Metabolism of acetanilide with hepatic microsomes and reconstituted cytochrome monoxygenase systems

Hepatic miscrosomes and reconstituted cytochrome monoxygenase systems from control rats and from rats that have been pretreated with phenobarbital or 3-methylcholanthrene convert radioactive acetanilide to ring-hydroxylated products, primarily 4-hydroxyacetanilide. 3-Methylcholanthrene pretreatment results in the greatest enhancement of activity: cytochrome fractions from 3-methylcholanthrene-pretreated rats have many-fold higher activity than cytochrome fractions from control or phenobarbital-treated rats. The percentage of migration and retention of tritium (NIH Shift) measured in 4-hydroxyacetanilide after enzymatic oxidation of 4-[³H]acetanilide is nearly identical using microsomes or the corresponding reconstituted system, but in both cases the percentage of migration and retention of tritum is markedly lower for preparations from 3-methylcholanthrene-treated animals with values of 25%, as compared to the values of 40–60% for preparations from control or phenobarbital-treated animals. High-pressure liquid chromatography was employed for separation and quantitation of radioactive products.     

Failure of 3-hydroxy-3-phenylpropanoic acid and cinnamic acid to serve as precursors of tropic acid in Datura innoxia

Datura innoxia plants were fed the R- and S-isomers of [3-¹⁴C]-3-hydroxy-3-phenylpropanoic acid, and [3-¹⁴C]cinnamic acid along with dl-[4-³H]phenylalanine. The hyoscyamine and scopolamine isolated from the plants 7 days later were labeled with tritium, but devoid of ¹⁴C, indicating that 3-hydroxy-3-phenylpropanoic acid and cinnamic acid are not intermediates between phenylalanine and tropic acid. The [³H] tropic acid obtained by hydrolysis of the hyoscyamine was degraded and shown to have essentially all its tritium located at the para position of its phenyl group, a result consistent with previous work.     

Cyclization and hydroxylation stereochemistry in the biosynthesis of gibberellic acid

In Gibberella fujikuroi cultures, ent-[3β-³H,17-¹⁴C]kaurene is converted to gibberellic acid with retention of the tritium label at the 3α-position. This evidence for the stereochemistry of 3-hydroxylation also permits the stereochemistry of the ‘proton-initiated’ cyclization step in gibberellic acid biosynthesis to be deduced.     

A highly sensitive radiometric assay for zoxazolamine hydroxylation by liver microsomal cytochrome P-450 and P-448: properties of the membrane-bound and purified reconstituted system.

A radiometric assay for the in vitro metabolism of zoxazolamine has been developed which combines high sensitivity and rapid determination of product. [4,6-³H]zoxazolamine was metabolized to 6-hydroxyzoxazolamine, and the tritium released as ³H₂O was determined after treating the incubation mixture with activated charcoal. This treatment efficiently removes labeled substrate (99.98%), permitting enzymatically released tritium to be measured directly in the aqueous medium. Since the preponderant in vitro product of zoxazolamine metabolism by rat liver microsomes and the purified reconstituted mixed function oxidase system is 6-hydroxyzoxazolamine, and since this aryl hydroxylation occurs without significant NIH shift, the subsequent release of tritium from the 6-position accurately represents metabolism of the molecule. The use of [4,6-³H]zoxazolamine for a tritium release assay of mixed function oxidase activity is ideal since this compound shows no significant isotope effect or NIH shift during metabolic conversion to 6-hydroxyzoxazolamine. 3-Methylcholanthrene treatment of rats resulted in a fourfold induction of zoxazolamine hydroxylation while phenobarbital or pregnenolone 16α-carbonitrile pretreatment caused only a 20–50% increase in zoxazolamine metabolism. The use of a purified reconstituted system revealed that cytochrome P-448 from 3-methylcholanthrene-treated rats was approximately 10- to 15-fold more efficient than cytochrome P-450 from phenobarbital-treated rats in catalyzing the hydroxylation of zoxazolamine.     

Monooxygenase activity in Cunninghamella bainieri: evidence for a fungal system similar to liver microsomes

The fungus Cunninghamella bainieri effects the oxidative N-demethylation of aminopyrine, O-demethylation of 4-nitroanisole and anisole, the aryl hydroxylation of anisole, aniline, and naphthalene, and the reduction of nitro and azo groups. The hydroxylation of 4-[²H]-anisole and 2-[²H]-anisole proceeds with migration and retention of isotopic hydrogen (NIH shift). The above reactions and the formation of the trans-dihydrodiol of naphthalene and the incorporation of oxygen-18 from ¹⁸O₂ into the trans-dihydrodiol and hydroxylated anisole are characteristic of reactions catalyzed by the cytochrome P₄₅₀ monooxygenases of hepatic microsomes. The product ratios in these hydroxylations are very similar to those obtained using liver microsomes providing further evidence that the C. bainieri monooxygenase enzymes are similar to the liver monooxygenases. Furthermore, an epoxide hydrase enzyme similar to that present in hepatic microsomes must also be present in C. bainieri.     

Metabolism of all-trans-retinyl acetate to retinoic acid in hamster tracheal organ culture

All-trans-[³H]retinyl acetate has been shown to be metabolized to all-trans-[³H]retinoic acid in a target tissue of vitamin A action, the hamster trachea in organ culture. That the compound produced is indeed all-trans-retinoic acid is demonstrated by chromatography of the biosynthetically produced retinoic acid with synthetic all-trans-retinoic acid in two different high-pressure liquid chromatographic systems, either as the free acids in a reverse-phase system or as the methyl esters in a normal-phase system. The all-trans-[³H]retinoic acid was also found in the tracheal epithelium and cartilage as well as in the medium. In addition the tracheal tissue also contained retinyl palmitate and other esters. Finally, further in vitro metabolism of [³H]retinyl acetate paralleled the metabolism of [³C]retinoic acid suggesting that these two compounds are being metabolized through similar pathways.     

Trichodiene biosynthesis and the stereochemistry of the enzymatic cyclization of farnesyl pyrophosphate

Cyclization of trans,trans-[1-³H₂,12,13-¹⁴C]farnesyl pyrophosphate (2a) by a preparation of trichodiene synthetase isolated from the fungus, Trichothecium roseum, gave trichodiene (5a), which was shown by chemical degradation to retain both tritium atoms of the precursor at C-11. Incubation of 1S-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2b) and 1R-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2c) with trichodiene synthetase and degradation of the resulting labeled trichodienes, 5b and 5c, established that the displacement of the pyrophosphate moiety from C-1 of the precursor and formation of the new C-C bond in the formation of trichodiene takes place with net retention of configuration. These results are accounted for by an isomerization-cyclization mechanism involving the intermediacy of nerolidyl pyrophosphate (4).     

Metabolism of [2-3H]- and [6-3H]-nicotinic acid in intact Nicotiana tabacum plants

Earlier observations of Dawson on the relative incorporation of [2-³H]- and [6-³H]-nicotinic acid into nicotine have been confirmed in intact Nicotiana tabacum plants. All the tritium in the nicotine derived from [2-³H]-nicotinic acid was located at C-2 of the pyridine ring. However the radioactive nicotine derived from [6-³H]-nicotinic acid was not labelled specifically at C-6 with tritium. By carrying out feeding experiments with [6-¹⁴-C, 2-³H]- and [6-¹⁴C, ³H]-nicotinic acids, it was established that there was very little loss of tritium from C-2 and C-6 of nicotinic acid during 5 days of metabolism in the tobacco plant.     

Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : III. Conversion of 4-Methoxycinnamic Acids into 4-Hydroxybenzoic Acids

Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid and 3,4,5-trimethoxycinnamic acid to cell suspension cultures of Vanilla planifolia resulted in the formation of 4-hydroxybenzoic acid, vanillic acid, and syringic acid, respectively. The homologous 4-methoxybenzoic acids were demethylated to the same products. It is concluded that the side chain degrading enzyme system accepts the 4-methoxylated substrates while the demethylation occurs at the benzoic acid level. The demethylating enzyme is specific for the 4-position. Feeding of [O-¹⁴C-methyl]-3,4-dimethoxycinnamic acid revealed that the first step in the conversion is the glycosylation of the cinnamic acid to its glucose ester. A partial purification of a UDP-glucose: trans-cinnamic acid glucosyltransferase is reported. 4-Methoxy substituted cinnamic acids are better substrates for this enzyme than 4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxy substituted cinnamic acids are intermediates in the biosynthetic conversion of cinnamic acids to benzoic acids in cells of V. planifolia.     

The 2-hydroxylation of trans-cinnamic acid by chloroplasts from Melilotus alba Desr

Chloroplasts isolated from sweetclover leaves contain an enzyme which converts trans-[3-¹⁴C]cinnamic acid to 2-hydroxy-trans-[3-¹⁴C]cinnamic (o-coumaric) acid. The identity of the product has been verified by recrystallization with unlabeled o-coumaric acid to constant specific activity, and by gas-liquid cochromatography of unlabeled o-coumaric acid and the radioactive product.The enzyme has an optimum of pH 7.0 and its activity can be enhanced ~ 4-fold by adding 4 mm glucose-6-phosphate to the reaction mixture. Light can replace glucose-6-phosphate, presumably as a source of reducing power required for the hydroxylation system. It was found that approximately 50% of the hydroxylase activity is bound to the lamellar membranes, from which it can be released by sonication.     

Metabolism of phenylpropanoids in Hydrangea serrata var. thunbergii and the biosynthesis of phyllodulcin

DL-Phenylalanine-[3-¹⁴C] and cinnamic acid-[3-¹⁴C] were fed to this plant and the label from cinnamic acid was incorporated into gallic acid, phyllodulcin and quercetin. By feeding p- coumaric acid-[U-³H], caffeic acid-[U-³H] and hydrangea glucoside A-[U-³H], it was possible to show that hydroxylation at C-3′in phyllodulcin occurs after the ring closure of dihydroisocoumarin. The biosynthetic pathway of phyllodulcin in this plant is thus: phenylalanine → cinnamic acid → p- coumaric acid → hydrangenol → phyllodulcin.     

Early stages of ecdysteroid biosynthesis: The role of 7-dehydrocholesterol

The synthesis of [4-¹⁴C]cholesta-4,6-dien-3-one and [4-¹⁴C]3β-hydroxy-5α-cholestan-6-one is described. Both [4-¹⁴C]cholest-4-en-3-one and [4-¹⁴C]cholesta-4,6-dien-3-one were not incorporated significantly into ecdysteroids compared to [1α,2α-³H]cholesterol in fifth instar and maturing adult female Schistocerca gregaria. Similarly, [4-¹⁴C]3β-hydroxy-5α-cholestan-6-one was not incorporated significantly in the latter system. The results suggest that none of the three ¹⁴C-substrates are intermediates in ecdysteroid biosynthesis from cholesterol, although possible complications from permeability barriers cannot be discounted. [4-¹⁴C, 7-³H]7-dehydrocholesterol has been synthesized and incorporated into ecdysteroids in adult female Schistocerca gregaria and in Spodoptera littoralis pupae. Although approximately half the tritium was eliminated during ecdysteroid synthesis in S. gregaria, there was essentially complete retention of the tritium in Spodoptera. The results support the direct incorporation of 7-dehydrocholesterol into ecdysteroids and not via cholesterol. A possible explanation for the loss of appreciable tritium in S. gregaria is discussed.     

Substrate stereochemistry of isovaleryl-CoA dehydrogenase elimination of the 2-pro-R hydrogen in biotin-deficient rats

(2R)-[³H]Isovaleric acid and (2S)-[³H]isovaleric acid (ammonium salts) have been synthesized. These substances, mixed with [1-¹⁴C]isovalerate, have been administered to biotin-deficient rats, which accumulate β-hydroxyisovaleric acid in their urine, the metabolite being formed via isovaleryl-CoA and β-methylcrotonyl-CoA. The results show that most of the tritium from (2R)-[³H]isovalerate was lost, and most of the tritium from (2S)-[³H]isovalerate retained in the conversion to β-hydroxyisovalerate. The stereochemistry of the isovaleryl-CoA dehydrogenase reaction is compared with the stereochemistry of other short-chain acyl-CoA dehydrogenase reactions.     

The use of a tritium release assay to measure 6-N-trimethyl-L-lysine hydroxylase activity: synthesis of 6-N-[3-3H]trimethyl-DL-lysine

6-N-[3-³H]Trimethyl-dl-lysine was synthesized from 6-N-acetyl-l-lysine by the following chemical scheme: 6-N-acetyl-l-lysine → 2-keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid oxime → 6-N-[3-³H]acetyl-dl-lysine → dl-[3-³H]lysine → 2-N-[3-³H]formyl-dl-lysine → 2-[3-³H]formyl-6-N-trimethyl-dl-lysine → 6-N-[3-³H]trimethyl-dl-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rat kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-l-lysine hydroxylase activity as measured by tritium release from 6-N-[3-³H]trimethyl-dl-lysine were: α-ketoglutarate, ferrous ions, l-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-[3-³H]trimethyl-dl-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-l-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.     

A criterion to determine whether cis-4-hydroxyproline is produced in animal tissues

Hydrolyzates of tissues that had been labeled with [¹⁴C]proline often contain significant amounts of cis-4-hydroxy[¹⁴C]proline. Since animal cells do not contain an enzyme which can effect formation of cis-4-hydroxyproline, there are only two possible explanations for its presence. Either it is formed during acid hydrolysis of trans-4-hydroxyproline (which is synthesized by cells and is a common constituent of connective tissues), or it is produced by a nonenzymatic mechanism such as attack by oxygen radicals. It is important to resolve this issue because if a nonenzymatic mechanism is active in connective tissues, then it will be necessary to reevaluate currently accepted ideas about production of hydroxyproline. This communication describes a method for distinguishing between the two alternate explanations. Tissues or cells are labeled with [¹⁴C]proline, and then a known amount of trans-4-hydroxy[³H]proline is added to each sample before hydrolysis; the relative amounts of [¹⁴C]- and [³H]-cis-4-hydroxyproline are compared after hydrolysis. It is known from a separate series of measurements with mixtures of [¹⁴C]- and [³H]-trans-4-hydroxyproline standards that there is a very high correlation (r = 0.998) between acid-induced formation of the [¹⁴C]- and [³H]-cis epimers. One can thus compare the amount of cis-4-hydroxy[¹⁴C]proline in a hydrolyzate from a biological system with the amount that would be expected if it were all formed during acid hydrolysis. This method was used to show that fibroblasts cultured under conditions commonly used to study collagen metabolism do not produce cis-4-hydroxyproline. This result strongly suggests that nonenzymatic hydroxylation does not normally occur in cell culture systems.     

Biosynthesis of azetidine-2-carboxylic acid in Convallaria majalis

Convallaria majalis plants were fed dl-methionine-[1-¹⁴C]. [1-¹⁴C, 4-³H], and [1-¹⁴C, 2-³H], S-adenosyl-l-methionine-[1-¹⁴C], and dl-homoserine-[1-¹⁴C], resulting in the formation of labeled azetidine-2-carboxylic acid (A-2-C). The complete retention of tritium relative to carbon-14 in the feeding experiment involving methionine-[1-¹⁴C, 4-³H] indicates that aspartic acid or aspartic-β-semialdehyde are not intermediates between methionine and A-2-C. However, since the A-2-C derived from methionine-[1-¹⁴C, 2-³H] had lost 95% of the tritium relative to the C-14, it is not considered that methionine or its S-adenosyl derivative are the immediate precursors of A-2-C. Our data and that of others is consistent with the intermediate formation of γ-amino-α-ketobutyric acid which on cyclization yields 1-azetine-2-carboxylic acid, A-2-C then being formed on reduction.     

Brief Report: Differential oxidations of estradiol-17β by the chimpanzee in vivo

The metabolism of estradiol-17β is primarily an oxidative process at either carbon-2 or carbon-16 in the human. The objective of this study was to determine the relative importance of these two oxygenation pathways in the chimpanzee. The rate of oxidation of estradiol-17β at each position was determined by measuring the release of tritium into body water from carbon-2 or carbon-16. [2-³H]-Estradiol-17β or [16-³H]-estradiol-17β was injected intravenously into three adult male chimpanzees, and blood samples were obtained at several time intervals between 1 and 48 hr. The blood was lyophilized, and the release of tritium from the specifically labeled estrogens into the body fluid pool was determined. The release of tritium from the 16α-position was very low and did not exceed 3% in any animal. The release of tritium from the carbon-2 was much faster, amounting to 29%, 34%, and 35%, respectively, by 24 hr. The ratio of tritium released from carbon-2:carbon-16 was 5.0, 13.2 and 16.9, respectively, at 24 hr after injection of the specifically labeled estradiol-17β. These results demonstrate clearly that the major pathway for oxidative metabolism of estradiol-17β in the chimpanzee is via oxygenation at carbon-2, with the formation of catechol estrogens, as in the human.     

The synthesis of tritiated ribosylzeatin with high specific activity

Ribosylzeatin tritiated on the purine ring with a specific activity of 829 × 10¹⁰ Bq (22 mCi) per millimole was easily prepared by allylic oxidation of N⁶-(Δ²-isopentenyl)[2,8-³H]adenosine which had been synthesized by alkylation of commercially available [2,8-³H]adenosine. This allylic oxidation gave mainly the trans-isomer which was obtained free of the cis-isomer and with a radiochemical purity of 99.8% by a one-step purification using reversed-phase HPLC. This simple procedure yields ribosylzeatin, the most common naturally occurring cytokinin, labelled with tritium at near maximum specific activity.     

DNA crosslinks,single-strand breaks and effects on bacteriophage T4 survival from tritium decay of [2-3H]adenine, [8-3H]adenine and [8-3H]guanine

Escherichia coli and bacteriophage T4 DNA containing [2-³H]adenine accumulated crosslinks between the complementary strands. For T4 DNA stored in frozen solution there were 0.41 to 0.54 crosslinks formed per tritium decay. The crosslinks were demonstrated both by an increased DNA sedimentation rate in alkaline sucrose gradients and by an increasing amount of DNA that renatured quickly after denaturation by heat or alkali. Single-strand breaks were also formed with an efficiency of 0.08 to 0.50 breaks per tritium decay. DNA containing both [8-³H]adenine and [8-³H]guanine showed no crosslinking but did undergo single-strand breaks at a rate of 0.08 per tritium decay. T4 bacteriophage containing [2-³H]adenine lost plaque-forming ability when stored at 4 °C, with 0.34 lethal hits per tritium decay, whereas the same phage labeled with a mixture of [8-³H]adenine and [8-³H]guanine sustained only 0.12 lethal hits per tritium decay. The loss of plaque-forming ability in the latter case is probably due to a radiation effect from the emitted beta particle; the high lethal efficiency for tritium decay at 2-adenine is probably caused either by crosslinks between complementary strands or from some undetected lesion produced in the DNA.     

Measurement of intracellular collagen degradation

Intracellular degradation of newly synthesized collagen is quantitated by incubating fibroblasts with [¹⁴C]proline and determining the percentage of total [¹⁴C]hydroxyproline that is present in a low molecular weight fraction. Several problems make this difficult. (1) Commercial [¹⁴C]proline is often contaminated with [¹⁴C]hydroxyproline and must be purified before use. (2) Salt and [¹⁴C]proline interfere with the determination of [¹⁴C]hydroxyproline in the low molecular weight fraction and must be removed by preparative ion-exchange chromatography. (3) Epimerization of trans- to cis-hydroxyproline during acid hydrolysis is variable and must be taken into account. (4) Loss of [¹⁴C]hydroxyproline during processing varies; [³H]hydroxyproline can be used as an internal measure of recovery, even though tritium may be lost during hydrolysis. An analytic cation-exchange resin is used for the final quantitation of [¹⁴C]hydroxyproline in the low and high molecular weight fractions. With these methods, degradation of newly synthesized collagen can be determined with a precision of ± 3%.