Trihydroxy Metabolites Research Articles

Escherichia coli biotransformation of daidzein fermentation products from soy‐based foods—relevance to food oestrogenicity‐based functionality

SummaryOestrogenic isoflavones (e.g. daidzein) present in soy‐based foods are likely to be important in food functionality. Biotransformation of daidzein during fermentation‐based soy‐containing food manufacture forms three trihydroxy metabolites, 6‐ortho‐hydroxydaidzein (6‐OHD), 8‐ortho‐hydroxydaidzein (8‐OHD) and 3′‐ortho‐hydroxydaidzein (3′‐OHD) which alters the oestrogenicity of the final food product. We report that Escherichia coli (a key component of the gut microbiome) metabolism converts 8‐OHD to 6‐OHD with a likely concomitant increase in oestrogenicity. This means that the functionality of 8‐OHD‐containing soy‐based foods is altered by gut microbiome metabolism. This change in oestrogenicity‐based food functionality might have benefits for postmenopausal women, while being a feminising health risk for males and could add to the risk of oestrogen‐mediated precocious puberty in girls.

Regiospecific and Stereospecific Triangulation of the Structures of Metabolites Formed by Sequential Metabolism at Multiple Prochiral Centers

Structures of in vivo secondary metabolites of a norbornane-containing drug candidate with multiple prochiral centers were triangulated, in a regio- and stereospecific fashion, using in vitro metabolism data from synthetic primary metabolites and in vivo metabolism data from the separate administration of a radiolabeled primary metabolite, [(14)C]-(S)-2-((1R,2S,4R,5S)-5-hydroxybicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (M1). A mass balance study on the 11β hydroxysteroid dehydrogenase type 1 enzyme inhibitor [(14)C]-(S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221) in rats was dosed at 2 mg/kg. Radioactivity was excreted mainly in urine. Metabolites of AMG 221 were quantified by high-performance liquid chromatography with radiometric detection and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS revealed at least 38 metabolites. Seven monohydroxylated metabolites mediated formation of the other 31 metabolites. Twenty-eight metabolites were identified regio- and stereo-specifically. Little parent drug was observed in urine or feces. Monohydroxy metabolite M1 was the major metabolite comprising 17 to 24% of excreted dose, and seven monohydroxy metabolites comprised 29 (male) and 37% (female) of dose. Of 11 quantifiable isobaric dihydroxy metabolites that comprised 8.3 (male) and 24% (female) of dose, 10 were identified regio- and stereospecifically by triangulation. A single trihydroxy metabolite comprised approximately 10% of dose. Complex secondary metabolism of drugs with multiple prochiral centers can be elucidated in a regio- and stereospecific fashion without NMR through synthesis and in vitro and in vivo studies on the metabolism of chiral primary oxidation products.

Detection of JWH-018 metabolites in smoking mixture post-administration urine

Smoking mixtures containing the cannabimimetic indoles may still be available over-the-counter in several countries. Due to the high affinity of these compounds to the cannabinoid receptors, their effective dose is lower than that of the marijuana products resulting in a low concentration of the excreted metabolites accompanied by a higher psychoactive potency. Up to now the in vivo metabolism of the cannabimimetic indoles seems to be insufficiently investigated and no data have been published on an assay of JWH-018 in urine. In this publication the urinary metabolites of JWH-018 are reported. Using gas and liquid chromatography combined with tandem mass spectrometry two main monohydroxylated metabolites were identified in the forensic urine samples. Based on the differences in their electron ionization MS/MS spectra it is supposed that one is formed by hydroxylation of the indole ring whilst the other by hydroxylation of the N-alkyl chain. The main metabolites are almost completely glucuroconjugated, whereas minor ones ( N-despentyl hydroxy-, carboxy-, dihydroxy-, and reduced di- and trihydroxy metabolites) were also present in the free fraction. The parent compound was not detected in urine.

Metabolism of Vitamin D2 to 17,20,24-Trihydroxyvitamin D2 by Cytochrome P450scc (CYP11A1)

As well as catalyzing the conversion of cholesterol to pregnenolone for steroid synthesis, cytochrome P450scc (P450scc) can also metabolize vitamins D2 (D2) and D3 (D3). Two products of D2 metabolism by P450scc, 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2, have been identified and shown to exert biological activity on cultured keratinocytes. The aim of this study was to fully characterize the metabolism of D2 by P450scc, including identifying additional products and determining the kinetics of D2 metabolism. Two new products were isolated by reverse-phase high-performance liquid chromatography: a dihydroxy metabolite with a hydroxyl group at C20 plus another unidentified position, and a trihydroxy metabolite identified by NMR as 17,20,24-trihydroxyvitamin D2. Kinetics of D2 metabolism was determined with substrate solubilized by 2-hydroxypropyl-beta-cyclodextrin or incorporated into phospholipid vesicles. In 2-hydroxypropyl-beta-cyclodextrin, D2 was hydroxylated at C20 with a k(cat)/K(m) 5-fold lower than that for cholesterol metabolism. 20-Hydroxyvitamin D2 was hydroxylated with a similar k(cat)/K(m) to D2, whereas 17,20-dihydroxyvitamin D2 was hydroxylated with a lower k(cat)/K(m) than that for D2 in 2-hydroxypropyl-beta-cyclodextrin. In vesicles, D2 displayed a high K(m) relative to that for cholesterol, but hydroxylation resulted in products that could be further hydroxylated with relatively low K(m) values. We conclude that P450scc catalyzes three sequential hydroxylations of D2 producing 20-hydroxyvitamin D2, 17,20-dihydroxyvitamin D2, and 17,20,24-trihydroxyvitamin D2, which dissociate from the active site of P450scc and accumulate in the reaction mixture. D2 metabolism occurs with lower efficiency (k(cat)/K(m)) than that observed for both cholesterol and D3 metabolism by P450scc.

A study of the serum carotenoids of eight cases of hypercarotenemia in Sri Lanka

Over-consumption of absorbable carotenoids causes hypercarotenemia. Although hypercarotenemia is detected in Sri Lanka, a detailed study on this condition has not been carried out previously. Two millilitres of venous blood was drawn from hypercarotenemic patients (n=8) and examined by high-performance liquid chromatography for carotenoids and vitamin A. A common high-performance liquid chromatographic pattern in serum was shown by six of the cases with β-carotene (9.9–35.7 µg/dl), β-cryptoxanthin and monohydroxy metabolites collectively (5.3–48.5 µg/dl), and six to eight metabolites of dihydroxy, trihydroxy and polyhydroxy metabolites (22.5–282.1 µg/dl). Vitamin A levels were within the normal range (32–61 µg/dl). However, two cases identified were abnormal. The first of these showed low β-carotene (3.5 µg/dl) and no β-cryptoxanthin and monohydroxy metabolites, but normal dihydroxy, trihydroxy and polyhydroxy metabolites (128.2 µg/dl). However, the vitamin A level was high (75.2 µg/dl). The other case showed high β-carotene (212.3 µg/dl) and β-cryptoxanthin (49.3 µg/dl) but no normal monohydroxy, dihydroxy, trihydroxy and polyhydroxy metabolites. Instead there was an atypical metabolite (343.9 µg/dl). According to the present study, excessive intake of boiled, homogenized carrot and ripe papaw is the main causative factor for hypercarotenemia. Over-consumption of carotenoids-rich plant foods may be complicated in the case of individuals having defects of either the control of the 15,15′-dioxygenase activity or metabolism of carotenoids.

Interaction of Methoxychlor and Related Compounds with Estrogen Receptor α and β, and Androgen Receptor: Structure-Activity Studies

We previously demonstrated differential interactions of the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane (HPTE) with estrogen receptor alpha (ERalpha), ERbeta, and the androgen receptor (AR). In this study, we characterize the ERalpha, ERbeta, and AR activity of structurally related methoxychlor metabolites. Human hepatoma cells (HepG2) were transiently transfected with human ERalpha, ERbeta, and AR plus an appropriate steroid-responsive luciferase reporter vector. After transfection, cells were treated with various concentrations of HPTE or structurally related compounds in the presence (for detecting antagonism) and absence (for detecting agonism) of 17beta-estradiol and dihydrotestosterone. The monohydroxy analog of methoxychlor, as well as monohydroxy and dihydroxy analogs of 2, 2-bis(p-hydroxyphenyl)-1,1-dichloroethylene, had ERalpha agonist activity and ERbeta and AR antagonist activity similar to HPTE. The trihydroxy metabolite of methoxychlor displayed only weak ERalpha agonist activity and did not alter ERbeta or AR activities. Replacement of the trichloroethane or dichloroethylene group with a methyl group resulted in a compound with ERalpha and ERbeta agonist activity that retained antiandrogenic activities. This study identifies some of the structural requirements for ERalpha and ERbeta activity and demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that simultaneously act as agonists or antagonists through one or more hormone receptors.

Neurokinin A-induced contraction of guinea-pig isolated trachea: potentiation by hepoxilins.

1. Hepoxilin A3 (8R and 8S isomers) (HxA3), hepoxilin A3-C (8R and 8S isomers) (HxA3-C) and trioxilin A3 (8S isomer) (TrXA3, the stable derivative of HxA3) were tested for their effects on helicoidal strips of guinea-pig isolated tracheae. 2. None of the compounds (10(-9)-10(-6) M) tested had a direct effect on resting tension of trachea. 3. HxA3 (8S) and HxA3-C (8R) (10(-8) M) produced a significant leftward shift of the log concentration-response curves to neurokinin A (NKA) (EC50 (nM), control = 29.0 +/- 2.8, HxA3 (8S) = 21.7 +/- 3.7, HxA3-C (8R) = 13.8 +/- 3.8, n = 6 for each). Also the maximal response to NKA was significantly increased when the tissues were exposed to these hepoxilins (% of the maximal response to NKA, control = 100, HxA3 (8S) = 114.5 +/- 5.3, HxA3-C (8R) = 139.0 +/- 6.2, n = 6 for each). The threshold concentrations for both hepoxilins was 10(-8) M and their effects were dose-related. 4. Stereochemical specificity was observed. The 8S-isomer of HxA3 was active in potentiating the NKA-induced contraction of the trachea while the 8R isomer was inactive. In contrast, the 8R isomer of HxA3-C was active while the 8S isomer was inactive. The trihydroxy metabolite of the active isomer of HxA3 (8S), i.e. TrXA3 (8S) (10(-6) M), was inactive in potentiating the NKA-induced contraction of the trachea. 5. It is concluded that hepoxilins sensitize the guinea-pig isolated trachea to the potent bronchoconstrictor, NKA.

Hepoxilin A 3 (HxA 3) is formed by the rat aorta and is metabolized into HxA 3-C, a glutathione conjugate

In this paper we describe the release of hepoxilin A 3 (HxA 3) by intact pieces of the rat thoracic aorta and its stimulation by exogenous arachidonic acid but not by the calcium ionophore A23187. Homogenates of the rat aorta metabolize HxA 3 via two competing pathways; one involves hepoxilin epoxide hydrolase to form the trihydroxy metabolite, trioxilin A 3 (TrXA 3), and a second pathway involves conjugation of HxA 3 with glutathione via glutathione S-transferase to form a glutathione conjugate, which we refer to as hepoxilin A 3-C (HxA 3-C), a name based upon the accepted nomenclature for the glutathione conjugate leukotriene C. The formation of HxA 3-C was dependent on the presence of reduced glutathione in the incubation medium. HxA 3-C formation was greatly enhanced in the presence of TCPO, an epoxide hydrolase inhibitor which blocks utilization of the substrate via hepoxilin epoxide hydrolase. Comparison of HxA 3-C formation by several arteries and veins indicated that glutathione conjugation was more evident in veins than arteries. The aorta from spontaneously hypertensive rats was essentially similar in HxA 3-C formation to aorta from local normotensive Wistar rats although the aorta from the normotensive Wistar Kyoto rats was much more active than aorta from either of the two other rat types. The biological activity of HxA 3 and HxA 3-C was investigated on isolated helicoidal strips of the rat aorta. While both compounds were inactive on their own, HxA 3 and to a lesser extent HxA 3-C potentiated the contractile response induced by norepinephrine. The present results provide evidence of the presence in rat aorta of a new pathway of arachidonic acid metabolism whose products may possess potential regulatory properties on vascular tissue.

Metabolism studies of the antischistosomal drug praziquantel using tandem mass spectrometry: Distribution of parent drug and ten metabolites obtained from control and schistosome-infected mouse urine

The collisionally activated dissociation spectrum of the antischistosomal drug praziquantel (PZQ) has many structure-specific fragmentations which permit identification of PZQ and seventeen of its hydroxylated metabolites in mouse urine. These fragmentations may also be used to quantify the metabolic pattern of PZQ. In the present study, a triple-quadrupole mass spectrometer system has been used to generate [M + H]+ ions for PZQ and its monohydroxy, dihydroxy and trihydroxy metabolites which yield daughter ions capable of quantifying PZQ and ten of these metabolites. The goal of these experiments was to evaluate the effect of this hepatic infection on drug metabolism. This was accomplished in two steps. First, the amount of unmetabolized, mono- and dihydroxylated PZQ was established from the [M + H]+ ions. Then the specific metabolites at each level of hydroxylation were determined from daughter ion spectra. The product of these two values produces the metabolite pattern. The reproducibility of these assays ranged from good, with a coefficient of variation of 3% for the most abundant metabolite, to poor (43%) for PZQ, which is only 1% of the total elimination pattern. The excretion of unchanged PZQ and two dihydroxylated metabolites was enhanced in animals bearing schistosomiasis compared to control mice, while a third dihydroxylated metabolite was depressed.

Metabolism of methoxychlor by hepatic P-450 monooxygenases in rat and human. 1. Characterization of a novel catechol metabolite

Previous investigations demonstrated that the incubation of the chlorinated hydrocarbon pesticide methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] with rat liver microsomes generates phenolic estrogenic metabolites. The current study shows that the incubation of liver microsomes from untreated and phenobarbital-treated rats and human donors, in the presence of NADPH, yields three phenolic metabolites. Identification of the metabolites was achieved by TLC, HPLC, GC/MS, and LC/MS and by hydrodynamic voltammetric analysis. These metabolites were identified as the mon- and didemethylated phenolic derivatives (mono-OH-M and bis-OH-M, respectively) and as a novel trihydroxy derivative (tris-OH-M). The tris-OH-M was demonstrated to be a catechol [1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3,4-dihydroxyphenyl)ethane]. Furthermore, the tris-OH-M becomes radiolabeled by [methyl-3H3]-S- adenosylmethionine (SAM) in a reaction catalyzed by catechol O-methyltransferase (COMT), indicating that tris-OH-M behaves like a catechol. Incubation of the monohydroxy metabolite with liver microsomes from phenobarbital-treated rats (PB microsomes) yields the dihydroxy and the trihydroxy metabolites. Furthermore, the time course of methoxychlor metabolism by PB microsomes demonstrated a rapid appearance and disappearance of the monohydroxy metabolite with the subsequent formation of the dihydroxy and trihydroxy metabolites. On the basis of these findings, it is proposed that the metabolic route of methoxychlor by mono-oxygenases involves sequential demethylations to the dihydroxy derivative and a subsequent ring hydroxylation.

24,25,28-Trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2: novel metabolites of vitamin D2

Understanding of the inactivation pathways of 25-hydroxyvitamin D2 and 24-hydroxyvitamin D2, the two physiologically significant monohydroxylated metabolites of vitamin D2, is of importance, especially during hypervitaminosis D2. In a recent study, it has been demonstrated that the inactivation of 24-hydroxyvitamin D2 occurs through its conversion into 24,26-dihydroxyvitamin D2 [Koszewski, N.J., Reinhardt, T.A., Napoli, J.L., Beitz, C.D., & Horst, R.L. (1988) Biochemistry 27, 5785]. At present, little information is available regarding the inactivation pathway of 25-hydroxyvitamin D2 except its further metabolism into 24,25-dihydroxyvitamin D2 [Jones, G., Rosenthal, A., Segev, D., Mazur, Y., Frolow, F., Halfon, Y., Rabinovich, D., & Shakked, Z. (1979) Biochemistry 18, 1094]. In our present study, we investigated the metabolic fate of 25-hydroxyvitamin D2 in the isolated perfused rat kidney and demonstrated its conversion not only into 24,25-dihydroxyvitamin D2 but also into two other new metabolites, namely, 24,25,28-trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2. The structure identification of the new metabolites was established by the techniques of ultraviolet absorption spectrophotometry and mass spectrometry and by the characteristic nature of each new metabolite's susceptibility to sodium metaperiodate oxidation. In order to demonstrate the physiological significance of the two new trihydroxy metabolites of vitamin D2, we induced hypervitaminosis D2 in a rat using [3 alpha-3H]vitamin D2 and analyzed its plasma for the various [3 alpha-3H]vitamin D2 metabolites on two different high-pressure liquid chromatography systems.(ABSTRACT TRUNCATED AT 250 WORDS)

L-ascorbic acid modulates 5-lipoxygenase activity in human polymorphonuclear leukocytes.

Leukotriene B4 (LTB4), a metabolite of the arachidonic acid pathway mediated by 5-lipoxygenase, is released by stimulated polymorphonuclear neutrophils (PMNL) and has been postulated to be an important mediator of the inflammatory response. Extracellular L-ascorbate at concentrations in the range of 0.5-5.0 mM effectively inhibits the biosynthesis of LTB4 and 5-hydroxyeicosatetraenoic acid (5-HETE) stimulated by the calcium ionophore A 23187. The ionophore-activated LTB4 production is reduced after incubation of PMNL with opsonized zymosan as a phagocytic stimulus. Extracellular L-ascorbate at concentrations above 0.1 mM reverses the zymosan-induced deactivation of 5-lipoxygenase, resulting in significantly higher LTB4 and 5-HETE yields. The inhibitory effect of zymosan preincubation on LTB4 production is independent of calcium or free arachidonic acid in the incubation medium. Interaction of L-ascorbate with the catabolism of LTB4 was excluded by measuring the trihydroxy metabolites which were unchanged. Furthermore, the formation of glutathione derivatives of leukotriene A4 can be excluded due to the lack of glutathione transferase activity in PMNL. In order to link the intracellular function of L-ascorbate with the serum level, the ascorbate uptake has been studied in more detail. The L-ascorbate transport into PMNL is stereospecific and can best be described by kinetics consisting of a saturation part, Km and Vmax being 39 microM and 0.28 nmol/10(8) cells.min, respectively, plus passive diffusion, the diffusion coefficient P being 1.75 microliter/10(8) cells.min. Furthermore, the uptake is inhibited by the isomers D-ascorbic acid and D-erythorbic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of selenium-deficient diets on the production of prostaglandins and other oxygenated metabolites of arachidonic acid and linoleic acid by rat and rabbit aortae

Selenium is an essential component of glutathione peroxidase, which reduces free and esterified hydroperoxides of polyunsaturated fatty acids. Adequate glutathione peroxidase activity could be important for the maintenance of prostacyclin synthesis by blood vessels, since hydroperoxides can inhibit the formation of this substance. We have investigated the effects of dietary selenium deficiency on glutathione peroxidase activity and the synthesis of 6-oxoprostaglandin F 1α and monohydroxy and trihydroxy metabolites of polyunsaturated fatty acids by aorta. The latter products can be formed either by the actions of cyclooxygenase or lipoxygenase or by lipid peroxidation. Aortic glutathione peroxidase activity was reduced by over 80% by feeding rats a selenium-deficient diet for 4 weeks, and to undetectable levels after 6 weeks. There were no appreciable differences in the levels of free and esterified oxygenated metabolites of linoleic acid or arachidonic acid between the control and treated groups after 4 weeks. However, after 6 weeks, there were modest, but statistically significant reductions in the formation of 6-oxoprostaglandin F 1α and monohydroxy products formed by cyclooxygenase. On the other hand, the amounts of esterified 18:2 metabolites appeared to be higher in aortae from animals on the selenium-deficient diet, although only the increase in esterified 9-hydroxy-10,12-octadecadienoic acid was statistically significant. These results suggest that selenium deficiency can affect the formation of prostacyclin and other oxygenated metabolites of polyunsaturated fatty acids by aorta, possibly by increasing lipid peroxidation. However, the differences between control and selenium-deficient rats after 6 weeks were not very dramatic, in spite of the fact that glutathione peroxidase activity was undetectable. It would therefore appear that additional mechanisms are also involved in controlling the levels of lipid hydroperoxides in aorta.

Release of prostaglandins and monohydroxy and trihydroxy metabolites of linoleic and arachidonic acids by adult and fetal aortae and ductus arteriosus.

The conversion of arachidonic acid (20:4) to prostaglandins by vascular tissue is important in the adult because of the antithrombotic effect of prostacyclin and in the fetus because of the vasodilatory effect of prostaglandin (PG) E2 on the ductus arteriosus. We have shown that vascular tissue converts various polyunsaturated fatty acids to monohydroxy and trihydroxy metabolites derived from hydroperoxides, which may be involved in regulating prostaglandin synthesis. We have now measured the amounts of these hydroperoxide metabolites, as well as those of prostaglandins, released from slices of rat, rabbit and bovine aortae, as well as from fetal calf aorta and ductus arteriosus. The major oxygenated polyunsaturated fatty acid metabolite formed by rat and bovine blood vessels was 6-oxo-PGF1 alpha. Fetal calf aorta and ductus arteriosus produced about five times as much 6-oxo-PGF1 alpha as adult bovine aorta. Much smaller amounts of the cyclooxygenase products, PGE2, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11-hydroxy-20:4), and 15-hydroxy-20:4, were released by aortae. Small amounts of the lipoxygenase product, 12-hydroxy-20:4, were also detected. Substantial amounts of free and esterified monohydroxy and trihydroxy metabolites of linoleic acid (18:2) were detected, especially in rat and rabbit aortae. Rabbit aorta, which had low cyclooxygenase activity, formed more oxygenated 18:2 metabolites than 20:4 metabolites. Indomethacin did not inhibit the formation of the 18:2 metabolites, indicating that cyclooxygenase was not involved. Neither exogenous 13-hydroxy-18:2 nor trihydroxyoctadecenoic acid was incorporated to a large extent into lipids from vascular endothelial or smooth muscle cells, suggesting that the esterified 18:2 oxygenation products had arisen mainly via direct oxygenation of lipids.

Metabolism of linoleic acid by prostaglandin endoperoxide synthase from adult and fetal blood vessels

Linoleic acid (18:2) is converted by prostaglandin endoperoxide synthase in paniculate fractions and homogenates of fetal calf aorta to its 9- and 13-hydroperoxy metabolites. These intermediates are then either dehydrated to the corresponding oxo compounds or reduced to monohydroxy products. Alternatively, the hydroperoxyoctadecadienoic acids can be converted to epoxyhydroxyoctadecenoic acids, which are hydrolyzed to trihydroxy metabolites by epoxide hydrolases present in both particulate and cytosolic fractions from aorta. Linoleic acid (Km, 442 μM) is a much poorer substrate for prostaglandin endoperoxide synthase than is arachidonic acid (20:4) (Km, 48 μM). However, the oxygenation of 18:2 by particulate fractions from aorta is linear with time for at least 5 min, whereas the oxygenation of 20:4 is linear for only 15 s. Arachidonic acid strongly inhibits the conversion of 18:2 to monohydroxy (ID50, 10 μM) and trihydroxy (ID50, 140 μM) products. Linoleic acid has a similar, but much weaker effect on the formation of 6-oxoprostaglandin F1 α from 20:4. Substantial amounts of both the monohydroxy (9-hydroxy-1O, 12-oc-tadecadienoic acid and 13-hydroxy-9, ll-octadecadienoic acid) and trihydroxy (9,10,ll-trihydroxy-12-oc-tadecenoic acid, 9,10,13-trihydroxy-ll-octadecenoic acid and 9,12,13-trihydroxy-lO-octadecenoic acid) metabolites of 18:2 were shown by gas chromatography-mass spectrometry to be formed from endogenous substrate during incubation of slices of fetal calf aorta in physiological medium. This raises the possibility that some of these products or their hydroperoxy precursors may have some biological significance.

(23S)-1,23,25-Trihydroxycholecalciferol, an intestinal metabolite of 1,25-dihydroxycholecalciferol.

(23S)-23,25-Dihydroxycholecalciferol was converted into a polar metabolite in a calciferol-deficient chick kidney homogenate. The metabolite was identified as (23S)-1,23,25-trihydroxycholecalciferol by absorbance spectroscopy and mass spectrometry, and by formation of derivatives. (23S)-1,23,25-Trihydroxycholecalciferol was also observed as a 1,25-dihydroxycholecalciferol metabolite in intestinal cells isolated from 1,25-dihydroxycholecalciferol-treated rat. The trihydroxy metabolite was 50-fold less potent than 1,25-dihydroxycholecalciferol in the chick intestinal 1,25-dihydroxycholecalciferol receptor assay.

Metabolism of ethynodiol diacetate in the rhesus monkey before and after administration of rifampicin

1. On administration of a single oral dose of [4-(14)C]ethynodiol diacetate (0.15 mg/kg) to rhesus monkey, plasma concn. of total 14C peaked after about 4 h. About 60% of the plasma radioactivity was present as glucuronide conjugates and no unchanged drug was detected. 2. Some 67 +/- 6% (mean +/- S.D.) of the dose of 14C was excreted in 4 days, 50 +/- 6% in urine and 18 +/- 2% in faeces. Most of the urinary excretion occurred within 24 h of dosage. 3. Glucuronide conjugates accounted for 60% of the urinary 14C, and 46% of the faecal 14C was free steroids. 4. Norethisterone and its tetrahydro metabolites were identified in the free, glucuronide and sulphate fractions of plasma and urine. Keto-4,5-dihydronorethisterones and trihydroxy metabolites were identified in the conjugated fractions of urine, and a complex mixture of polar metabolites was detected in faeces. 5. Rifampicin treatment (7.5 mg/kg/day, orally) for 8 days decreased the half-life of total 14C in plasma following a single oral dose of 4-[14C]ethynodiol diacetate (0.15 mg/kg) from 44 +/- to 24 +/- 2 h. 6. Faecal elimination of total 14C was significantly increased to 29 +/- 5% of the dose following rifampicin treatment, but urinary excretion was unchanged. 7. Rifampicin treatment increased the amount of polar metabolites and decreased the amount of norethisterone in the free and conjugated fractions of plasma and urine. The amounts of sulphate and non-hydrolysed conjugates in faeces were increased after rifampicin treatment.

Disposition and metabolism of 16β-ethyl-17β-hydroxy-4-estren-3-one (TSAA-291), a new antiandrogen, in rats

Metabolic fate of a new antiandrogen, l6β-ethyl-l7β-hydroxy-4-estren-3-one (TSAA-291), was studied in rats. 14C-TSAA-291 intramuscularly injected as an aqueous suspension was absorbed gradually to give an increase in the plasma level which attained a plateau at 0.5 h, persisted till 8 h and then declined with an approx. half-life of 3.6 days. The drug was widely distributed in tissues, with the concns. almost equal to or higher than that in the plasma. The 14C-drug was eliminated mostly as metabolites within 10 days after dosing with higher activities found in the feces than in urine. Biliary 14C effectively underwent enterohepatic cycling. Biliary metabolites of TSAA-291 were characterized by the combined use of deuterium labeling and GLC-MS analysis. The metabolites identified were as follows: the parent drug, monohydroxy TSAA-291 having the additional hydroxy function in the steroid skeleton, 17β-hydroxy-16β-(1ξ-hydroxyethyl)-4-estren-3-one, l6β-ethyl-l7β-hydroxy-5β-estran-3-one, l6β-ethyl-17β-hydroxy-5α-estran-3-one, l6β-ethyl-5β-estrane-3α,17β-diol, l6β-ethyl-5α-estrane-3α,17β-diol, l6β-ethyl-3α-hydroxy-5β-estran-17-one and 16β-ethyl-3α-hydroxy-5α-estran-17-one. Monoketodihydroxy and/or trihydroxy metabolites were also detected in the bile.

The metabolism of 4,4′-dichlorobiphenyl in rats and frogs

The metabolism of 4,4′-dichlorobiphenyl in the rat has been investigated in detail and four monohydroxy-, four dihydroxy- and two trihydroxy metabolites were detected. The structures of the metabolites - except for the trihydroxy compounds - were ascertained by GC-MS studies and by comparison with synthetic compounds. Metabolism experiments in frog resulted in the isolation and characterisation of four metabolites. The structure of the major metabolites in both the rat and the frog are consistent with epoxidation of the biphenyl nucleus followed by epoxide ring opening accompanied by a 1,2-chlorine shift (NIH-shift). The formation of one minor rat metabolite, 4-chloro-3t́-biphenylol can only be explained by reductive dechlorination.