, alpha-naphthoflavone, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, and 10-undecynoic acid) were used to dissociate the three LA hydroxylase activities. These experiments suggest that the site-specific hydroxylation [omega-, (omega-1)-, (omega-2)-] of LA may be catalyzed by different isozymes of cytochrome P-450.

Animals↗
PubMed1988

Beneficial effects of the peptidoleukotriene receptor antagonist, SK&F 104353, on the responses to experimental endotoxemia in the conscious rat.

The purpose of this study was to examine the effects of the peptidoleukotriene receptor antagonist, SK&F 104353, on the responses to endotoxin in conscious male Sprague-Dawley rats. Administration of Salmonella enteritidis endotoxin (30 mg/kg i.v.; LD90) resulted in a decrease in the number of circulating platelets, leukopenia, an increase in hematocrit, and 0% survival at 24 hr. Pretreatment with SK&F 104353 (1 mg/kg, i.v. bolus followed by 3 mg/kg/hr, i.v. infusion for 6 hr) 5 min before injection of endotoxin produced steady state plasma drug levels of 1.6 micrograms/ml in naive animals and levels of approximately 3.4 micrograms/ml in endotoxemic animals (P less than 0.05). SK&F 104353 significantly attenuated the endotoxin-induced thrombocytopenia (P less than 0.05) but had no effect on either the endotoxin-induced early leukopenia or late leukocytosis. Additionally, SK&F 104353 significantly reduced the endotoxin-induced hemoconcentration (P less than 0.05) and improved survival to 30% at 48 hr (P less than 0.05). A higher dose of SK&F 104353 (2 mg/kg, i.v. bolus followed by 10 mg/kg/hr, i.v. infusion for 6 hr) did not produce any further benefit. These data indicate clearly the pathophysiologic role of peptidoleukotrienes in endotoxemia and suggest SK&F 104353 could be useful for ameliorating some of the deleterious sequelae associated with this condition.

Animals↗
PubMed1988

Inhibition by ranitidine of acetaminophen conjugation and its possible role in ranitidine potentiation of acetaminophen-induced hepatotoxicity.

Pretreatment with ranitidine (RA) potentiates the hepatotoxicity of acetaminophen (APAP) in male Fischer 344 rats. The present studies were undertaken to investigate the role of APAP metabolism in this potentiation. Administration of RA (50 mg/kg p.o.) to male Fischer 344 rats 30 min before [3H]APAP (750 mg/kg p.o.) increased the plasma concentrations of acetaminophen at 2 hr (193%) and 4 hr (277%) after APAP. Covalent binding of [3H]APAP-related material to hepatic macromolecules in RA-pretreated animals was similar to APAP alone values up to 12 hr after treatment; however, 24 hr after APAP, binding in the RA-pretreated animals was twice that observed in animals given [3H]APAP alone. Urinary excretion (0-24 hr) of APAP and APAP glucuronide were reduced in ranitidine-pretreated animals to 64 and 66% of control, respectively, indicating that in vivo RA altered APAP conjugation with glucuronic acid. APAP uridine diphosphoglucuronyltransferase activity in rat hepatic microsomes was competitively inhibited by RA (0.1-2 mM). The Ki apparent for RA inhibition of APAP uridine diphosphoglucuronyltransferase was 0.04 mM. In contrast, neither APAP nor 4-nitrophenol sulfotransferase activity in rat hepatic cytosol was inhibited by RA at concentrations up to 5 mM. Together, these results support the suggestion that RA-mediated alterations of APAP conjugation may explain the potentiation of APAP-induced hepatotoxicity by RA in rats.

Acetaminophen↗
PubMed1987

Biochemical characterization of hepatic microsomal leukotriene B4 hydroxylases.

omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins.

Animals↗
PubMed1986

Metabolism and excretion of a glutathione conjugate of acetaminophen in the isolated perfused rat kidney.

Acetaminophen-glutathione (APAP-GSH) is the initial sulfur-containing metabolite of APAP produced by the liver. However, little, if any, APAP-GSH is found in the urine of intact animals. Rather, the cysteine (APAP-CYS) and N-acetylcysteine (APAP-NAC) conjugates are the predominant sulfur-containing metabolites of APAP excreted in the urine. To define more precisely the role of the kidney in total body disposition of APAP, the metabolism and excretion of each of these metabolites was quantified in the isolated perfused rat kidney (IPK). With perfusate concentrations of 0.031, 0.125 and 0.250 mM APAP-GSH, the IPK metabolized APAP-GSH to APAP-CYS rapidly. Further metabolism of APAP-CYS to APAP-NAC proceeded at a much slower rate. Consequently, at 0.031 mM APAP-GSH, negligible amounts of APAP-CYS were found in the urine. However, as the concentration of APAP-GSH was increased so did the excretion of APAP-CYS. In contrast, the excretion of APAP-NAC did not exhibit dependence on APAP-GSH concentration. APAP-NAC was excreted by a probenecid sensitive transport mechanism whereas APAP-CYS excretion appeared to be related only to glomerular filtration. In addition, the disappearance of APAP-GSH was much greater than could be accounted for by glomerular filtration. These data indicate that the IPK is an effective model for the study of metabolism and excretion of xenobiotics that have undergone conjugation with GSH.

Acetaminophen↗
PubMed1986

Purification, characterization, and structural properties of a single protein from rat basophilic leukemia (RBL-1) cells possessing 5-lipoxygenase and leukotriene A4 synthetase activities.

Arachidonate 5-lipoxygenase of rat basophilic leukemia (RBL-1) cells was purified more than 1000-fold by gel filtration and anion exchange protein-high performance liquid chromatography (HPLC). Physical properties of the purified 5-lipoxygenase such as molecular weight (74,000-76,000), N-terminal sequence (30 amino acids), and amino acid composition were determined. The purified enzyme converted [14C]arachidonic acid at 20 degrees to [14C] 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and to [14C]dihydroxyeicosatetraenoic acids (diHETEs). Utilizing [14C] 5(S)HPETE as substrate, the purified enzyme also converted the hydroperoxy acid to [14C]diHETES. The [14C]diHETE reaction products were identified primarily (greater than 80% of recovered radioactivity) as the nonenzymatic hydrolysis products of leukotriene A4 (i.e., 6-trans-leukotriene B4 and 12-epi-6-trans-leukotriene B4) by reverse phase HPLC, scanning spectrophotometry, and gas chromatography-mass spectrometry. The bioconversion of [14C] arachidonate and [14C]5(S)HPETE to reaction products by the purified enzyme was dependent on the presence of both Ca2+ and ATP. The enzymatic activities were inhibited in a similar manner by the lipoxygenase inhibitors nordihydroguaiaretic acid, diphenyldisulfide, and SK&F 86002. The data provide evidence that RBL-1 cell 5-lipoxygenase and leukotriene A4 synthetase activities reside on a single monomeric protein with a free N-terminus and that they possess similar biochemical characteristics.

Amino Acids↗
PubMed1985

Metabolism of leukotriene B4 in hepatic microsomes.

Leukotriene B4 was metabolized in rat hepatic microsomes to two products. Mass spectral analysis of these two metabolites indicated that the major metabolite was the 20-hydroxy metabolite while the minor metabolite was the 19-hydroxy metabolite. The formation of these metabolites required NADPH and was linear with time (20 min) and protein (1.6 mg/ml). The Km apparent and Vmax for omega hydroxylation of LTB4 was 14 uM and 0.138 nmol/min/mg protein. In contrast, the km and Vmax for omega minus one hydroxylation was 54 uM and 0.093 nmol/min/mg protein. These results suggest that omega and omega minus one hydroxylations of LTB4 may be mediated by different isozymes of hepatic P-450.

Animals↗