A comparative catabolism study of isoleucine by insect and mammalian tissues.

The metabolism of [U-14C]isoleucine was examined in different tissues of five species of lepidopteran and four species of non-lepidopteran insects. Slices of fat body, epidermis, Malpighian tubule, gut, and muscle were incubated in a culture medium containing [U-14C]isoleucine; the medium was analyzed by ion-exclusion LC to quantify labeled metabolites. Tissues of lepidopteran insects secrete high levels of metabolites including 2-keto-4-methylvalerate, 2-methylbutyrate, propionate, and acetate. Tissues of non-lepidopteran insects secrete low amounts of these acids. Analysis of isoleucine transaminase activity in selected tissues of non-lepidopteran insects indicated that those tested contain significant activity. These results demonstrate that tissues of lepidopteran insects have a unique ability to secrete short chain acids, derived from isoleucine, into the medium. The secretion of propionate correlates with the ability to synthesize ethyl-branched juvenile hormones and indicates the presence of an efficient transport system for short chain acids. We also monitored the secretion of acidic metabolites of isoleucine by different tissues of the rat. Muscle was most active in secreting keto acid whereas heart secreted high levels of 2-methylbutyrate. Negligible quantities of metabolites of isoleucine were secreted by the liver.

Formation of cyclic products from the diepoxide of long-chain fatty esters by cytosolic epoxide hydrolase.

Since diepoxides are known metabolites of polyunsaturated fatty acids, the action of the cytosolic epoxide hydrolase purified from liver tissue was examined on these diepoxides. Diepoxymethylstearate was metabolized to the corresponding tetraol by high concentrations of affinity-purified cytosolic epoxide hydrolase. When the enzyme was diluted (1000- to 2000-fold), disappearance of the tetraol metabolite occurred simultaneously with formation of other hydration products with GC retention times and chromatographic mobilities different from those of the tetraol. The hydration products were identified as tetrahydrofuran diols based on comparison of chromatographic properties and mass spectral information with the properties and spectra of chemically generated products. Also, a mixture of diepoxymethylarachidonates was hydrated to tetraols using concentrated enzyme. As the enzyme was diluted (1000- to 2000-fold), a decrease in tetraol formation occurred along with the elevation of other hydration products whose mass spectra were consistent with tetrahydrofuran diol structures. These data are consistent with the epoxide hydrolase at low concentrations acting to open one epoxide followed by nonenzymatic cyclization to the tetrahydrofuran diols. The data also suggest that oxygenated lipids may be endogenous substrates for the cytosolic epoxide hydrolase. Since some oxylipins are known chemical mediators, the in vivo presence and role of these novel diols and tetrahydrofuran diols should be examined.

Reversed-phase liquid chromatographic separation of juvenile hormone and its metabolites, and its application for an in vivo juvenile hormone catabolism study in Manduca sexta.

A convenient reversed-phase liquid chromatographic method was developed to separate juvenile hormone (JH) and its metabolites. The known metabolites including JH acid, JH diol, and JH acid-diol, as well as an unknown metabolite, were efficiently separated within 25 min on a 50 X 4.6 mm polymer column using a linear gradient of acetonitrile:5 mM Hepes (pH 7.4) buffer. Use of the polymer column diminished tailing observed for the diol metabolite on a C18 silica column, and allowed use of slightly basic buffers without concern of column instability. Use of buffer was essential to give good peak shape and reproducible retention behavior for the acidic metabolites. Using this method, an in vivo JH catabolism study was performed in fifth stadium larvae of Manduca sexta. Injected (10R)-[3H]JH III was rapidly converted to JH acid-diol and to an unknown compound(s) indicating that, in addition to JH esterase, epoxide hydrolase and other reactions play an important role in the catabolism of JH.

Heterocyclic derivatives of 3-substituted-1,1,1-trifluoro-2-propanones as inhibitors of esterolytic enzymes.

A series of (alkylthio)trifluoropropanones containing a heterocyclic moiety was synthesized. The compounds were tested for in vitro inhibition of four hydrolytic enzymes including insect juvenile hormone esterase (JHE), eel acetylcholinesterase (AChE), yeast lipase (LP), and bovine alpha-chymotrypsin. The I50 values ranged from 10(-3) to 10(-7) M. 3-(2-Pyridylthio)-1,1,1-trifluoro-2-propanone was found to be the most potent inhibitor as compared to the other tested heterocyclic analogues with an I50 value of 98 nM against JHE from the fifth-instar larvae of Trichoplusia ni. Results from X-ray crystallography showed that the compound exists in a tetrahedral gem-diol form stabilized by an intramolecular hydrogen bond in the solid state. X-ray crystallography of a less potent inhibitor, 3-(4-pyridylthio)-1,1,1-trifluoro-2- propanone, showed that it also exists in the hydrated form, but it lacks an intramolecular hydrogen bond. These results provide indirect support that trifluoromethyl ketones are transition-state mimic inhibitors of esterases, and the bearing of the results on the transition-state mimic theory is discussed. The I50 values against AChE were in the micromolar range. Compounds containing a imidazolyl, triazolyl, and pyrimidyl moiety showed the highest inhibition of this enzyme. Differential selectivity of inhibition was associated with the bond distances between the nitrogen and the carbonyl group as in the natural substrate, when measured in the molecules in their minimal energy conformations. Inhibition of LP was moderate to weak, when compared to JHE and AChE. None of the tested compounds showed significant inhibition of alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Catabolism of epoxy fatty esters by the purified epoxide hydrolase from mouse and human liver.

Epoxymethylsterate, 9,10- and 12,13-epoxymethyloleates, and a mixture of isomers of epoxymethylarachidonate and diepoxymethylstearate were synthesized, and their metabolic rates were measured using crude and purified cytosolic epoxide hydrolase. Hepatic epoxide hydrolase was purified from human samples and clofibrate-fed mice by affinity chromatography. The major metabolites under these conditions of all the epoxy fatty esters were their vicinal diols whose structures were confirmed by GC-MS. 12,13-Epoxymethyloleate was metabolized faster than 9,10-epoxymethyloleate and other epoxy fatty esters, but all substrates were turned over rapidly. This rapid turnover suggests that epoxy fatty acids may be endogenous substrates for the cytosolic epoxide hydrolase.

Microsomal and cytosolic epoxide hydrolase and glutathione S-transferase activities in the gill, liver, and kidney of the rainbow trout, Salmo gairdneri. Baseline levels and optimization of assay conditions.

Microsomal and cytosolic epoxide hydrolase (mEH and cEH respectively) and glutathione S-transferase (GST) activities were measured in the liver, kidney, and gills of rainbow trout. Assays were optimized for time, pH, and temperature, using trans-stilbene oxide (TSO) and cis-stilbene oxide (CSO) as substrates for cEH and mEH, respectively. Optimal pH values for mEH, cEH, and GST were similar to mammalian values (i.e. 8.5, 7.5, and 9). Temperature optima differed between tissues and cell fractions. Specific activity of cEH-TSO was 3-14 times greater than mEH-CSO for all three tissues, and 8-60 times greater on a tissue weight basis. Liver and, to a lesser extent, kidney mEH were active against benzo[a]pyrene 4,5-oxide, whereas gill mEH was not active against this substrate. Liver cytosolic GST was active against CSO and 1-chloro-2,4-dinitrobenzene (CDNB) but not TSO, whereas gill and kidney cytosolic GST were active only against CDNB. Liver and kidney microsomal GST were active against CDNB, but no activity was found in gill microsomes. The results are discussed in relation to possible endogenous substrates and uninduced xenobiotic metabolizing capacities of different trout tissues.

Comparative aspects of propionate metabolism.

1. The catabolism of propionate has been studied extensively in vertebrates and the major pathway has been shown to be its derivatization to propionyl-CoA, carboxylation to D-methylmalonyl-CoA, isomerization to L-methylmalonyl-CoA and then conversion to succinyl-CoA via a vitamin B12 dependent methylmalonyl-CoA mutase. 2. By contrast, in all insect species studied to date, many of which do not contain detectable levels of vitamin B12, the major metabolic pathway of propionate is its conversion to 3-hydroxypropionate and then to acetate. Carbon-3 of propionate becomes the carboxyl carbon of acetate and carbon-2 of propionate becomes the methyl carbon of acetate. 3. A number of species of non-insect arthropods and other invertebrates contain relatively high levels of vitamin B12 and catabolize propionate by the same pathway as that of vertebrates. Under anoxic conditions, some invertebrates, including bivalves, convert succinate to propionate. 4. In plants, evidence has been presented for the metabolism of propionate to both acetate and succinate. Micro-organisms possess a myriad of pathways by which they produce and catabolize propionate.

Metabolism of propionate to acetate in the cockroach Periplaneta americana.

Carbon-13 NMR and radiotracer studies were used to determine the precursor to methylmalonate and to study the metabolism of propionate in the cockroach Periplaneta americana. [3,4,5-13C3]Valine labeled carbons 3, 4, and 26 of 3-methylpentacosane, indicating that valine was metabolized via propionyl-CoA to methylmalonyl-CoA and served as the methyl branch unit precursor. Potassium [2-13C]propionate labeled the odd-numbered carbons of hydrocarbons and potassium [3-13C]propionate labeled the even-numbered carbons of hydrocarbons in this insect. This labeling pattern indicates that propionate is metabolized to acetate, with carbon-2 of propionate becoming the methyl carbon of acetate and carbon-3 of propionate becoming the carboxyl carbon of acetate. In vivo studies in which products were separated by HPLC showed that [2-14C]propionate was readily metabolized to acetate. The radioactivity from sodium [1-14C]propionate was not incorporated into succinate nor into any other tricarboxylic acid cycle intermediate, indicating that propionate was not metabolized via methylmalonate to succinate. Similarly, [1-14C]propionate did not label acetate. An experiment designed to determine the subcellular localization of the enzymes involved in converting propionate to acetate showed that they were located in the mitochondrial fraction. Data from both in vivo and in vitro studies as a function of time indicated that propionate was converted directly to acetate and did not first go through tricarboxylic acid cycle intermediates. These data demonstrate a novel pathway of propionate metabolism in insects.