Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)−1, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which leftmyo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus nigerpH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger,Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, theAspergillus fumigatus, Emericella nidulans, andMyceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of themyo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatusphytase. In contrast, phosphate liberation by A. niger orA. terreus phytase decreased with incubation time, and themyo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of eitherA. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.

Previous investigations in the rat have shown that the non-provitamin A carotenoid astaxanthin is metabolized into 3-hydroxy-4-oxo-beta-ionone and 3-hydroxy-4-oxo-7,8-dihydro-beta-ionone and, in addition, is a potent CYP1A gene inducer. Here we investigated the metabolism of this compound as well as its capacity to induce CYP genes in primary cultures of human hepatocytes. Free metabolites of 14C-astaxanthin produced in this cellular model were purified by high pressure liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC-MS) analyses as 3-hydroxy-4-oxo-beta-ionol and 3-hydroxy-4-oxo-beta-ionone. In addition, deconjugation of polar compounds by glusulase and further analyses with HPLC and GC-MS revealed four radiolabeled metabolites including: 3-hydroxy-4-oxo-beta-ionol, 3-hydroxy-4-oxo-beta-ionone, and their reduced forms, 3-hydroxy-4-oxo-7, 8-dihydro-beta-ionol and 3-hydroxy-4-oxo-7,8-dihydro-beta-ionone. The same four metabolites were identified in human plasma from two volunteers who had orally taken 100 mg astaxanthin 24 h before blood collection. In cultured hepatocytes, astaxanthin was a significant inducer of the major cytochrome P450 enzyme, CYP3A4 as well as of CYP2B6, but not of other CYPs, including those from CYP1A and CYP2C families. The lack of autoinduction of astaxanthin metabolism in human hepatocytes suggests that neither CYP3A4 nor CYP2B6 contribute to the formation of metabolites. We conclude that metabolism of astaxanthin and its CYP-inducing capacity are different in humans and in rats. The novel methodology used in our studies could be extended to evaluating the role of metabolites of more important carotenoids such as beta-carotene in differentiation and carcinogenicity.

The structure of the urinary metabolites formed after moclobemide administration in humans was elucidated, and the pattern compared with that in the plasma. The metabolic pathways of moclobemide were also compared with those of structurally related substances. After oral moclobemide administration, on average 95% of the dose was recovered in the urine within 4 days, with a mean of 92% being excreted during the first 12 h. The drug is extensively metabolized: less than 1 % of the dose was excreted unchanged. A total of 19 metabolites, accounting together for about 64% of the dose, was isolated and all metabolites accounting for more than 170 of the dose were identified. Consistent with other morpholinecontaining compounds, metabolic pathways of moclobemide include mainly oxidative attack on the morpholine moiety, leading to a multitude of oxidation products. Four primary metabolic reactions were identified: morpholine N-oxidation, aromatic hydroxylation, morpholine C-oxidation and deamination. The major metabolites in urine are 4 carboxylic acids (M7A and M7B, M8, M9) that account for 49% of the dose. Only 2 metabolites (M3, MlO) were found to be hydroxylated on the aromatic nucleus. They were excreted completely as conjugates of glucuronic and/ or sulfuric acid. Conjugation in general, however, seems to be of minor importance in the overall biotransformation of the drug. The metabolite pattern in plasma was found to be qualitatively but not quantitatively similar to that observed in urine. Almost all of the main urinary metabolites were found in plasma as well. The unchanged parent compound and 2 primary oxidation products of the morpholine ring (Ml, MlS), which were present in urine only in trace amounts, could easily be detected in I plasma.Moclobemide belongs to a new generation of monoamine oxidase (MAO) inhibitors of the benzamide type and contains a morpholine ring as a characteristic part of its structure.The aim of this study was to elucidate the structures of the urinary metabolites formed from moclobemide after its administration to humans and to compare the metabolite pattern in urine and plasma. In addition, a comparison of metabolic pathways between moclobemide and structurally related drugs was attempted. Material and methods Drug administration and sample collectionTwo healthy young volunteers received 50 mg 14Clabelled moclobemide (2 pCi/mg; position of radiolabel, see Fig. 1) orally in a hard gelatine capsule. Blood was taken by puncture of an arm vein at different time points up to 10 h after administration and plasma was obtained by centrifugation.Urine and faeces were collected quantitatively in fractions up to 96 h after administration. Isolating and identifying the metabolitesMetabolites were isolated from pooled 0-12 h urine by chromatography on Amberlite XAD-2 resin and subsequent extractions with ethyl acetate at pH 9 and pH 3. These extractions were made before and after enzymatic hydrolysis with a mixture of beta-glucuronidase and arylsulfatase. Separation and purification of the metabo...

Summary24-Isopropylcholesterol (1) and 22-dehydro-24-isopropylcholesterol2 have been isolated as the only sterols from an Australian sponge of the genus Pseuduxinyssa. Structures have been deduced from spectroscopic data.Sterols of sponges are characterized by a great variety of unusual structural features, such as modified ring systems and polyalkylated side chains [ 11. They also frequently occur as mixtures of very complex composition. We now report the isolation of two novel sterols, 1 and 2, as the only two sterols of a new species of the sponge-genus Pseudaxinyssa (class Demospongia, order Axinellida) which was collected at various localities on the Australian Great Barrier ReeJThe sterols were isolated by Soxhlet extraction of sun dried sponge samples with petrol ether and purified by chromatography on preparative silica gel plates. GC. analysis showed the presence of only two components, whose molecular formulae were obtained from high resolution mass spectra as C3&20 and C3,,Hso0. Catalytic hydrogenation transformed the mixture to one single sterol, C,d,,O, proving that both sterols have the same carbon framework, one being mono-unsaturated, the other di-unsaturated. Since peaks in the mass spectrum of the mono-unsaturated component at m/e 273,255,231 and 213 gave a clear indication of the presence of a mono-unsaturated, intact steroid nucleus [2], the strong peak at m/e 271 in the spectrum of the di-unsaturated sterol had to be taken as evidence for the presence of one double bond in its side chain [3]. Mass spectra of the silylated sterols were very similar to those of ,8-sitosterol and stigmasterol, with prominent peaks at mle 129 and M-129 which are typical for &-sterols [2] [4]. It therefore could be concluded that both sterols possess normal cholesterol-like structures with three 'extra-carbon atoms' in the side chain. The nature of the side chains could be derived from NMR. spectra. Samples (0.2-0.3 mg) of pure sterols were obtained by GC. separation. The NMR. spectrum of sterol 1 shows in addition to the signals of five methyl groups [C(18) 0.68; C(19) 1.01; C(21) 0.94(d); C(26) and C(27) 0.85 ppm(d)] a six-proton doublett at 0.87 ppm indicating the presence of a second isopropyl group. A similar observation was made in the NMR. spectrum of sterol 2 (doublet at 0.77 ppm), which was, furthermore, almost superimposable onto the spectrum of

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1. Ornidazole, labelled with 14C in the imidazole ring, administered orally to rats, dogs and men was largely excreted in the urine, predominantly as metabolites, with less than 4% of the drug being excreted unchanged. Free and conjugated metabolites were found in the ratio of approx. 1 : 2. 2. The pattern of free ornidazole and metabolites was different in the three species: while ornidazole predominated in man, ornidazole and metabolite M1 in the dog, the most extensive metabolic pattern was found in the rat. 3. The following metabolites were identified: M1, 1-chlorlo-3-(2-hydroxymethyl-5-nitro-1-imidazolyl)-2-propanol; M2, 2-methyl-5-nitroimidazole; M3, N-(3-chloro-2-hydroxypropyl)acetamide: M4, 3-(-2-methyl-5-nitro-1-imidazolyl)-1, 2-propanediol; M5, acetamide. 4. The formation of metabolite M3 and M5 indicated cleavage of the imidazole ring between N-1/C-5 and C-2/C-3. Other ring scissions were not observed. Metabolites carrying a free amino group were not detected. On the basis of the structures identified, a scheme is suggested for the metabolism of ornidazole.

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The thermal and hydrolytic degradation of tetrahydrolipstatin (THL, 1) was investigated. All main degradation products were isolated, characterized, and synthesized. Labile intermediates unavailable to isolation were detected and identified by GC/MS analysis of their silylated derivatives, and whenever possible, compared with independently prepared reference compounds. The identified degradation products represent at least 97 % of the total degradation mixture. Two main reaction pathways are proposed. Pharmacological data are reported for the degradation mixture and the main degradation products. Introduction. -Tetrahydrolipstatin (THL; 1; N-formyl-L-leucine (S)-1-{[(2S,3S)- 3-hexyl-4-oxooxetan-2-yl]methyl}dodecyl