Asymmetric metabolic N-oxidation of N-ethyl-N-methylaniline by purified flavin-containing monooxygenase

The prochiral tertiary amine N-ethyl-N-methylaniline (EMA) is known to be metabolically N-oxygenated in vitro with microsomal preparations. This biotransformation is thought to be mediated predominantly by the flavin-containing monooxygenase (FMO) enzyme system. Microsomal N-oxygenation of EMA is known to be stereoselective and varies between species. In order to further characterise this metabolic transformation, we have examined the in vitro metabolism of EMA using purified porcine hepatic FMO. Following incubation of EMA with purified FMO, EMA N-oxide, the only metabolite detected, was found to be produced stereoselectively [ratio (?)-(S):(+)-(R), ca. 4:1]. The enantiomeric ratio of the N-oxide product did not change markedly with respect to time, enzyme or substrate concentration. Determination of the kinetics of formation of the N-oxide indicated a single affinity for the prochiral substrate with differential rates of formation of the enantiomers. The extent of EMA N-oxide formation was shown to be affected by activators and inhibitors of FMO and pH, but its stereoselectively was unaltered. © 1994 Wiley-Liss, Inc.     

Species variability in the stereoselective N-oxidation of pargyline

The monoamine oxidase inhibitor pargyline (N-benzyl-N-methyl-2-propynylamine) is known to undergo extensive in vitro microsomal N-oxidation, thought to be mediated predominantly by the flavin-containing monooxygenase (FMO) enzyme system. Formation of the pargyline N-oxide (PNO) metabolite creates a chiral nitrogen centre and thus asymmetric oxidation is possible. This study describes a reverse-phase high-performance liquid chromatographic (HPLC) method for the quantitation of PNO and a chiral-phase HPLC method for the determination of the enantiomeric ratio of PNO. In vitro microsomal N-oxidation of pargyline was found to be highly steroselective in a number of species, with the (+)-enantiomer being formed preferentially. This metabolic transformation was stereospecific when purified porcine hepatic FMO was used as the enzyme source. © 1994 Wiley-Liss, Inc.     

N-Demethylation and N-oxidation of imipramine in rat thoracic aortic endothelial cells

The aim of this study was to examine whether cultured rat thoracic aortic endothelial cells (TAECs) have the ability to metabolize the tertiary amine, imipramine. In rat TAECs, imipramine was biotransformed into N-demethylate and N-oxide by cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO), respectively. The intrinsic clearance (V _max/K _m) for the N-oxide formation was approximately five times as high as that for the N-demethylate formation, indicating that oxidation by CYP was much higher than that by FMO. Moreover, we suggest that CYP2C11 and CYP3A2 are key players in the metabolism to N-demethylate in rat TAECs using the respective anti-rat CYP antibodies (anti-CYP2C11 and anti-CYP3A2). The presence of CYP2C11 and CYP3A2 proteins was also confirmed in cultured rat TAECs using a polyclonal anti-CYP antibody and immunofluorescence microscopy. In contrast, the formation rate of N-oxide at pH 8.4 was higher than that at pH 7.4. Inhibition of N-oxide formation by methimazole was found to be the best model of competitive inhibition yielding an apparent K _i value of 0.80 μmol/L, demonstrating that N-oxidation was catalyzed by FMO in rat TAECs. These results suggest that rat TAEC enzymes can convert substrates of exogenous origin such as imipramine, indicating that TAECs have an important function for metabolic products, besides hepatic cells.     

Assimilatory reduction of trimethylamine N-oxide in the yeast Sporopachydermia cereana

Summary Washed microsomal preparations (100 000 xg sediment) from the yeast Sporopachydermia cereana that had been grown on trimethylamine N-oxide as sole nitrogen source catalysed the NAD(P)H-dependent reduction of trimethylamine N-oxide to trimethylamine. Under anaerobic conditions, this was the sole reaction product, but under aerobic conditions only small amounts of trimethylamine accumulated, most being further metabolized to methylamine and formaldehyde (no detectable dimenthylamine accumulated due to its rapid turnover). In the absence of NAD(P)H, no formation of amines or formaldehyde from trimethylamine N-oxide was detected. The trimethylamine N-oxide reductase activity was inhibited by quinacrine, Cu²⁺ ions, triethylamine N-oxide (apparent K _i 0.43 mM) and dimethyl sulphoxide (K _i 0.94 mM). Chlorate and nitrate failed to inhibit the enzyme. The K _m for trimethylamine N-oxide was 29 M. Triethylamine N-oxide was also reduced by the microsomal preparation with the formation of acetaldehyde, and this reduction was sensitive to the same inhibitors as trimethylamine N-oxide, suggesting that both amine oxides are metabolized by the same enzyme(s). It is concluded that trimethylamine N-oxide is metabolized in this yeast via an NAD(P)H-dependent reductase.Abbreviations TMAO triemthylamine N-oxide     

Microsomal formation and chemical decomposition of pargyline N-oxide

Pargyline undergoes metabolic N-oxidation in rat and rabbit liver microsomal preparations. The reaction requires oxygen and is NADPH dependent. N-oxidation and N-demethylation are equal in both control and induced rat liver microsomes, while N-oxidation is more dominant in rabbit tissue. Experiments investigating the CO-sensitivity and the effects of metyrapone suggest that cytochrome P-450 systems are involved in both reactions in the rat while an additional enzyme is responsible for the N-oxidation in the rabbit. Pargyline N-oxide is characterized by chemical instability and undergoes two consecutive rearrangements to yield propenal and Schiff bases, the latter undergoing hydrolysis to aldehydes and primary amines. Accordingly, due to the inherent instability of the N-oxide, metabolic N-oxidation of pargyline is, in addition to α-carbon oxidation, indicated as a metabolic route to benzaldehyde. Similarly the ease with which pargyline N-oxide generates propenal implicates N-oxidation as a metabolic route to be considered when evaluating the toxicity of pargyline.     

Flavin-containing monooxygenase mediated metabolism of benzydamine in perfused brain and liver

Benzydamine (BZY) N-oxidation mediated by flavin-containing monooxygenase (FMO) was evaluated in perfused brain and liver. Following 20 min of perfusion with modified Ringer solution, the infusion of BZY into brain or liver led to production of BZY N-oxide. BZY N-oxide, a metabolite of BZY oxidized exclusively by FMO, was mostly recovered in the effluent without undergoing further metabolism or reduction back to the parent substrate. The BZY N-oxide formation rate increased as the infusion concentration of BZY increased both in perfused brain and perfused liver. BZY N-oxidation activities in perfused rat brain and liver were 4.2 nmol/g brain/min and 50 nmol/g liver/min, respectively, although the BZY N-oxidation activity in brain homogenates was one 4000th that in liver homogenates. This is the first study of FMO activity in brain in situ.     

The flavin-containing monooxygenase in mouse lung: Evidence for expression of multiple forms

The flavin-containing monooxygenase (FMO) was purified from mouse lung microsomes. On SDS-PAGE, the purified enzyme separated as two bands, a major band of 58,000 daltons and a minor band of 59,000 daltons. Antibodies to mouse liver FMO cross-reacted with both bands in the purified preparations, whereas antibodies to rabbit lung FMO cross-reacted only with the major band. In microsomal preparations the major band was recognized by both antibodies, but neither antibody detected the minor band in microsomes. A cDNA encoding the pig liver FMO hybridized with mRNA isolated from mouse liver, kidney, and lung, whereas cDNA encoding the rabbit lung FMO hybridized only with mouse lung and kidney mRNA. Thermal stability studies showed that the FMO preparation purified from mouse lung consisted of a heat-stable and a heat-labile component. The heat-labile component of lung FMO was inhibited competitively by imipramine, whereas the heat-stable component was insensitive to the presence of imipramine. Immunoprecipitation of purified mouse lung FMO with anti-rabbit lung FMO completely removed the protein band reactive to anti-rabbit lung FMO while leaving reactivity to anti-liver FMO. The catalytic and immunochemical differences seen between FMO from rabbit lung and mouse lung appear to result from the expression of at least two forms of FMO in the mouse lung, one similar to the rabbit pulmonary form and one similar to the major mouse liver form of FMO.     

Pyrrolic and N-oxide metabolites formed from pyrrolizidine alkaloids by hepatic microsomes in vitro: Relevance to in vivo hepatotoxicity

An analytical method of improved sensitivity has enabled measurements to be made of N-oxide as well as pyrrolic metabolites formed from a range of unsaturated pyrrolizidine alkaloids in hepatic microsome preparations. Using microsomes from livers of phenobarbitone-pretreated male Fischer rats, all 13 alkaloids tested were metabolised to both N-oxides and pyrroles. The most lipophilic alkaloids gave enhanced rates of metabolism. No consistent relationship existed between rates of N-oxide and of pyrrole formation. The two pathways appeared to be independent. The ratio of N-oxide to pyrrolic metabolites varied, depending on the type of ester: it was highest for ‘open’ diester alkaloids, lowest for 12 membered macrocyclic diesters and for monoesters. Steric hindrance by the acid moiety could account for these differences, by affecting the balance between microsomal oxidation of the amino alcohol moiety at the nitrogen and C8 positions respectively and could explain the high pyrrole yields given by some macrocyclic diesters. The levels of pyrrolic metabolites bound to liver tissues and responsible for hepatotoxicity in rats given pyrrolizidine alkaloids, did not necessarily reflect the rates of formation of such metabolites measured in vitro. In the animal additional factors could influence the formation and tissue binding of pyrrolic metabolites, including the detoxication of alkaloids by hydrolysis and the chemical reactivity and stability of the toxic metabolites. A comparison of heliotridine esters with retronecine esters showed that the 7-hydroxyl or -ester configuration had a relatively small influence on the balance between formation of pyrrolic metabolites and detoxication by N-oxidation. The results did not support any hypothesis that heliotridine esters should generally be more hepatotoxic than analogous retronecine esters. The structure of the acid moiety was likely to have at least as much influence on toxicity as the base configuration.     

EVIDENCE FOR THE BIOSYNTHESIS OF MANNOSYLPHOSPHORYLDOLICHOL AND N-ACETYLGLUCOSAMINYLPYROPHOSPHORYLDOLICHOL BY AN AXOLEMMA-ENRICHED MEMBRANE PREPARATION FROM BOVINE WHITE MATTER

An axolemma-enriched membrane fraction prepared by an improved procedure from bovine white matter catalyzes the enzymatic transfer of [¹⁴C]mannose and N-acetyl[¹⁴C]glucosamine from their nucleotide derivatives into a mannolipid and an N-acetylglucosaminyl lipid in the presence of exogenous dolichyl monophosphate. The labeled glycolipid products have the chemical and chromatographic characteristics of mannosylphosphoryldolichol and N-acetylglucosaminylpyrophosphoryldolichol. The initial rates of synthesis of the glycolipids by the axolemma-enriched membrane fraction have been compared with the initial rates of glycolipid formation catalyzed by a microsomal preparation and myelin in the presence or absence of dolichyl monophosphate. Essentially no glycolipid synthesis was observed when either GDP-[¹⁴C]mannose or UDP-N-acetyl[¹⁴C]glucosamine were incubated with myelin in the presence or absence of exogenous dolichyl monophosphate. A comparison of the initial rates of synthesis of the glycolipids using endogenous acceptor lipid revealed that the rate of formation of mannolipid was 7 times faster for the microsomal membranes than the axolemma-enriched membranes. In the presence of an amount of dolichyl monophosphate approaching saturation the initial rate of glycolipid synthesis was markedly enhanced for both membrane preparations. However, due to a more dramatic enhancement in the axolemma-enriched membranes the initial rate of mannolipid synthesis was only approx. 2.5 times greater in the microsomal membranes. A similar observation was made when the initial rates of N-acetylglucosaminyl lipid synthesis were compared for axolemma-enriched and microsomal preparations in the presence and absence of exogenous dolichyl monophosphate. These studies indicate that the axolemma-enriched membranes have a relatively lower content of dolichyl monophosphate than the microsomal membranes although the difference in the amount of mannosyltransferase is only two to three-fold lower. The presence of a sugar nucleotide pyrophosphatase activity capable of degrading GDP-mannose and UDP-N-acetylglucosamine has also been demonstrated in the axolemma-enriched membrane fraction.     

Distinct pulmonary and hepatic forms of flavin-containing monooxygenase in sheep

1. Flavin-containing monooxygenase (FMO) in pulmonary and hepatic microsomes from sheep was analyzed by western blotting by probing with antibodies raised against FMO purified from rabbit lung and pig liver. 2. Pulmonary microsomes from sheep contain a single major protein which cross-reacts with the antibody to rabbit lung FMO, but no band can be observed when probed with the antibody to the pig liver enzyme. Likewise, sheep liver microsomes contain a protein which cross-reacts with the antibody to pig liver FMO, but no significant staining is observed following incubation with antibody to the lung enzyme. 3. Sheep pulmonary and hepatic microsomal FMO also display a difference in activity toward chlorpromazine and n-dodecylamine. 4. Preliminary evidence suggests that sheep FMO may be induced (liver) or repressed (lung) during pregnancy. 5. Sheep are similar to rodents (rat, mouse, guinea pig, hamster and rabbit) in having distinct forms of pulmonary and hepatic FMO. The immunochemical and catalytic difference between sheep liver and lung FMO is similar to that of rabbit.     

Toxicity of the heterocyclic amine batracylin: investigation of rodent N-acetyltransferase activity and potential contribution of cytochrome P450 3A

The heterocyclic amine, batracylin (BAT), is genotoxic and several lines of evidence suggest that acetylation is one step in the formation of a DNA-damaging product. The variation in susceptibility to BAT toxicity observed between rats and mice has also been linked to the acetylated product. BAT N-acetyltransferase (NAT) activity was determined in rat and mouse hepatic cytosols. Formation of acetylbatracylin (ABAT) was 6 times greater in F-344 hepatic samples compared to either mouse strain, while hepatic BAT NAT activities were similar in C57B1/6 and A/J mice. No deacetylation of ABAT was detected. In contrast, 2-aminofluorene NAT activity in C57B1/6 hepatic cytosol was twice that of the A/J strain and activities in both strains of mice were greater than in rat. Deacetylation of 2-acetylaminofluorene was detected in both species with enzyme activities in C57B1/6>A/J>F-344. Hepatocytes from the F-344 rats, the species most sensitive to BAT toxicity, were used to investigate the contribution of other biotransformation reactions to BAT cytotoxicity. Leakage of cellular lactate dehydrogenase was greater in hepatocytes from male rats than from females, increased on in vivo exposure to dexamethasone, and decreased in the presence of troleandomycin, suggesting that CYP3A-mediated biotransformation of BAT is involved in the formation of a cytotoxic product. When phenol red, a substrate for UDP-glucuronsyltransferase (UDPGT), was absent from the medium, BAT cytotoxicity was reduced. These data are consistent with a role for NAT, CYP, and UDPGT in the biotransformation of BAT. This revised version was published online in July 2006 with corrections to the Cover Date.     

Liver metabolic reactions: tertiary amine N-dealkylation, tertiary amine N-oxidation, N-oxide reduction, and N-oxide N-dealkylation. II. N,N-dimethylaniline

Rat liver microsomal preparations enzymatically catalyze the N-demethylation and N-oxidation of dimethylaniline as well as the N-demethylation of dimethylaniline-N-oxide. Both compounds were used as substrates and the formation of formaldehyde and N-oxide were determined.Both demethylation and N-oxidation of dimethylaniline are dependent on NADPH. This cofactor also increases the demethylation of dimethylaniline-N-oxide, although it is not an absolute requirement. Nicotinamide increases the rate of formation of formaldehyde and N-oxide from dimethylaniline by a factor of about 4 and decreases the N-oxide demethylation by the same factor. The cofactor optimum consists of NADPH, nicotinamide, and magnesium ions for the demethylation and N-oxidation of dimethylaniline, and of NADPH alone for the demethylation of its N-oxide. The kinetic constants of the three test reactions have been determined under these optimal cofactor requirements.Various agents strongly influence the rates of product formation of the three test reactions studied. SH-blocking agents, the chelating agent EGTA, as well as nicotinamide influence the rates of formaldehyde formation from dimethylaniline and N-oxide demethylation in an opposite way. This demonstrates that, in the tertiary amine demethylation of dimethylaniline, a C-oxidation pathway is operative in addition to an N-oxidation pathway with subsequent N-oxide demethylation. The following influences on the actual metabolic reactions could be deduced from the effects of agents on the test reactions: SKF 525-A inhibits and phenobarbital pretreatment stimulates N-oxide demethylation; EDTA inhibits both the latter reaction and N-oxidation; EGTA and nicotinamide stimulate C-oxidation and inhibit N-oxide demethylation; SH-blocking agents inhibit C-oxidation and stimulate both N-oxidation and N-oxide demethylation.Quantitative and qualitative species differences with respect to cofactor requirement and effect of SKF 525-A have been observed between rat and pig liver microsomes. In addition, profound differences in subcellular localization and metabolic rates between dimethylaniline and other substrates are known. Thus it is unlikely that the three metabolic reactions dealt with in this report are characteristic of tertiarr amine N-dealkylation in general.     

Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis

We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions.     

Phosphinyliminodithiolane insecticides: Novel addition reactions of phosfolan and mephosfolan sulfoxides and sulfones

2-(Diethoxyphosphinylimino)-1,3-dithiolane (phosfolan) and its 4-methyl analog (mephosfolan) are proinsecticides as determined by microsomal mixed-function oxidase (MFO) activation to potent acetylcholinesterase (AChE) inhibitors. They are similarly activated by peracid oxidation which yields the sulfoxide and sulfone derivatives. The hydrolytically unstable S-oxides are irreversible AChE inhibitors that are 160- to 47,000-fold more potent than phosfolan and mephosfolan. MFO S-oxidation is indicated for both proinsecticides by (a) NADPH-dependent increases in potency as AChE inhibitors to an extent expected of sulfoxides, and (b) formation of the S-oxide hydrolysis product diethyl phosphoramidate.     

Conversion of codeine to morphine in isolated capsules of Papaver somniferum

The biotransformation of codeine to morphine was studied in isolated capsules of Papaver somniferum. Cofactors such as nicotinamide adenine dinucleotide, adenosine 5′-triphosphate, S-acetyl coenzyme A and pyridoxal phosphate were not required in the conversion of codeine to morphine. Reducing agents such as dithiothreitol, glutathione and β-mercaptoethanol strongly promoted codeine and morphine degradation, while morphine formation remained at a constant level. Hydrogen peroxide (concentration > 0.25 mM) caused the conversion of codeine and morphine to N-oxides by non-enzymatic oxidation. Isolated capsules of P. somniferum provide a method of studying the biotransformation of codeine to morphine.     

Biosynthesis of glycosoaminoglycans by microsomal preparations from cultured mastocytoma cells

Neoplastic mast cells of mice (including long-established and newly derived lines) were grown in large-volume suspension cultures to provide enough cells for preparation of microsomal fractions. Microsomal preparations from P815Y and P815S cells synthesized ¹⁴C-labelled glycosaminoglycan when incubated with UDP-[¹⁴C]glucuronic acid and UDP-N-acetylgalactosamine. No significant amount of ¹⁴C-labelled glycosaminoglycan was formed when UDP-N-acetylglucosamine was substituted for the UDP-N-acetylgalactosamine. Microsomal preparations from X163 cells synthesized ¹⁴C-labelled glycosaminoglycan when incubated with UDP-[¹⁴C]glucuronic acid and either UDP-N-acetylgalactosamine or UDP-N-acetylglucosamine. The ¹⁴C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylgalactosamine was degradable by testicular hyaluronidase, indicating that it was chondroitin-like. The ¹⁴C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylglucosamine was not degradable by testicular hyaluronidase. Microsomal preparations from P815S cells were tested for sulphating activity by incubation with adenosine 3′-phosphate 5′-sulphatophosphate, as well as UDP-[¹⁴C]glucuronic acid, and UDP-N-acetylgalactosamine. The resulting newly synthesized polysaccharide was shown by chondroitinase ABC digestion to be 70% chondroitin 4-sulphate and 30% chondroitin. The molecular size of this newly synthesized glycosaminoglycan was determined by gel filtration to be larger than 40000 mol.wt. In general, the glycosaminoglycan-synthesizing ability of the microsomal preparations appeared to reflect glycosaminoglycan synthesis by the intact cells.     

Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 μM and the calibration curve was linear at least up to 5 μM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 μM and from 3.5 to 6.7 at 5 μM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 μM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.     

Microsomal activation of thioacetamide-S-oxide to a metabolite(s) that covalently binds to calf thymus DNA and other polynucleotides

In the presence of NADPH liver microsomes isolated from phenobarbital-pretreated rats catalyze the conversion of [³H]thioacetamide-S-oxide to a reactive intermediate(s) which covalently binds to calf thymus DNA, calf liver RNA, polyguanylic acid (poly(G)) and polyadenylic acid (poly(A)). The highest level of binding of radioactivity was obtained with poly(G), followed by poly(A), RNA and DNA. The incorporation of radioactivity into DNA was linear for 30 min and there was a requirement for NADPH for time-dependent covalent binding to occur. Performing the microsomal incubations in an atmosphere of 80% CO/20% O₂ or adding partially purified anti cytochrome P-450 immune serum to the microsomal incubations inhibited the total metabolism of thioacetamide-S-oxide and had a small, but insignificant, inhibitory effect on binding of radioactivity to calf thymus DNA. Using a reconstituted monooxygenase system containing cytochrome P-450 purified from phenobarbital-treated rats we were unable to detect any metabolism of thioacetamide-S-oxide. Only background levels of radioactivity were incorporated into calf thymus DNA when microsomes isolated from phenobarbital-treated rats were incubated with [³H]thioacetamide in the presence of NADPH. These results suggest that thioacetamide-S-oxide is an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s) which binds to calf thumus DNA.     

(−)-Epilamprolobine and its N-oxide,lupin alkaloids from Sophora tomentosa

From the fresh leaves of Sophora tomentosa, three new lupin alkaloids, (?)-epilamprolobine, (+)-epilamprolobine N-oxide and 5-(3′-methoxycarbonylbutyroyl)aminomethyl-trans-quinolizidine N-oxide, have further been isolated along with (+)-matrine, (+)-matrine N-oxide, (+)-sophocarpine N-oxide, (?)-anagyrine, (?)- baptifoline, (?)-cytisine, (?)-N-methylcytisine, (?)-N-formylcytisine, (?)-N-acetylcytisine and (±)-ammodendrine. The absolute configurations of (+)-epilamprolobine N-oxide (1R:5R:6S) and (?)-epilamprolobine (5R:6S) have also been established by spectroscopic data and by comparison with synthetic (+)-epilamprolobine (5S:6R)derived from (?)-lupinine (5R:6R). (?)-Epilamprolobine is a diastereomer of (+)-lamprolobine (5R:6R) in Lamprolobium fruticosum and 5-(3′-methoxycarbonylbutyroyl) aminomethyl-trans-quinolizidine N-oxide is presumed to be an artefact. A biosynthetic pathway for the formation of (?)-epilamprolobine is also proposed.     

Modulations in the biotransformation of tobacco extract and N′-nitrosonornicotine under differential dietary protein status

The modulation of the phase I and phase II biotransformation enzymes upon treatment with tobacco extract (TE) and N'-nitrosonornicotine (NNN) was investigated using male Sprague-Dawley rats fed differential protein diets. It was observed that the animals fed a low protein diet showed an overall decrease in the basal levels of hepatic and pulmonary phase I and II enzymes. TE and NNN significantly decreased the detoxifying system in the low-proteinfed animals. Animals fed 20% protein, however, showed significant increases in glutathione and glutathione S-transferase upon treatment. Furthermore, TE and NNN treatment brought about a significant depletion in the hepatic pool of vitamin A with a concomitant increase in the vitamin C levels.