Rat Liver Microsomal Fraction Research Articles

CHANGES IN INOSITOL 1,4,5-TRIPHOSPHATE BINDING IN MICROSOMAL FRACTIONS FROM THE RAT LIVER DURING SEPSIS

Inositol 1,4,5-triphosphate has been proposed as a second messenger for calcium mobilization. The addition of inositol 1,4,5-triphosphate at a low concentration has been shown to cause calcium release from intracellular microsomal stores in rat hepatocytes. The effects of sepsis on the inositol 1,4,5-triphosphate binding from microsomal fractions of rat liver were investigated. Sepsis was induced by cecal ligation and puncture (CLP). Control rats were sham operated. Three microsomal fractions (rough, intermediate, and smooth I) were isolated from the rat liver. The study of inositol 1,4,5-triphosphate receptor binding was performed with tritium-labeled inositol 1,4,5-triphosphate. Our results showed that the Bmax of inositol 1,4,5-triphosphate binding in early septic, late septic, and control groups was 14.9 +/- .9 fmol/mg, 9.8 +/- 1.0 fmol/mg, and 17.2 +/- 1.3 fmol/mg, respectively. The binding activity was unaffected during early sepsis but was significantly depressed by 40-50% (p < .05, vs. control) during late sepsis (18 h after CLP) in all three subfractions of endoplasmic reticulum. Because the inositol 1,4,5-triphosphate binding plays an important role in the regulation of intra-cellular calcium homeostasis in hepatocytes, an impairment of the calcium release due to depressed inositol 1,4,5-triphosphate binding in the endoplasmic reticulum may have a pathophysiological significance in contributing to altered hepatic metabolism during septic shock.

Intracellular Sorting of Lysosomal β-Glucuronidase Is Altered Due to Administaration of Dibutyl Phosphate1

Organophosphate compounds are known to cause a selective increase of beta-glucuronidase activity in rat serum. Previous data suggested that increase of serum beta-glucuronidase activity was well correlated with decrease of that activity in rat liver microsomal fraction, thereby, suggesting a role of the microsomal enzyme in mediating the organophosphate effect. To investigate further the intracellular sorting pathway of beta-glucuronidase in dibutyl phosphate-treated rats, liver subcellular fractions were prepared at 12 or 48 h after in vivo administration of [3H]leucine and it was established that microsomal beta-glucuronidase was the origin of the increased serum enzyme. To characterize the intracellular secretory pathway of beta-glucuronidase in dibutyl phosphate-treated rats, Golgi subfractions were isolated and a time course study was carried out. At 30 min after administration of dibutyl phosphate, specific activity of beta-glucuronidase in GF-2 (Golgi intermediate fraction) and GF-3 (Golgi heavy fraction) was significantly increased to the maximum. Furthermore, colchicine pretreatment of rats caused a delay of the peak of specific activity for 30 min in GF-2 and GF-3, and accumulation of enzyme activity in Golgi subfractions was observed. Colchicine pretreatment also had an inhibitory effect on release of beta-glucuronidase into serum until 30 min after dibutyl phosphate injection. The electrophoretic pattern of microsomal beta-glucuronidase on polyacrylamide gel was found to show two major bands of microsomal enzyme type and lysosomal enzyme type in dibutyl phosphate-treated rats. Taken together, these findings indicate that microsomal beta-glucuronidase follows the intracellular secretory pathway and is secreted into serum via Golgi complex in response to dibutyl phosphate.

The influence of glutathione and detoxifying enzymes on DNA damage induced by 2-alkenals in primary rat hepatocytes and human lymphoblastoid cells.

The reaction of 2-alkenals with GSH to form GSH conjugates by Michael addition is a major detoxification pathway. The reaction proceeds at a much higher rate under catalysis by glutathione S-transferase (GST) than the non-enzymatic reaction. Oxidation of 2-alkenals to the corresponding acids by cytosolic and microsomal fraction of rat liver also contributes to detoxification. Primary rat hepatocytes rich in GSH and proficient for GST and other metabolizing enzymes consume much more alkenal than human lymphoblastoid cells (Namalva cells), that are poor in GSH and in metabolic activities. In Namalva cells DNA single strand breaks were induced by much lower concentrations of acrolein, crotonaldehyde and (E)-2-hexenal than in primary rat hepatocytes. In both cell systems intracellular GSH depletion by 2-alkenals proceeds in a dose dependent manner, approaching about 20% of pretreatment level before DNA damage becomes detectable. GSH conjugates of (E)-2-hexenal and (2E,6Z)-2,6-nonadienal induce DNA damage in Namalva cells at high concentrations (1.5 mM). In the absence of GSH these conjugates decompose slowly into aldehyde and GSH. Although the rate of decomposition is only about 10(-4) times that of Michael adduct formation, such GSH conjugates could potentially function as transport molecules for 2-alkenals, if they reach tissues low in GSH and GST.

Bacterial ribonuclease: mutagenic effect in microbial test-systems.

Pure enzyme samples of ribonuclease from Bacillus intermedius 7P (known commercially as 'binase') were investigated for genotoxicity in four microbial tests: the Ames plate incorporation method, AraR-assay; the prophage induction test; and the DNA-repair test. The weak mutagenic effect of binase at high concentrations (0.1 mg/plate, 1 mg/plate) was established by induction of forward AraR-mutations and histidine-reverse mutations (both frameshift mutations and base pair substitution). Metabolic activation with rat or chicken liver, human placenta or plant (from tulip bulbs) microsomal fractions in vitro was seen to abolish the binase mutagenicity. Bacillus intermedius 7P ribonuclease appears to possess DNA damaging activity in uvrA- and polA- mutants, but not in the recA-deficient Escherichia coli strain, and exhibits an induction of recA-dependent mutagenesis detected by the 8-fold increase of the prophage-induction level in lysogenic Bacillus subtilis culture and by the 5-fold increase of this level in the Streptomyces lavendulae 3 lysogenic strain. The importance of the roles of both of enzyme catalytic activity and native structure is emphasized. A proposed mechanism for exogenous ribonuclease action is discussed. Bacillus intermedius 7P ribonuclease probably does not act as a direct genotoxic agent interacting with DNA, but could provoke nucleotide imbalance through its catalytic action on membrane-associated RNAs, which results in alteration of DNA replication and, as a consequence, in recA-dependent mutagenesis.

Differences in the kinetic properties, effect of calcium and sensitivity to inhibitors of paraoxon hydrolase activity in rat plasma and microsomal fraction from rat liver

The properties of a rat hepatic microsomal enzyme that hydrolyses O, O-diethyl- p-nitro-phenylphosphate (paraoxon) were studied and compared to the paraoxon hydrolase activity found in rat plasma. The pH stability for both enzyme activities was optimum between pH 6.0 and 9.0. An overall analysis of the data showed that the microsomal fraction was less resistant to the effect of the pH than plasma. The kinetic constants for heat inactivation evaluated for paraoxonase in rat plasma and liver microsomal fraction indicate that paraoxonase tends to inactivate faster in rat liver microsomes than in rat plasma. The apparent activation energies of the heat inactivation process were 77.7 and 61.1 kcal/mol for rat plasma and microsomal fraction, respectively. Enzyme activity was lost after both dialysis and incubation with EDTA and partially restored by the addition of calcium. In rat plasma samples the requirement for calcium was absolute (essential activator) while in the microsomal fraction the reaction may occur, to a minimum extent, in the absence of the activator (non-essential activator). Calcium restored 85% activity when added immediately after EDTA; restored activity decreased when the time interval between addition of EDTA and calcium was increased. Other metals were not able to restore activity previously inhibited by EDTA or dialysis. The response to several inhibitors (EDTA, Mn, Co, Zn, Ba, Mg, Cu, La, Hg and p-hydroxy-mercuribenzoate) of rat plasma and microsomal fraction was studied, determining the type of inhibition and the inhibition constants. Plasma enzyme was always more resistant than liver sample to the effect of the inhibitors and showed different types of inhibition than the liver microsomal fraction. In general we found more differences than analogies between the rat plasma and liver enzymes which suggests the presence of two enzymes or two different forms of the same enzyme. Furthermore the existence of an EDTA-resistant fraction in rat liver microsomes suggests that more than one enzyme capable of hydrolysing paraoxon is present in the microsomal fraction of rat liver.

Direct detection of radical generation in rat liver nuclei on treatment with tumour-promoting hydroperoxides and related compounds

EPR spin trapping has been employed to directly detect radical production in isolated rat nuclei on exposure to a variety of hydroperoxides and related compounds which are known, or suspect, tumour promoters. The hydroperoxides, in the absence of reducing equivalents, undergo oxidative cleavage, generating peroxyl radicals. In the presence of NADPH (and to a lesser extent NADH) reductive cleavage of the OO bond generates alkoxyl radicals. These radicals undergo subsequent rearrangements and reactions (dependent on the structure of the alkoxyl radical), generating carbon-centred radicals. Acyl peroxides and peracids appear to undergo only reductive cleavage of the OO bond. With peracids this cleavage can generate aryl carboxyl (RCO 2·) or hydroxyl radicals (HO·); with acyl peroxides, aryl carboxyl radicals are formed and, in the case of t-butyl peroxybenzoate, alkoxyl radicals (RO·). The radicals detected with each peroxide are similar in type to those detected in the rat liver microsomal fraction, although the extent of radical production is lower. The subsequent reactions of the initially generated radicals are similar to those determined in homogenous chemical systems, suggesting that they are in free solution. Experiments with NADPH/NADH, heat denaturation of the nuclei and various inhibitors suggest that radical generation is an enzymatic process catalysed by haemproteins, in particular cytochrome P-450, and that NADPH/cytochrome P-450 reductase is involved in the reductive cleavage of the OO bond. The generation of these radicals by the rat liver nuclear fraction is potentially highly damaging for the cell due to the proximity of the generating source to DNA. Several previous studies have shown that some of the radicals detected in this study, such as aryl carboxyl and aryl radicals, can damage DNA, via various reactions which results in the generation of strand breaks and adducts to DNA bases: these processes are suggested to play an important role in the tumour promoting activity of these hydroperoxides and related compounds.

Mutagenicity tests of polyoxyethylene hydrogenated castor oil 60 (HCO-60).

The genotoxicity of polyoxyethylene hydrogenated castor oil 60 (HCO-60) was examined in reverse mutation test in bacteria, chromosome aberration test in vitro and micronucleus test in mice. The in vitro tests were done with and without metabolic activation using rat liver microsomal fraction. HCO-60 did induce neither reverse mutation in Salmonella typhimurium TA100, TA98, TA1535, and TA1537 and in Escherichia coli WP2uvrA, nor chromosome aberrations in Chinese hamster V79 cells. In addition, no increase in micronucleated polychromatic erythrocytes was elicited in the bone marrow of BDF1 male and female mice. It is concluded that HCO-60 was not genotoxic in these in vitro and in vivo assays.

Polycyclic aromatic hydrocarbons (PAHs) and mutagenicity of soot particulates in air emissions from two-stage incineration of polystyrene

Polycyclic aromatic hydrocarbons (PAHs) and mutagenicity of particulates from incineration of polystyrene were analyzed by GC/MSD and TA-98 Ames test, respectively. The results of this investigation indicate that a critical temperature exists of 1000 °C–1100 °C in the stage- two incinerator for the formation of PAHs and soot particulates. The major precursor of PAHs formation (i.e., C 6H 6) is produced in large amounts in nitrogen atmosphere when the stage-two temperature rises to 900 °C. The mutagenicity of particulates and polyurethane foam (PUF) extracts increased as the pyrolysis temperature was increased from 900 to 1200 °C. The mutagenicity of PUF extracts is weaker than the muta- genicity of particulates extracts. Moreover, the mutagenicity of samples is much higher in the presence of rat liver microsomal fraction (S9 mixture) than in its absence.

Comparison of co-cultivation of V79 cells with rat hepatocytes and rat H4IIE hepatoma cells for studying nitrosamine-induced hprt gene mutations.

The induction of mutations by nitrosamines in the hprt locus of V79 Chinese hamster cells was examined after metabolic activation in a co-cultivation system using either freshly isolated rat hepatocytes or H4IIE rat hepatoma cells and the results obtained were compared with systems which employ the rat liver microsomal fraction (S9-mix). This study was also designed as a first approach to investigating the induction of point mutations by tobacco-specific nitrosamines in mammalian cells in order to obtain information about the significance of these compounds in connection with the carcinogenicity of tobacco smoke. The mutagenicity of two tobacco-specific nitrosamines, 4-(methylnitroso)-1-(3-pyridol)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), were investigated and compared to two extensively investigated nitrosamines, i.e. dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). DMN was activated to mutagenic species by primary hepatocytes at mumolar concentrations, i.e. 1/100 of the concentrations required for mutagenesis by DEN and NNK. NNN was not activated to mutagenic species by liver S9 or primary hepatocytes. The findings shown here on the mutagenicities of NNK and NNN with liver preparations are in agreement with their relative carcinogenic potencies. When the established liver cell line H4IIE was used for metabolic activation, DMN and was found to be mutagenic, whereas the results for NNN were borderline and for DEN and NNK were without effect. The fate of these compounds via different metabolic pathways is discussed in terms of systems for detection of mutagenic metabolites and type of mutation induced.

Substrate stereoselectivity and enantiomer/enantiomer interaction in propranolol metabolism in rat liver microsomes

The substrate stereoselectivity and enantiomer/enantiomer interaction of ( S)- and ( R)-propranolol for the formation of their metabolites were investigated in rat liver microsomal fractions. The enantiomers of primary metabolites of propranolol, 4-, 5-, 7-hydroxy- and N-desisopropylpropranolol were separated and assayed by an HPLC method employing a chiral ovomucoid column. Regioselective substrate stereoselectivity ( R < S for 4- and 5-hydroxylations; R > S for - 7-hydroxylation; R = S for N-desisopropylation) was observed in the formation of propranolol metabolites when the individual enantiomers or a racemic mixture of propranolol were used as substrates. Concentration-dependent metabolic inhibition of propranolol enantiomers by their optical isomers was also observed. In addition, the inhibition of propranolol 4-, 5- and 7-hydroxylations between the enantiomers showed a typical competitive nature. These findings suggested that the propranolol enantiomers competed for the same enzyme, probably a cytochrome P450 isozyme in the CYP2D subfamily.

Effect of the Ionophore A23187, Thapsigargin, Caffeine and Heparin on Phosphatidylserine Synthesis in Rat Liver Microsomal Fraction

It has been shown that the incorporation of [ 14C]serine into phosphatidylserine in rat liver microsomal fractions is distinctly stimulated by ATP and Mg 2+ at low (10 μM) Ca 2+ concentration. This stimulation occurred in untreated microsomes, but did not occur in fully disrupted microsomal vesicles. The stimulatory effect of ATP and Mg 2+ on phosphatidylserine synthesis was reduced by calcium ionophore A23187 and by thapsigargin, which is a specific blocker of Ca 2+-ATPase. It was also diminished by caffeine and enhanced by heparin, agents known to modulate Ca 2+ release by receptor channels. It is therefore postulated that the synthesis of phosphatidylserine occurs on the luminal side of the endoplasmic reticulum and can be regulated by events responsible for Ca 2+ release and entry into these structures.

Radical production from peroxide and peracid tumour promoters: EPR spin trapping studies

EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals from a number of organic peroxides and peracids which are known or suspected tumour promoters. All of the compounds when incubated with rat liver microsomal fractions in the presence of NADPH or NADH are metabolised to radical species which can be detected, and in most cases identified definitively, as the corresponding spin adducts; the assignment of particular signals to certain spin adducts has been confirmed by photolytic experiments. In the majority of cases, the predominant species are arenecarbonyloxyl [RC(O)O·] and hydroxyl radical adducts. The mechanism of formation of the former species is shown to be enzymatic and cytochrome P-450 dependent and requires the presence of reducing equivalents. This type of radical is shown to undergo ready decarboxylation to give aryl radicals in aggreement with previous chemical studies. The detection of these radical species, which are known to cause DNA strand breaks and be cytotoxic, with all the compounds tested, provides strong supportive evidence for the theory that it is the generation of radical species from these compounds which is the cause of their tumour-promoting activity.

Studies on the Metabolic Fate of Leukotriene Antagonist ONO-1078. (4). Metabolism and Protein Binding.

ONO-1078(4-oxo-8-[4-(4-phenylbutoxy)benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate)のラット，モルモットおよびイヌにおける代謝について検討した．また，血漿(あるいは血清)蛋白との結合性について検討を行うとともに，併用を予想される相手薬剤との蛋白結合の相互作用についても検討した． 1．ラットでの代謝物は未吸収で糞中へ排泄された未変化体を除いて，ほとんどすべてアミド結合が水解された代謝物であった．また，ラットの代謝物とイヌの代謝物は異なっており，種差が認められた． 2．ラットの主たる代謝物としてアミド結合が水解されたMC-0およびMH-0の他，MH-0がタウリン抱合されたMH-1，そしてMH-0が水酸化後にタウリン抱合されたMH-2が同定された．また，MH-0の一部はトリグリセリドとして脂肪組織中へ取り込まれた． 3．モルモットではラットと同様にアミド結合が水解された代謝物を認めた． 4．イヌではアミド結合は水解されず，主たる代謝物はフェニル基の水酸化されたM-1およびM-2，そしてこれらの硫酸抱合体のM-3であった． 5．ONO-1078のアミド結合は，ラットおよびモルモットの肝ミクロゾーム分画で速やかに加水分解を受けた．また，ラット肝臓中に存在する水解酵素はcarboxylesteraseであると推定した．しかし，イヌでは肝ミクロゾーム分画において水解されなかった． 6．ラット肝ミクロゾーム分画にcarboxylesterase阻害剤の存在下，イヌの代謝物であるM-1およびM-2の生成を認めた． 7．経口投与後の血漿(あるいは血清)中放射能の蛋白結合率は，ラットでは97.1～98.9%，イヌでは76.1～84.1%であった．ONO-1078の蛋白結合率はラット，イヌ，ヒトのいずれの血清蛋白とも99.7%以上であった． 8．ヒト血清蛋白を用いた薬物相互作用の検討で，ONO-1078は併用を予想される他剤により蛋白結合率の変化を受けず，他剤の結合率にも影響を与えなかった．

Glycyrrhetinic acid bound to 11β-hydroxysteriod dehydrogenase in rat liver microsomes

A binding protein which exibits high affinity to [ 3H]glycyrrhetinic-acid in the rat liver microsomal fraction was solubilized with 0.2% Triton DF-18 and then purified to homogeneity. The equilibrium dissociation constant of the [ 3H]glycyrrhetinic-acid binding reaction and the maximal concentration for the binding of the purified protein, as determined by Scatchard plot analysis, were 27.6 nM and 7.79 nmol/mg protein, respectively. The molecular mass of the subunit (34 kDa) and 30 amino acids of N-terminal sequence of the purified protein were entirely the same as those of the reported 11β-hydroxysteriod dehydrogenase (11β-HSD). In each purification step, the recovery and purification (fold) of the glycyrrhetinic-acid binding activity corresponded to the values of 11β-HSD activity. These results show that the purified [ 3H]glycyrrhetinic-acid binding protein is 11β-HSD. From the molecular mass of 11β-HSD (135 kDa) and the maximal concentration of the binding site, it was calculated that one glycyrrhetinic acid molecule binds to one 11β-HSD molecule. The inhibitory effects of various glycyrrhetinic-acid derivatives on [ 3H]glycyrrhetinic acid binding and 11β-HSD activity indicate that the C 30-carboxyl and C 11-carbonyl groups of glycyrrhetinic acid are the principal structures for the 11β-HSD inhibition.

Acetylene, a mammalian metabolite of 1,1,1-trichloroethane.

1,1,1-Trichloroethane (TCE) is a widely used industrial solvent of low acute toxicity. It is slowly oxidized to trichloroethanol and trichloroacetic acid by cytochrome P-450-dependent mono-oxygenases. Increased inhalative uptake by rats under hypoxia and spin-trapping experiments indicate that TCE is also reductively metabolized to a radical intermediate. Acetylene is formed as a metabolite, suggesting transfer of an additional electron to form the corresponding carbene. Hypoxia and induction of mixed-function mono-oxygenases accelerate the formation of acetylene. Experiments performed in vitro with rat liver microsomal fractions yield analogous results.

The topography of trifluoroacetylated protein antigens in liver microsomal fractions from halothane treated rats

Sera from patients with halothane hepatitis contain immunoglobulin G (IgG) antibodies to trifluoroacetylated liver microsomal proteins of 100, 76, 59, 57 and 54 kDa, which are produced as a consequence of metabolism of halothane to trifluoroacetyl halide by cytochrome(s) P450. In the present study, the membrane topographies of the various antigens in rat liver microsomal fractions were investigated. Liver microsomal fractions from rats treated with halothane in vivo, and rat liver microsomal fractions which had been incubated with halothane in vitro, were used as the source of tri-fluoroacetyl antigens. The antigens were detected by immunoblotting. Whereas the 100, 76, 59 and 57 kDa antigens were solubilized from the microsomal membrane by either 0.1 M sodium carbonate or 0.1% (w/v) sodium deoxycholate, the 54 kDa antigen was not solubilized by 0.1% (w/v) sodium deoxycholate. In intact microsomal fractions, the 100, 76, 59 and 57 kDa antigens were not degraded appreciably by trypsin unless detergent was added to permeabilize the microsomal membrane. These results indicate that the 54 kDa antigen is an integral membrane protein, whereas the 100, 76, 59 and 57 kDa antigens are peripheral membrane proteins situated within the lumen of microsomal vesicles, and hence presumably located within the lumen of the endoplasmic reticulum in vivo.

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 μg 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.

Immobilization of solubilized UDP-glucuronosyltransferase from rat liver microsomes to Sepharose 4B

A method for the covalent binding of rat liver UDP-glucuronosyltransferase to a cyanogen bromide-activated agarose matrix is described. the rat liver microsomal fraction was solubilized with 8 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); 90% of the microsomal protein was solubilized. Some 50–60% of this protein became bound covalently to the activated agarose matrix. the immobilized UPD-glucuronosyltransferase remained completely active for 50 days when stored at 4° in a 20% (v/v) glycerol buffer (pH7.4). the immobilized enzyme has a temperature optimum around 37°, and a broad pH optimum (pH 5–7.4). the enzyme displayed linear kinetics over a period of 1 hr; it conjugates a large variety of substrates.

Detection of radicals produced by reaction of hydroperoxides with rat liver microsomal fractions

EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals produced on reaction of a number of primary, secondary and lipid hydroperoxides with rat liver microsomal fractions in both the presence and absence of reducing equivalents. Two major mechanisms of radical generation have been elucidated. In the absence of NADPH or NADH, oxidative degradation of the hydroperoxide occurs to give initially a peroxyl radical which in the majority of cases can be detected as a spin adduct to DMPO; these radicals can undergo further reactions which result in the generation of alkoxyl and carbon-centered radicals. In the presence of NADPH (and to a lesser extent NADH) alkoxyl radicals are generated directly via reductive cleavage of the hydroperoxide. These alkoxyl radicals undergo further fragmentation and rearrangment reactions to give carbon-centered species which can be identified by trapping with DBNBS. The type of transformation that occurs is highly dependent on the structure of the alkoxyl radical with species arising from β-scission, 1,2-hydrogen shifts and ring closure reactions being identified; these processes are in accord with previous chemical studies and are characteristic of alkoxyl radicals present in free solution. Studies using specific enzyme inhibitors and metal-ion chelators suggest that most of the radical generation occurs via a catalytic process involving haem proteins and in particular cytochrome P-450. An unusual species (an acyl radical) is observed with lipid hydroperoxides; this is believed to arise via a cage reaction after β-scission of an initial alkoxyl radical.

NMR assignments of territrems A, B, and C and the structure of MB2, the major metabolite of territrem B by rat liver microsomal fraction.

The 1H- and 13C-nmr assignments of territrems A, B, and C [1-3] were made by using nOe and 2D nmr techniques. Following the same methods, the structure of MB2 [4], the major product of territrem B incubated with rat liver microsomal fraction, was determined as a hydroxylation product at the pro S methyl group of C-4 of territrem B.