Competitive inhibition of sparteine oxidation in human liver by beta-adrenoceptor antagonists and other cardiovascular drugs

The rate of oxidation of sparteine by the 9000 x g supernatant fraction of a human liver was measured in the presence of various drugs which exert cardiovascular effects. Hexamethonium, ouabain, caffeine and isoproterenol had no effect on this rate, while alprenolol, metoprolol, oxprenolol, propranolol, timolol, pindolol, lidocaine, mexiletine, 17-n-pentyl-sparteine, tolazoline, quinine, quinidine, cinchonine and cinchonidine inhibited the in vitro reaction competitively. Stereoselective inhibition was observed between quinine (Ki = 15 microM) and quinidine (Ki = 0.06 microM). Genetic evidence suggests that the primary metabolism of sparteine depends on a single species of cytochrome P450. In vitro competitive inhibition of sparteine oxidation by a drug indicates that this drug is capable of occupying the same enzymatic site as sparteine. This may mean that the competing drug is also metabolized at that site and thereby subject to the same genetic variation as sparteine's oxidation; absence of inhibition excludes this possibility.     

New mammalian metabolites of sparteine

Sparteine is reportedly metabolized in mammals with the formation of an N-oxide which undergoes dehydration to delta 2 and delta 5-dehydrosparteine. In our studies male Sprague-Dawley rats were found to metabolize sparteine and alpha-isosparteine to lupanine and alpha-isolupanine respectively in vivo. Metabolic conversion of sparteine in vitro in the presence of microsomal and 9000 x g supernatant fractions of the rat liver homogenate did not produce detectable lupanine. The in vivo studies were conducted by pretreating rats with inducers and inhibitors of microsomal enzymes. Inducers did not increase levels of lupanine in the rat urine but a significant decrease was observed in the presence of the inhibitor SFK 525A. Disulfiram reduced lupanine levels in the urine. The bioconversion of sparteine to lupanine appears to be mediated by microsomal enzymes and may proceed via an aldehyde intermediate. The conversion of sparteine to lupanine may parallel the mammalian metabolism of nicotine to cotinine.     

Gas chromatography/mass spectrometry assays for the determination of debrisoquine and sparteine metabolites in microsomal fractions of rat liver.

Debrisoquine and sparteine are prototype substrates of a genetic deficiency in cytochrome P450-dependent drug metabolism. Sensitive assays of in vitro oxidation of sparteine and debrisoquine are required for evaluation of this polymorphism. The activities were measured by quantitative analysis of 2-dehydrosparteine and 4-hydroxydebrisoquine production, respectively, using capillary column gas chromatography coupled with mass selective ion detection. With a single extraction, separation of parent drug, metabolite, and a suitable internal standard was readily achievable. Time-dependent production of both metabolites could be detected from as little as 40 micrograms of microsomal protein. Both activities showed a maximal activity with a 240-min incubation period. The ability to simultaneously quantify the parent drug and its metabolite suggests it would also be useful for evaluation of in vivo metabolism.     

Polymorphic drug oxidation in humans

Genetic polymorphisms in the oxidative metabolism of debrisoquine, mephenytoin, phenformin, sparteine, and tolbutamide have been discovered during recent years. Among these pharmacogenetic conditions, polymorphic oxidation of debrisoquine and sparteine has been intensively studied. Two phenotypes, the extensive (EM) and the poor (PM) metabolizers, have been observed in all populations so far investigated. The PM phenotype exhibits a grossly impaired or nearly absent capacity to metabolize these drugs. The incidence of the PM phenotype in European populations ranges from 5 to 9%. Pronounced variations in the incidence of the PM phenotype have been demonstrated among different ethnic groups. The metabolism of debrisoquine and sparteine is determined by two alleles at a single gene locus; PMs are homozygous for an autosomal recessive gene. Because of markedly impaired metabolism, the PM phenotype develops side effects if normal doses of debrisoquine and sparteine are administered. Defective metabolism in the PM phenotype is not restricted to debrisoquine and sparteine. Impaired metabolism of guanoxan , phenformin, perhexiline, methoxyamphetamine, phenacetin, encainide, metoprolol, alprenolol, bufuralol, nortriptyline, and desipramine have been described. As a consequence of impaired metabolism of these drugs, toxicity and therapeutic failure are observed in the PMs. With regard to molecular mechanisms, studies with microsomes from human liver provide evidence that in the PM phenotype a cytochrome P-450 isozyme is either missing or functionally inadequate.     

In vitro anticholinesterase activity of various alkaloids

In the current study, a number of alkaloids including retamine, cytisine, and sparteine (quinolizidine-type), yohimbine and vincamine (indole-type), scopolamine and atropine (tropane-type), colchicine (tropolone-type), allantoin (imidazolidine-type), trigonelline (pyridine-type) as well as octopamine, synephrine, and capsaicin (exocyclic amine-type) were tested in vitro for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at 1 mg/ml concentration by the Ellman method using an ELISA microplate reader. Among the alkaloids tested, only capsaicin exerted a remarkable inhibitory effect towards both AChE and BChE [(62.7 +/- 0.79)% and (75.3 +/- 0.98)%, respectively]. While the rest of the alkaloids did not show any significant inhibition against AChE, three of the alkaloids, namely retamine, sparteine, and yohimbine, exerted a noteworthy anti-BChE effect as compared to galanthamine, the reference drug.     

A human cytochrome P-450 characterized by inhibition studies as the sparteine-debrisoquine monooxygenase

The present study compares the debrisoquine monooxygenase and the sparteine monooxygenase activities of human liver microsomes. In the presence of 14 competitive inhibitors, apparent inhibition constants (Ki) as determined by these two activities ranged over four orders of magnitude with a correlation coefficient 0.99. These in vitro results represent the strongest evidence to date that the debrisoquine monooxygenase and the sparteine monooxygenase are identical and involve a single isozyme of cytochrome P-450.     

Clinical significance of oxidation and acetylation genetic polymorphism in patients with hyperthyreosis

INTRODUCTION: The relationship between genetically determined polymorphic oxidation and acetylation and susceptibility to some disease was aroused much interest. The aim of our study was to evaluate whether patients with hyperthyreosis differ from healthy persons in their ability to oxidize sparteine and acetylate sulphadimidine as model drugs. Oxidation and acetylation were estimated in 48 patients with hiperthyreosis. MATERIAL AND METHODS: The control group consisted of 160 healthy volunteers for comparison of oxidation phenotype and 60 healthy volunteers for comparison of acetylation phenotype. The phenotyping of oxidation revealed two distinct populations among 40 patients with hyperthyreosis: 38 persons (95%) were extensive metabolizers (EM) of sparteine and 2 persons (5%) was poor metabolizers (PM). In 160 healthy persons (91.2%) were EM and 14 persons (8.8%) were PM. The difference between frequency distribution of PM and EM in healthy persons and in patients with hyperthyreosis was not statistically significant. RESULTS: The phenotyping of acetylation showed among 48 patients with hyperthyreosis 8 persons (13%) were rapid acetylators (RA) and 40 persons (87%) were slow acetylators (SA). In 60 healthy volunteers the phenotype of rapid acetylation was observed in 31 persons (51%) and slow acetylation in 29 persons (49%). Relative risk (odds ratio) of development of thyroid diseases was 5.34 times higher for SA in comparison to RA. The prevalence of SA among patients with hyperthyreosis in comparison to healthy volunteers was statistically significant (p < 0.0002). CONCLUSIONS: The results of our study may suggest that slow acetylation phenotype is associated with increased risk of the development of hyperthyreosis.     

A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver

A sensitive method for the assay of sparteine oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km PM = 3033 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).     

Metal-catalyzed oxidation of the Werner syndrome protein causes loss of catalytic activities and impaired protein-protein interactions

Metal-catalyzed oxidation reactions target amino acids in the metal binding pocket of proteins. Such oxidation reactions generally result in either preferential degradation of the protein or accumulation of a catalytically inactive pool of protein with age. Consistently, levels of oxidized proteins have been shown to increase with age. The segmental, progeroid disorder Werner syndrome results from loss of the Werner syndrome protein (WRN). WRN is a member of the RecQ family of DNA helicases and possesses exonuclease and ATP-dependent helicase activities. Furthermore, each of the helicase and exonuclease domains of WRN contains a metal binding pocket. In this report we examined for metal-catalyzed oxidation of WRN in the presence of iron or copper. We found that WRN was oxidized in vitro by iron but not by copper. Iron-mediated oxidation resulted in the inhibition of both WRN helicase and exonuclease activities. Oxidation of WRN also inhibited binding to several known protein partners. In addition, we did not observe degradation of oxidized WRN by the 20 S proteasome in vitro. Finally, exposure of cells to hydrogen peroxide resulted in oxidation of WRN in vivo. Therefore, our results demonstrate that WRN undergoes metal-catalyzed oxidation in the presence of iron, and iron-mediated oxidation of WRN likely results in the accumulation of a catalytically inactive form of the protein, which may contribute to age-related phenotypes.     

Effect of sparteine sulfate on insulin secretion in normal men

This study aimed at evaluating the influence of sparteine sulfate either upon basal plasma glucose and insulin or glucose-induced insulin secretion in normal man. Thirteen overnight fasted volunteers took part in this study; five of them were submitted to sparteine sulfate bolus (15 mg in 10 ml of saline solution) followed by a slow infusion (90 mg/100 ml X 60 min) and eight subjects underwent two different glucose pulses (20 gr. i.v.) in absence or in presence of sparteine, infused as described above. In basal conditions, along with sparteine infusion, plasma glucose showed a progressive and significant decrease (P less than 0.0001) and plasma insulin was significantly higher from min 10 to 120' (P less than 0.0005-0.001). Even during the glucose-induced insulin secretion, in the presence of sparteine infusion, plasma glucose levels were significantly lower while plasma insulin levels were significantly higher when compared to those observed after glucose alone. The acute insulin response (AIR) was 42 +/- 10 microU/ml after glucose alone vs 67 +/- 9 microU/ml after glucose plus sparteine (P less than 0.05). Total insulinemic areas were significantly different being 1410 +/- 190 vs 2250 +/- 310 microU/ml/min (P less than 0.001) during glucose and glucose plus sparteine infusion, respectively. This study thereby, demonstrates that in normal man sparteine sulfate, administrated by intravenous infusion, is able to increase either basal or glucose-induced insulin secretion.     

Methionine oxidation inhibits assembly and promotes disassembly of apolipoprotein C-II amyloid fibrils

Methionine residues are linked to the pathogenicity of several amyloid diseases; however, the mechanism of this relationship is largely unknown. These diseases are characterized, in vivo, by the accumulation of insoluble proteinaceous plaques, of which the major constituents are amyloid fibrils. In vitro, methionine oxidation has been shown to modulate fibril assembly in several well-characterized amyloid systems. Human apolipoprotein (apo) C-II contains two methionine residues (Met-9 and Met-60) and readily self-assembles in vitro to form homogeneous amyloid fibrils, thus providing a convenient system to examine the effect of methionine oxidation on amyloid fibril formation and stability. Upon oxidation of the methionine residues of apoC-II with hydrogen peroxide, fibril formation was inhibited. Oxidized apoC-II molecules did not inhibit native apoC-II assembly, indicating that the oxidized molecules had a reduced ability to interact with the growing fibrils. Single Met-Val substitutions were performed and showed that oxidation of Met-60 had a more significant inhibitory effect than oxidation of Met-9. In addition, Met-Gln substitutions designed to mimic the effect of oxidation on side chain hydrophilicity showed that a change in hydrophobicity at position 60 within the core region of the fibril had a potent inhibitory effect. The oxidation of preformed apoC-II fibrils caused their dissociation; however, mutants in which the Met-60 was substituted with a valine were protected from this peroxide-induced dissociation. This work highlights an important role for methionine in the formation of amyloid fibril structure and gives new insight into how oxidation affects the stability of mature fibrils.     

Interaction of alkaloids with plant transfer ribonucleic acids. Effect of sparteine on lupin arginyl-tRNA formation

The effect of the alkaloid sparteine on arginyl-tRNA formation was studied. It was demonstrated that sparteine sulfate in the concentration range 10–60 mM inhibits the charging reaction when amino acid, ATP and tRNA are used as variable substrates. The mode of action is different for all substrates studied. It was concluded that at high sparteine concentration the pattern of inhibition for all varied substrates is generally uncompetitive. A non-competitive mechanism for amino acid and tRNA was observed at low sparteine concentration, but in the case of ATP it is also uncompetitive.     

Inhibition of Peroxynitrite-Mediated Oxidation of Dopamine by Flavonoid and Phenolic Antioxidants and Their Structural Relationships

The interaction between peroxynitrite and dopamine and the inhibition of this reaction by plant-derived antioxidants have been investigated. Peroxynitrite promoted the oxidation of dopamine to 6-hydroxyindole-5-one as characterised by HPLC and photodiode array spectra, akin to the products of the tyrosinase-dopamine reaction, but no evidence of dopamine nitration was obtained. Although peroxynitrite did not cause nitration of dopamine in vitro, the catecholamine is capable of inhibiting the formation of 3-nitrotyrosine from peroxynitrite-mediated nitration of tyrosine. The plant-derived phenolic compounds, caffeic acid and catechin, inhibited peroxynitrite-mediated oxidation of dopamine. This effect is attributed to the ability of catechol-containing antioxidants to reduce peroxynitrite through electron donation, resulting in their oxidation to the corresponding o-quinones. The antioxidant effect of caffeic acid and catechin was comparable to that of the endogenous antioxidant, glutathione. In contrast, the structurally related monohydroxylated hydroxycinnamates, p-coumaric acid and ferulic acid, which inhibit tyrosine nitration through a mechanism of competitive nitration, did not inhibit peroxynitrite-induced dopamine oxidation. The findings of the present study suggest that certain plant-derived phenolics can inhibit dopamine oxidation.     

Cyclosporin A does not increase the oxidative susceptibility of low density lipoprotein in vitro

Accelerated atherosclerosis is the leading cause of morbidity in renal transplant recipients. The pathogenic mechanisms responsible for the progression of atherosclerosis in renal transplant recipients have not been elucidated. Cyclosporin A (CsA) is an immunosuppressive agent used post-transplant and may contribute to increased oxidative susceptibility of low density lipoprotein (LDL). There is a paucity of data testing the effect of CsA on LDL oxidation. Hence, the aim of this study was to test the effect of in vitro enrichment of LDL with CsA on LDL oxidation. LDL oxidation in presence of different concentrations of CsA was tested using metal-dependent (copper), metal-independent (AAPH) and cell-mediated (macrophages) oxidation systems. In all 3 systems, CsA had no significant effect on LDL oxidation. Also, pre-incubation of LDL with CsA did not affect LDL oxidation and LDL alpha tocopherol levels. Thus, the results of our studies with CsA indicate that it is not a direct pro-oxidant.     

Effect of dichloroacetate on mechanical performance and metabolism of compromised diaphragm muscle.

We investigated the effect of dichloroacetate (DCA) on tension generation and carbohydrate metabolism of the rat diaphragm in vitro. Isolated diaphragms were placed in individual organ chambers and were hooked to force-displacement transducers. Net lactate production and glucose and lactate oxidation were measured in vitro. Diaphragmatic fatigue was precipitated by in vivo endotoxemic shock, by in vitro hypoxia, or by in vitro repetitive tetanic stimulation. In diaphragms isolated from endotoxemic rats, DCA increased tension generation by 30 and 20% at stimulation frequencies of 20 and 100 Hz, respectively. Associated with changes in mechanical performance, DCA reduced net lactate production by 53% after 60 min of incubation and increased glucose oxidation 54% but had no effect on lactate oxidation. During in vitro hypoxia, DCA reduced net diaphragmatic lactate production by 30% and increased glucose oxidation by 45% but did not attenuate hypoxic fatigue. DCA had no effect on tension generation during repetitive tetanic stimulation. We conclude that DCA improves in vitro diaphragmatic fatigue due to endotoxicosis but not due to hypoxia or repetitive stimulation.     

Human debrisoquine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22

The enzyme P450db1 (db1) is responsible for the common human defect in drug oxidation known as the "debrisoquine/sparteine polymorphism." Polyclonal antibody against the rat db1 protein was used to screen a human liver lambda gt11 library for the db1 cDNA clone. A cDNA containing the full protein coding sequence was isolated; the deduced NH2-terminal sequence of this cDNA was identical to that derived from direct sequencing of the purified human db1 protein. Comparison of the human db1 with rat db1 revealed 71 and 73% similarities of nucleotides and amino acids, respectively. By use of human-rodent somatic cell hybrids the db1 gene was localized to human chromosome 22 (CYP2D locus).     

Serum non-transferrin-bound iron and low-density lipoprotein oxidation in heterozygous hemochromatosis

Non-transferrin-bound iron (NTBI) is implicated in lipid peroxidation but the relation with oxidative modification of low-density lipoprotein (LDL) is not known. We assessed variables reflecting in vitro and in vivo LDL oxidation in two age- and sex-matched groups (n=23) of hereditary hemochromatosis heterozygotes (C282Y), characterized by a clear difference in mean serum NTBI (1.55+/-0.57 micromol/L vs 3.70+/-0.96 micromol/L). Plasma level of oxidized LDL (absolute and relative to plasma apolipoprotein B), and IgG and IgM antibodies to oxidized LDL, markers of in vivo LDL oxidation, did not differ between the groups with low and high serum NTBI. Mean lag-phase of in vitro LDL oxidation was also not significantly different between both study groups. Conclusion: these findings do not support the hypothesis that NTBI promotes oxidative modification of plasma LDL.     

Purified human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH

Purified serum paraoxonase (PON1) had been shown to attenuate the oxidation of LDL in vitro. We critically reevaluated the antioxidant properties of serum PON1 in the in vitro assays initiated with copper or the free radical generator 2,2'-azobis-2-amidinopropane hydrochloride (AAPH). The antioxidant activity of different purified PON1 preparations did not correlate with their arylesterase (AE), lactonase, or phospholipase A2 activities or with the amounts of detergent or protein. Dialysis of three of these preparations resulted in a 30-40% loss of their AE activities but in a complete loss of their antioxidant activities. We also followed the distribution of the antioxidant activity during human serum PON1 purification by two purification methods. The antioxidant activity of the anion-exchange chromatography fractions did not copurify with PON1 using either method and could largely be accounted for by the "antioxidant" activity of the detergent present. In conclusion, using the copper or AAPH in vitro assays, no PON1-mediated antioxidant activity was detected, suggesting that the removal of PON1 from its natural environment may impair its antioxidative activity and that this assay with highly purified PON1 may be an inappropriate method with which to study the antioxidative properties of the enzyme.     

Vanadate-mediated oxidation of NADH: description of an in vitro system requiring ascorbate and phosphate

Oxidation of NADH has been observed in an in vitro system requiring NADH, vanadate, ascorbate, and phosphate. Similar results were observed with NADPH. Ascorbate provides the reducing equivalents necessary to reduce vanadate to vanadyl. Vanadyl autoxidizes producing superoxide which initiates a free radical chain reaction resulting in oxidation of NADH. Oxidation is inhibited by superoxide dismutase but not by catalase or ethanol. Ascorbate functions to initiate the free radical chain reaction but is not required in stoichiometric concentrations. At higher concentrations, ascorbate inhibits NADH oxidation. Inorganic phosphate was required for NADH oxidation. Dialysis of phosphate buffers against solutions containing apoferritin or conalbumin or addition of transition metal cations or chelators to the reaction medium did not alter dependence on phosphate. Phosphate and vanadate were interchangeable in their effects on kinetic parameters of NADH oxidation except that vanadate was 100 times more potent than phosphate. Vanadate participates directly in the initiating and propagating redox reactions of NADH oxidation. Phosphate may be important in lowering the energy of activation for the necessary transfer of hydronium ion and water in the transition state between vanadate anion and vanadyl cation.     

Mechanistic Analysis of Ammonium Inhibition of Atmospheric Methane Consumption in Forest Soils

Methane consumption by forest soil was studied in situ and in vitro with respect to responses to nitrogen additions at atmospheric and elevated methane concentrations. Methane concentrations in intact soil decreased continuously from atmospheric levels at the surface to 0.5 ppm at a depth of 14 cm. The consumption rate of atmospheric methane in soils, however, was highest in the 4- to 8-cm depth interval (2.9 nmol per g of dry soil per day), with much lower activities below and above this zone. In contrast, extractable ammonium and nitrate concentrations were highest in the surface layer (0 to 2 cm; 22 and 1.6 μmol per g of dry soil, respectively), as was potential ammonium-oxidizing activity (19 nmol per g of dry soil per day). The difference in zonation between ammonium oxidation and methane consumption suggested that ammonia-oxidizing bacteria did not contribute significantly to atmospheric methane consumption. Exogenous ammonium inhibited methane consumption in situ and in vitro, but the pattern of inhibition did not conform to expectations based on simple competition between ammonia and methane for methane monooxygenase. The extent of ammonium inhibition increased with increasing methane concentration. Inhibition by a single ammonium addition remained constant over a period of 39 days. In addition, nitrite, the end product of methanotrophic ammonia oxidation, was a more effective inhibitor of methane consumption than ammonium. Factors that stimulated ammonium oxidation in soil, e.g., elevated methane concentrations and the availability of cosubstrates such as formate, methanol, or β-hydroxybutyrate, enhanced ammonium inhibition of methane oxidation, probably as a result of enhanced nitrite production.