In vivo formation of sigma-methyl- and sigma-phenyl-ferric complexes of hemoglobin and liver-cytochrome P-450 upon treatment of rats with methyl- and phenylhydrazine

Ferric sigma-phenyl complexes of hemoglobin and liver cytochrome P-450 are formed in vivo upon administration of C6H5NHNH2 to rats. Small amounts of the sigma-methyl complex of hemoglobin were also detected in vivo upon treatment of rats with CH3NHNH2. At the doses used for CH3NHNH2 (25 and 50 mg/kg) the states and levels of hemoglobin in the blood and spleen, and of cytochrome P-450 in the liver were almost unchanged. On the contrary, C6H5NHNH2 (25-100 mg/kg) led to a decrease of the HbO2 blood level (10-50%), together with an increase in the HbFe(III) level and the appearance of the HbFe(III)-C6H5 complex. The concentration of this complex reaches its maximum value (2 mM) 1 h after C6H5NHNH2 administration (20% of total hemoglobin). At the same time large amounts of HbO2, HbFe(III) and HbFe(III)-C6H5 appeared in the spleen, and remained high up to 24 h after treatment. Treatment of rats with C6H5NHNH2 (25-100 mg/kg) led to a significant decrease in the level of liver cytochrome P-450 (a 70% decrease 2 h after treatment with 100 mg/kg C6H5NHNH2). About 15% of the remaining cytochrome P-450 existed as a cyt.-P-450-Fe(III)-C6H5 complex, a new example of cytochrome P-450-Fe-metabolite complex which is stable in vivo.     

Role of cytochrome P-450 in endogenous antipyresis

In previous reports, we (15, 18) and others (29) demonstrated data showing that various inhibitors of cytochrome P-450/epoxygenase augment fever in rats and mice, indicating that the enzyme may be involved in endogenous antipyresis. The aim of this study was to further test the hypothesis that the P-450-dependent epoxygenase pathway of arachidonic acid is part of the homeostatic system to control the height of fever. Sprague-Dawley rats were implanted with biotelemeters to monitor body temperature. Fever was induced by intraperitoneal injection of lipopolysaccharide (LPS; 80 microg/kg). We demonstrate that intraperitoneal administration of P-450 inducers (bezafibrate and dehydroepiandrosterone, 10 and 100 mg/kg) before LPS reduced fever in rats in a dose-dependent manner. In complementary experiments, rats were implanted with brain cannulas in addition to the biotelemeters. Various isomers of epoxyeicosanoids were administered into the lateral ventricle at doses of 0.01 to 10 microg/rat to test their influence on LPS-induced fever in rats. Four of five isomers were antipyretic in a dose-dependent manner. The most potent antipyretic isomers were 11, 12-epoxyeicosatrienoic acid (EET) followed by 14,15-EET, 8,9-EET, and 12(R) hydroxyeicosatetraenoic acid. These data support the hypothesis that the cytochrome P-450/epoxygenase pathway of arachidonate metabolism is part of the endogenous antipyretic system.     

Biphasic effect of methadone on hepatic drug metabolism

When methadone HCl (30 mg/kg, po) was given acutely to mice, it was found to inhibit drug metabolism as evidenced by a prolongation of hexobarbital sleeping time and zoxazolamine paralysis time. Pharmacokinetic studies revealed that this acute dose of the narcotic analgesic could also prolong the plasma half-life of aminopyrine without any change in its volume of distribution. When added to the incubation mixture containing 10,000 g mouse liver supernatant fraction and a complete system for measuring aminopyrine N-demethylase or aniline hydroxylase, methadone showed a dose-dependent inhibition of the enzymes; the former enzyme was inhibited to a greater extent than the latter one. However, subacute treatment of mice with methadone HCl (30 mg/kg, po, twice daily for 3 days) resulted in increases in liver weight, microsomal protein, and cytochrome P-450 content in consonant with the increased activities of four hepatic drug-metabolizing enzymes: aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole, O-demethylase, and benzphetamine N-demethylase. Moreover, both hexobarbital sleeping time and zoxazolamine paralysis time were shortened. The plasma half-life of aminopyrine was decreased. These changes were prevented by simultaneous administration of puromycin diHCl (80 mg/kg, ip). Methadone thus seems to act in a manner very similar to that of propoxyphene or SKF-525A, acting as a potent inhibitor of hepatic drug metabolism when given acutely and as an inducer when given subacutely.     

Cysteamine in combination with N-acetylcysteine prevents acetaminophen-induced hepatotoxicity.

N-Acetylcysteine (NAC) is protective against acetaminophen-induced hepatotoxicity primarily by providing precursor for the glutathione synthetase pathway, while cysteamine has been demonstrated to alter the cytochrome P-450 dependent formation of toxic acetaminophen metabolite. Mice administered acetaminophen (500 mg/kg) had elevations of serum alanine aminotransferase (ALT) to 273.0 +/- 37.5 and 555.8 +/- 193.4 U/mL at 12 and 24 h, respectively, after injection. Administration of cysteamine (100 mg/kg) or NAC (500 mg/kg) significantly reduced serum ALT activity (p less than 0.001). Reducing the dose of NAC or cysteamine by 50% greatly reduced their hepatoprotective effect while the co-administration of the reduced doses of NAC (250 mg/kg) and cysteamine (50 mg/kg) following acetaminophen overdose prevented elevation of serum ALT activity (39.2 +/- 1.17 and 32.5 +/- 5.63 U/mL at 12 and 24 h post-injection, p less than 0.001) and preserved normal mouse hepatic histology. Neither NAC (500 mg/kg), cysteamine (100 mg/kg), or the lower doses in combination of both agents were found to alter the half-life or peak levels of acetaminophen. Liver microsomal aryl hydrocarbon hydroxylase activity measured 24 h after drug administration was not significantly different between treatment groups and controls receiving only saline. These results indicate a possible role for the concomitant use of NAC and cysteamine in the prevention of hepatic necrosis following toxic doses of acetaminophen. Neither decrease in plasma acetaminophen levels nor depression of cytochrome P-450 enzyme activity appears to be the mechanism of protection when these doses of NAC, cysteamine, or both drugs together are administered with a toxic dose of acetaminophen in mice.     

Purification of cytochrome P-450 catalyzing 25-hydroxylation of vitamin D3 from rat liver microsomes

Cytochrome P-450 catalyzing 25-hydroxylation of cholecalciferol (cytochrome P-450 cc25 ) was purified from rat liver microsomes based on its catalytic activity at each purification step. The specific cytochrome P-450 content of the final preparation was 15.1 nmol/mg of protein. Reconstituted activity of 25-hydroxylation of cholecalciferol with the purified enzyme was 2.3 nmol/min/mg of protein, which was 4,300 times as high as that in microsomes. The minimum molecular weight of the enzyme was 50,000 based on SDS-polyacrylamide gel electrophoretogram. Amino terminal sequence of the P-450 cc25 was H2N-Met-Asp-Pro-Val-Leu-Val-. Immunochemical study showed that the purified P-450 cc25 was homogeneous and the cytochrome was immunochemically different from either cytochrome P-450(PB-1) or cytochrome P-448(MC-1).     

Effects of two oral antimycotics, ketoconazole and fluconazole, upon steroidogenesis in rat adrenal cells in vitro

Rat adrenal cells were incubated with various concentrations of two orally active azole antimycotics in order to evaluate the effects on steroidogenesis. The first compound was ketoconazole, a well-known inhibitor not only of fungal cytochrome P-450 but at higher concentrations also of mammalian cytochrome P-450 dependent enzymes. The second was fluconazole, a newly developed oral antimycotic with a triazole structure, which likewise inhibits fungal cytochrome P-450. The influence of both drugs on mammalian cytochrome P-450 dependent enzymes was investigated in this study. Ketoconazole inhibited ACTH-stimulated corticosterone (IC50 = 0.9 microM) and aldosterone secretion (IC50 = 1.4 microM) and enhanced 11-deoxycorticosterone output at low concentrations but reduced it at higher concentrations. Radiotracer experiments with [3H]pregnenolone or [3H]11-deoxycorticosterone as exogenous substrates revealed a 50% inhibition of the oxidative substrate metabolism at about 1 microM ketoconazole. These effects could also be observed with fluconazole but occurred at concentrations approximately two orders of magnitude higher as compared to ketoconazole. We conclude that fluconazole has a much higher selectivity for fungal cytochrome P-450 than ketoconazole. The order of sensitivity of the cytochrome P-450 dependent enzymes of rat adrenal steroidogenesis to ketoconazole was the 11 beta/18-hydroxylase, the cholesterol side chain cleavage enzyme and the 21-hydroxylase with decreasing sensitivities.     

Aflatoxin B1-4-hydroxylase is associated with cytochrome P3-450 in C57BL/6 mouse liver

Messenger RNA from the livers of Aroclor 1254 treated mice was used to produce a cDNA library. cDNA clones corresponding to cytochromes P1-450 and P3-450 were isolated from this library by screening with a probe for the rat cytochrome P-450c gene. Specific non-cross hybridizing probes for P1-450 and P3-450 were prepared from unique restriction fragments. The radiolabeled probes were hybridized to RNA from mice treated with a low (15 mg/kg) and high (150 mg/kg, 400 mg/kg) doses of beta-naphthoflavone. The low dose of beta-naphthoflavone was found to induce only P3-450 mRNA, whereas higher doses induced both P1-450 and P3-450 mRNA. Similarly, a low dose of beta-naphthoflavone induced aflatoxin B1-4-hydroxylase, whereas higher doses induced both aflatoxin B1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. These results suggest that P3-450 mRNA codes for the cytochrome that is associated with aflatoxin B1-4-hydroxylase activity.     

Purification and comparative characterization of cytochrome P-450scc from porcine adrenocortical mitochondria.

Cytochrome P-450scc (cholesterol side-chain cleavage enzyme) was purified from porcine adrenocortical mitochondria. 2. The purified cytochrome P-450scc was found to be homogeneous on SDS-polyacrylamide gel electrophoresis. 3. The heme content of the purified enzyme was 20.6 nmol/mg protein. 4. The enzymatic activity of the reconstituted cytochrome P-450scc-linked monooxygenase system amounted to 7.8 nmol of pregnenolone formed per nmole of P-450 per minute, with cholesterol as a substrate. 5. The amino acid sequence of the amino-terminal region of the cytochrome P-450scc and the amino acid residue at the carboxyl terminal were determined and compared with those of other mammalian cytochromes P-450scc.     

Attenuation of low dose phenobarbital induction of hepatic microsomal aminopyrine N-demethylase activity in rats by cimetidine

Cimetidine, a substituted imidazole, is an inhibitor of hepatic cytochrome P-450-mediated drug metabolism in rats and humans. We investigated the effect of cimetidine on phenobarbital induction of hepatic microsomal aminopyrine N-demethylase activity in the rat. Phenobarbital induction of aminopyrine N-demethylase was log-linear in the range of 1-6 mg/kg/day and the ED50 was approximately 3 mg/kg/day. Cimetidine 75 mg/kg (four times a day) attenuated the induction of aminopyrine N-demethylase activity by 58% in low dose (3 mg/kg/day) but not in high dose (40 mg/kg/day) phenobarbital treated rats. This result could not be explained by residual inhibition of enzyme activity by cimetidine and suggests that cimetidine affects the induction of hepatic cytochrome P-450 by low dose phenobarbital.     

The effect of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes]

Effects of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes have been studied. Pyridine (200 mg/kg) increased total cytochrome P-450 content and activated metabolism of some specific substrates 24 hours after injection. There was an increase in the degree of p-nitrophenol and chlorzoxazone hydroxylation due to increasing ethanol-induced cytochrome P-450IIE1 content. Pyridine was also able to induce cytochrome P-450IIB1 in rat microsomes; this reaction was accompanied by acceleration of 7-pentoxyresorufin 0-dealkylation. Cytochrome P-450IA1 appearance in liver microsomes was associated with increasing content of cytochrome P-450IA2. Dealkylation rates for specific substrates (7-ethoxyresorufin and 7-methoxyresorufin) were also increased. Similar to pyridine, pyridine-7-oxide induced cytochromes P-450IIE1, P-450IIB1/B2, and P-450IA1/A2, resulting in activation of specific substrate metabolism. Hence, pyridine and its derivative pyridine-N-oxide can be regarded as effective inducers of cytochrome P-450.     

Musk xylene is a novel specific inducer of cytochrome P-450IA2.

The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.     

Inducers of cytochrome P-450d: influence on microsomal catalytic activities and differential regulation by enzyme stabilization

The interaction of isosafrole, 3,4,5,3',4',5'-hexabromobiphenyl (HBB) and hexachlorobiphenyl (HCB) with cytochrome P-450d was evaluated by characterization of estradiol 2-hydroxylase activity. Displacement of the isosafrole metabolite from microsomal cytochrome P-450d derived from isosafrole-treated rats resulted in a 160% increase in estradiol 2-hydroxylase. The increase was fully reversed by incubation with 1 microM HBB. Although isosafrole is capable of forming a complex with many different cytochrome P-450 isozymes, it appears to bind largely to cytochrome P-450d in vivo as was demonstrated by measuring the enzymatic activity of microsomal cytochromes P-450b, P-450c, and P-450d from isosafrole-treated rats. When estradiol 2-hydroxylase was measured in rats treated with increasing doses of HCB, there was a gradual decrease in microsomal enzyme activity despite a 20-fold increase in cytochrome P-450d. The ability of cytochrome P-450d ligands to stabilize the enzyme was investigated in two ways. First, cytochromes P-450c and P-450d were quantitated immunochemically in microsomes from rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a dose which maximally induced total cytochrome P-450, followed by a single dose of a second inducer. The specific content of cytochrome P-450d was significantly increased when isosafrole or HCB was the second inducer but not when 3-methylcholanthrene was the second inducer. Second, the relative turnover of cytochrome P-450d was measured by the dual label technique. Following TCDD treatment, microsomal protein was labeled in vivo with [3H]leucine, the second inducer was given and protein was again labeled 3 days later with [14C]leucine. A higher ratio of 3H/14C in the cytochrome P-450d from isosafrole + TCDD- and HCB + TCDD-treated rats relative to TCDD (control)-treated rats suggested that isosafrole and HCB were able to retard the degradation of cytochrome P-450d, presumably by virtue of being tightly bound to the enzyme.     

Treatment with poly I.C. enhances lipid peroxidation and the activity of xanthine oxidase, and decreases hepatic P-450 content and activities in mice and rats

Treatment of mice and rats with polyriboinosinic acid-polyribocytidylic acid (poly I.C., 5 mg/kg i.p.), a potent interferon inducer, decreased hepatic cytochrome P-450 system content and activities without influencing P-450-independent xenobiotic metabolizing enzymes. Treatment with poly I.C. decreased the content of P-450 by 28% in mice (P less than 0.05) and 30% in rats (P less than 0.05) but did not alter the activity of cytochrome c reductase. With treatment of poly I.C., the activity of XO increased 87% in mice (P less than 0.01) and 30% in rats (P less than 0.01). Lipid peroxidation was enhanced by 82% in mice (P less than 0.01) and 95% in rats (P less than 0.05). These results raise the possibility that a part of the depression of P-450 system content and activities by poly I.C. might be caused by enhanced lipid peroxidation associated with increased activity of XO.     

Microphotometric analysis of cytochrome P-450 in periportal, midzonal, and perivenular hepatocytes of mice treated with phenobarbital

To obtain detailed information on the increase of cytochrome P-450 (P-450) content in periportal, midzonal, and perivenular hepatocytes after phenobarbital (PB) administration, and to study the mechanism of increased P-450 in the endoplasmic reticulum (ER), we estimated microphotometrically the P-450 content and morphometrically the area of ER in hepatocytes of three zones from mice injected with 35, 50, 100, or 150 mg/kg of PB for 3 days. The amount of P-450 per unit cytoplasmic volume and the number of P-450 molecules per unit ER area (P-450 number) were increased by injection of 50, 100, or 150 mg/kg, and the ER area per unit cytoplasmic volume was increased by injection of 100 or 150 mg/kg, in hepatocytes from all three zones. Thus, the amount of P-450 in hepatocytes appeared in general to increase multiplicatively by simultaneous increases in both the P-450 number and the ER area. Furthermore, we could recognize two general types of relationship in the P-450 number and ER area between the patterns of change and the increasing doses: (a) increase in the P-450 number without ER proliferation (active type) in periportal and perivenular hepatocytes after injection of low doses; and (b) increase in ER proliferation without increase in the P-450 number (passive type) in hepatocytes of all three zones after injection of high doses.     

Induction of cytochrome P-450 isozymes by mirex and chlordecone

The effect of the insecticides, mirex and chordecone (Kepone), on the cytochrome P-450 monooxygenase system in C57BL/6N mouse liver microsomes was studied. Mice were treated intraperitoneally with low (6 mg/kg) and high (30 mg/kg) doses of mirex and chlordecone in corn oil for 2 days. For comparison, mice were also treated with either phenobarbital (PB) or 3-methylcholanthrene (3-MC). All treatments significantly increased the hepatic microsomal P-450 content over that of controls. Benzphetamine N-demethylase, ethoxyresorufin O-deethylase, benzo[a]pyrene hydroxylase, and acetanilide hydroxylase activities were also determined. Mirex and chlordecone resembled phenobarbital with respect to the induction of monooxygenase activities. Immunoquantitation with antibodies to purified P-450 IIB1 (Pb-induced P-450) and P-450 IA1 (3-MC-induced P-450) indicated that mirex and chlordecone induced P-450 IIB1 in a dose-dependent manner. The high dose of mirex also induced a small amount of a protein cross reacting with the antibody to IA1. The induction of this isozyme did not, however, contribute significantly to the monooxygenase activities measured.     

Drug-induced protoporphyria in the olfactory mucosa of the hamster

Administration of 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine (4-ethyl-DDC) to hamsters resulted in a marked loss of cytochrome P-450-dependent reactions (peroxidase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase) in both liver and olfactory epithelium within 2 hr. This inactivation of cytochrome P-450 was accompanied by inhibition of ferrochelatase (FK), stimulation of 5-aminolevulinate synthase (ALA-S), and accumulation of protoporphyrin both in the liver and to a lesser degree, in the olfactory epithelium. These results suggest that the mechanism of induction of protoporphyria in nasal tissues is similar to that occurring in the liver, namely, suicidal metabolism of 4-ethyl DDC by cytochrome P-450 resulting in formation of N-ethylprotoporphyrin, a potent inhibitor of FK. The consequent depletion of heme leads to stimulation of ALA-S and, thus, porphyrin accumulation. Investigation of the dose-response to 4-ethyl DDC demonstrated that, in liver, maximal inhibition of FK and accumulation of protoporphyrin occurred at a dose of 50 mg/kg while ALA-S activity continued to increase up to a dose of 100 mg/kg. This is compatible with an additional effect of the drug on ALA-S involving induction of cytochrome P-450 and, thus, further depletion of heme. In the olfactory epithelium, stimulation of ALA-S was significantly less marked, suggesting that this secondary effect does not operate in nasal tissue. This is consistent with reports that olfactory cytochrome P-450s are noninducible.     

Comparison of the inducing effect of triphenyldioxan, bis-(dichloropyridyloxy)benzene and phenobarbital on the liver monooxygenase

The induction by triphenyldioxane (TPD) of cytochrome P-450 in rat liver microsomes was studied. It was demonstrated that TPD injection in a single dose (10 mg/kg of body mass) is associated with a marked induction of cytochromes P-450 b/e (cytochrome PB-forms) in rat liver microsomes and a significant increase in the benzphetamine-N-demethylase activity typical of cytochrome P-450b. In other words, TPD is a potent inducer of PB-type, the inducing effect being attained by an injection of a single dose of TPD which is by one order of magnitude less than that of phenobarbital. It can be assumed that this compound shows a high affinity for the hypothetical receptor responsible for cytochrome P-450b synthesis. It was shown also that TPD does not induce the monooxygenase system of mouse liver, whereas 1,4-bis[2-(dichloropyridyloxy)]benzene (DPB) is a potent inducer of PB-type in mice, being fairly ineffective in rats. Hence, the species-specific effect of TPD and DPB appears to be opposite.     

Isolation of a cytochrome P-450 that catalyzes the 25-hydroxylation of vitamin D3 from rat liver microsomes

A molecular species of cytochrome P-450 that catalyzes the 25-hydroxylation of cholecalciferol (P-450cc25) was purified from rat liver microsomes on the basis of its catalytic activity. The purification procedure consisted of polyethylene glycol fractionation, and column chromatographies on octylamino Sepharose 4B, hydroxylapatite, DEAE-Sepharose CL-6B, and CM-Sepharose CL-6B. The specific cytochrome P-450 content of the final preparation was 17.0 nmol/mg of protein. The enzymatic activity was reconstituted with the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, an NADPH-generating system, and dilauroylglyceryl-3-phosphorylcholine, the specific activity obtained being 3.7 nmol/min/mg of protein, which was 4,000 times as high as that in microsomes. The apparent molecular weight of the P-450cc25 was 50,000, based on the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis. The absorption spectra of the oxidized form of the enzyme showed a Soret band at 416 nm, which is typical of the low spin state of cytochrome P-450, and alpha and beta bands at 570 and 536 nm, respectively. The Soret peak of the reduced cytochrome P-450-CO complex was at 450 nm. The purified enzyme not only catalyzed the 25-hydroxylation of cholecalciferol but also showed hydroxylation activity toward a variety of substrates, i.e. 1 alpha-hydroxycholecalciferol (at 25), testosterone (at 2 alpha and 16 alpha) and dehydroepiandrosterone (at 16 alpha). Amino terminal sequence of the purified cytochrome P-450 was determined by the manual sequence method to be H2N-Met-Asp-Pro-Val-leu-Val-Leu-Val-. The antibody elicited against the purified enzyme in a rabbit inhibited the cholecalciferol 25-hydroxylation activity by more than 90% with a concentration of 2 mg of immunoglobulin per nmol of cytochrome P-450.     

Responses to insulin by two forms of rat hepatic microsomal cytochrome P-450 that undergo major (RLM6) and minor (RLM5b) elevations in diabetes

Total cytochrome P-450 levels rise in diabetic rats. Two specific forms of cytochrome P-450 that are elevated have been isolated from liver microsomes of streptozotocin-induced idabetic male rats. One enzyme, termed RLM6, metabolizes aniline and acetol, but not testosterone, in a reconstituted system with NADPH-cytochrome P-450 reductase. RLM6 is isolated as a high spin cytochrome with a minimum molecular weight of 53,500. It has a unique amino-terminal amino acid sequence lacking methionine at the amino-terminal position. Polyclonal antibodies to RLM6 recognized most other forms of cytochrome P-450 in Western blots, but could be made monospecific by adsorption to cross-reacting proteins coupled to Sepharose 4B. Using the monospecific antibodies, RLM6 was estimated to be present in microsomes of untreated male rats at 0.04 nmol/mg protein (5% of total P-450). In chronically diabetic rats this level rose to 0.35 nmol/mg protein and 24% of the P-450 content. Immunoreactive protein of molecular weight identical to RLM6 was elevated in microsomes of non-diabetic rats treated with ethanol, acetone, or isoniazid as well as in rats starved for 48 h. Insulin treatment of diabetic rats for 1 week lowered the immunologically detectable levels of RLM6 to levels found in the untreated rat. The other form of cytochrome P-450, RLM5b, does not metabolize aniline and only poorly metabolizes acetol and testosterone. This 52.5-kDa protein is isolated as a predominantly (60%) high spin enzyme. It has a unique NH2-terminal amino acid sequence with methionine as the terminal residue, and is present in untreated male rat liver microsomes at 0.16 nmol/mg protein. It is elevated in diabetes, like RLM6, but treatment with insulin for 1 week does not completely restore the microsomal content to that of the non-diabetic rat.