Bioconversion of isosorbide dinitrate into isosorbide mononitrate by the protozoan Tetrahymena thermophila: relationship to glutathione transferase levels

Summary Growing cells of Tetrahymena thermophila (T. t.) metabolized, after 72 h, 80% of isosorbide dinitrate (ISDN) to isosorbide 5-mononitrate (5-ISMN) and isosorbide 2-mononitrate (2-ISMN) in a ratio 5-ISMN/2-ISMN=2.6 as evaluated by HPLC. The level of glutathione S-transferases (GSH-ST) determined by following the reactions with of 1-chloro-2,4-dinitrobenzene (CDNB), o-dinitrobenzene (o-DNB) or ISDN, showed the inductive effect of ISDN (0.5 mg/ml) on the level of this enzyme. The enzymatic activity, evaluated at 72 h, showed a twofold increase compared with the control. The GSH-ST activity correlated well with the rate of ISDN bioconversion.     

Patterns of isosorbide dinitrate and glyceryl trinitrate metabolites formed by selected segments of the rabbit gastrointestinal tract

Homogenates of selected segments of the rabbit gastrointestinal tract (GIT) were studied for their ability to biotransform isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN) to their mono- and di-nitrate metabolites, respectively. In addition, preferential formation of certain metabolites was investigated by examination of the patterns of metabolites formed by the various homogenates. After a 30-min incubation of ISDN with GIT homogenates (pH 7.4, 37 degrees C), the percent disappearance of ISDN and the ratio of isosorbide-2-mononitrate (2-ISMN) to isosorbide-5-mononitrate (5-ISMN) were as follows: stomach, 32%, 0.8; duodenum, 65%, 0.1; jejunum, 59%, 0.2; ileum, 38% , 1.2; cecum, 33%, 2.7; and colon, 32%, 3.4. After a 5-min incubation of GTN with GIT homogenates, the percent disappearance of GTN and the ratio of glyceryl-1,3-dinitrate (1,3-GDN) to glyceryl-1,2-dinitrate (1,2-GDN) were as follows: duodenum, 54%, 0.65; ileum, 73%, 0.68; and colon, 61%, 0.17. Incubation of 2 x 10(-7) M ISDN with mucosal and muscularis homogenates of duodenum, jejunum, and ileum resulted in significant losses of ISDN with an equimolar formation of the mononitrate metabolites. Most of the metabolic activity for ISDN resided in the mucosal layer of each section. The ratio of 2-ISMN to 5-ISMN varied in each section (stomach to colon) and cross section (mucosal versus muscularis) of the GIT. We conclude that the metabolism of ISDN and GTN by the GIT may contribute to the high clearance of these organic nitrates, and the low oral bioavailability of ISDN. Also, multiple mechanisms appear to be involved in the biotransformation of ISDN and GTN in the rabbit GIT.     

Sex- and species-related differences in the biotransformation of isosorbide dinitrate by various tissues of the rabbit and rat

The biotransformation of isosorbide dinitrate (ISDN) by various tissues of the rabbit and rat was examined. Incubation of 2 X 10(-7) M ISDN at 37 degrees C with tissue homogenates of liver, lung, kidney, intestine, skeletal muscle, aorta, and erythrocytes from the rabbit and rat resulted in a significant disappearance of ISDN after a 30-min incubation (also, 5-min incubation for liver). The disappearance of ISDN in each tissue homogenate was accompanied by an equimolar production of the mononitrate metabolites, isosorbide-2-mononitrate (2-ISMN) and isosorbide-5-mononitrate (5-ISMN), with the exception of liver homogenates where the loss of ISDN could not be accounted for by mononitrate formation. The relative rate of ISDN disappearance in various tissue homogenates was for the male rabbit, liver greater than lung approximately intestine greater than kidney greater than erythrocytes approximately skeletal muscle approximately aorta; for the female rabbit, liver greater than kidney approximately lung approximately intestine greater than erythrocytes approximately skeletal muscle approximately aorta; and for the male rat, liver greater than intestine greater than erythrocytes greater than skeletal muscle greater than lung approximately kidney. A sex difference in the percent disappearance of ISDN was observed in homogenates of lung and intestine from male and female rabbits. In addition, a sex difference in the ratio of metabolite (2-ISMN/5-ISMN) formed by denitration of ISDN was seen in homogenates of lung, skeletal muscle, and erythrocyte lysate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of isosorbide 5-mononitrate in human plasma by liquid chromatography-tandem mass spectrometry using atmospheric pressure photoionization

Isosorbide 5-mononitrate (5-ISMN) is an organic nitrate widely used for its vasodilating properties in the treatment of angina pectoris. In the present study, an efficient, sensitive, robust method was developed for the determination and quantification of isosorbide 5-mononitrate, in human plasma, by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), using photospray ionization. Isosorbide 5-mononitrate was extracted from 0.5 mL human plasma by liquid-liquid extraction (LLE). The method had a chromatographic run of 2.0 min using a C(8) analytical column (100 mm x 2.1 mm i.d.) and the linear calibration curve over the range was linear from 20 to 2000 ng mL(-1) (r(2)>0.995). The between-run precision, based on the relative standard deviation replicate quality controls, was 7.9% (60 ng mL(-1)), 5.2% (300 ng mL(-1)) and 7.0% (1800 ng mL(-1)). The between-run accuracy was 94.9%, 94.1% and 88.8% for the above-mentioned concentrations, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of isosorbide 5-mononitrate 40 mg.     

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5–83 ng/ml for ISDN, 2.6–208 ng/ml for 2-ISMN and 2.3–1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.     

Effect of 5-isosorbide mononitrate on isosorbide dinitrate-induced relaxation of rabbit aortic rings

The ability of isosorbide dinitrate (ISDN) and its two metabolites, 5-isosorbide mononitrate (5-ISMN) and 2-isosorbide mononitrate (2-ISMN), to relax phenylephrine-contracted rabbit aortic rings was compared. The three organic nitrates demonstrated similar efficacy. ISDN was found to be the most potent (median effective dose (ED50); 1.5 X 10(-7) +/- 1.1 X 10(-7) M), followed by 2-ISMN (ED50, 1.8 X 10(-6) +/- 9 X 10(-7) M) and 5-ISMN (ED50, 8.2 X 10(-6) +/- 3.6 X 10(-6) M). The log dose-response curve of ISDN in rabbit aortic rings was constructed in the absence and presence of three fixed concentrations of 5-ISMN (5 X 10(-6), 10(-5), and 3 X 10(-5) M). No shift in the ISDN dose-response curve at high ISDN concentrations was noted in the presence of 5-ISMN. Using the isobolographic method with fixed ISDN/5-ISMN ratio mixtures, no evidence for an antagonistic effect of 5-ISMN on ISDN-induced vasodilation was obtained. Analysis of the fixed ISDN/5-ISMN ratio mixture responses by the median-effect plot showed no antagonistic effect. It is concluded that in rabbit aortic rings 5-ISMN, the major metabolite of ISDN, is not an antagonist of ISDN at a "nitrate receptor," and no support is provided for the hypothesis that the accumulation in plasma of metabolites (e.g., 5-ISMN) with longer half-lives than the parent drug explains tolerance to organic nitrates.     

Isosorbide dinitrate bioconversion by Beauveria strains: implication of glutathione transferase levels

Summary The level of glutathione S-transferase (GSH0ST) activity was determined in growing cultures and in washed resting cells of Beauveria strains with and without addition of isosorbide dinitrate (ISDN), by following the reaction with o-dinitrobenzene (o-DNB). The level of GSH-ST varied according to the pH changes of the medium and decreased during culture. The enzymatic activity measured with o-DNB did not correlate with ISDN bioconversion carried out either with B. sulfurescens or B. tenella. Immediately after starting incubation of the resting cells with ISDN, the level of GSH-ST activity initially increased, but declined afterwards, whereas the bioconversion process continued and reached 500 mg/l isosorbide 5-mononitrate. When 1-chloro-2,4-dinitrobenzene was used as a substrate for the evaluation of GSH-ST activity using B. tenella, a conjugation product having a UV absorption at 410 nm was formed.     

High performance liquid chromatography-electrospray ionization mass spectrometric determination of isosorbide 5-mononitrate in human plasma

A novel, selective and sensitive high performance liquid chromatography-mass spectrometric (HPLC-MS) method has been developed for the determination of isosorbide 5-mononitrate (5-ISMN) in human plasma. With acetaminophen as internal standard, sample pretreatment involved one-step extraction with diethyl ether of 0.5 mL plasma. Analysis was performed on an ACQUITY UPLC BEH C(18) column (100 mm x 2.1mm, 1.7 microm) with mobile phase consisting of acetonitrile-water (20:80, v/v). The detection was carried out by means of electrospray ionization mass spectrometry in negative ion mode with selected ion recording (SIR). Standard curves were linear (r(2)> or =0.99) over the concentration range of 1.04-1040 ng/mL. The lower limit of quantification (LLOQ) was 1.04 ng/mL. The intra- and inter-day precisions (RSDs) were less than 8.6% and 13.4%, respectively, and the accuracy (RE) was within +/-0.45%. The method herein described was fully validated and successfully applied to the pharmacokinetic study of 5-ISMN in compound extended-release tablets in 18 healthy male volunteers after oral administration.     

Conversion of Nicotine into Nornicotine and N-Methylmyosmine by Fungi

Several species of fungi were tested for their abilities to degrade (S)-nicotine, of which Pelliculariafilamentosa JTS-208, the pathogen of tobacco damping off disease, and Cunninghamella echinulata IFO-4444, a saprophyte, were found to be able to degrade nicotine. P. filamentosa JTS-208 accumulated nornicotine only in the nicotine medium. C. echinulata IFO-4444 accumulated nornicotine and N-methylmyosmine, the first fungal metabolite, and three unidentified compounds.     

Lipid production and fatty acid composition of selected strains belonging to Mucorales

The lipid accumulation, fatty acid composition and γ-linolenic acid (GLA) production by 28 strains belonging to Mucorales were investigated. The lipid content varied from 5 to 30% on dry biomass and the percentage of γ-linolenic acid in total intracellular lipid was in a range from 2.5 to 15.4% (w/w). The best yield of γ-linolenic acid (expressed as mg GLA per 1 g biomass) was found for Mucor mucedo CCF – 1384 (28.4) and Cunninghamella echinulata CCF – 103 (25.1).     

Packed-column supercritical fluid chromatography for the analysis of isosorbide-5-mononitrate and related compounds in bulk substance and tablets

We describe a packed-column supercritical fluid chromatographic method that can be used for the analysis of isosorbide-5-mononitrate (5-ISMN) bulk substance and the 5-ISMN content of Imdur tablets. The method is based on methanol-modified carbon dioxide as the mobile phase and porous graphitized carbon (PGC, Hypercarb) as column support at 40 degrees C and 100 bar back pressure. The method makes it possible to simultaneously determine 5-ISMN and related compounds. In order to elute NO(3)(-) with acceptable retention time a quarternary ammonium hydrogen sulfate salt is added to the methanol modifier. An almost linear increase of the retention time with increasing carbon content of the counter ion was found. Tetramethyl ammonium hydrogen sulfate 5 mM in methanol was used in the final method as polar modifier for the simultaneous determination of possible degradation products within 12 min. The present method can separate and detect related compounds such as isosorbide-2, 5-dinitrate, isomannidemononitrate and isosorbide-2-mononitrate at the 0.1% (w/w) level as required by regulatory guidelines. Nitrate can be detected down to about 0.02% (w/w). Repeated analyses of ground tablet powder gave an assay precision for isosorbide-5-mononitrate of 1.4% (R.S.D., eight samples and two injections of each). For related substances at an area percent of 0. 1 the precision was less than 10%.     

刺孢小克银汉霉原变种和轮生刺孢小克银汉霉变种(新组合)

刺孢小克银汉霉[Cunninghamella echinulata(Thaxt.) Thaxt. ex Blakeslce]是小克银汉霉属的模式种,多年来因不同作者是否将贝尼尔小克银汉霉(C. bainieri Naum.)包含在内而对此种持广义(sensu lato)或狭义(scnsu stricto)的不同理解。我们在国内分离到51株刺孢小克银汉霉(广义)菌种,经与获自css的7株菌(包括刺孢小克银汉霉的模式菌种在内)和IMI的1株菌(原定名称均为刺孢小克银汉霉)一起研究后,根据它们的无性型形态特征有明显不同但在配合试验中又可互相配合形成可育的接合孢子的结果,认为将它们处理为同一个种的两个不同变种较为合理.其中,13株中国的菌和3株CBS及IMI的菌被鉴定为刺孢小克银汉霉原变种(C. echinulata var. echinulata);其余的38株中国的菌和5株CBS的菌被鉴定为轮生刺孢小克银汉霉变种(新组合)[C. echinulata var. verticillata (F. S. Paine.) comb. nov.j.以‘轮生(verticillata)’作为新组合变种的加词是根据与贝尼尔小克银汉霉同物异名的‘轮生布拉小克银汉霉(C. blakesleeana var. verticillata (F. S. Paine) Baijal & B. S. Mchrotra)’而来的.在前人的工作中,对刺孢小克银汉霉持狭义理解的作者往往根据孢子枝的分枝方式、巨型暗色孢子的有无、以及40℃能否生长来区分这两个分类群.对刺孢小克银汉霉持广义理解的作者则认为这些特性毫无意义.我们同意后面两个特性意义不大,因为二者的许多菌系都能形成巨型暗色袍子并在40℃生长,但认为孢子枝分枝方式确实是区别这两个分类群的重要依据.此外,根据产孢构造的种类、孢子枝主枝的直径、孢子枝第一次分枝的数目、分枝长度、孢子枝主枝的顶生泡囊形状以及其直径等性状综合考虑,这两个分类群是不难明确区分的,对12株刺孢小克银汉霉原变种和13株轮生刺孢小克银汉霉相互之间所进行的配合试验结果表明,在具一定性亲和力的(+)、(一)菌系同时存在时,两个变种内和变种之间均可形成可育的接合孢子。对上述三种配合的各六对菌所形成的有性型形态的研究结果则表明,除轮生刺孢小克银汉霉变种内形成的接合孢子的壁往往较厚外,各种配合形成的有性型形态基本上是一致的.我们研究组的其他成员最近曾对小克银汉霉的22株菌进行了rDNA的ITS区的PCR产物长度测定,其中包括了刺孢小克银汉霉原变种和轮生刺孢小克银汉霉变种的各3株菌,它们的平均长度(bp)分别为890±7.5和915±7.6.这个结果也支持了我们在这两个分类群可以互相配合的情况下仍将它们作为两个不同变种的处理.这两个分类群虽然已被发表多次,但它们的描述和绘图均过于简单而未能充分显示出它们的区别性特征,因此我们在本文中再次作了描述并再次提供了详尽的线条图和显微照相.     

Characterization of Genes Involved in D-Sorbitol Oxidation in Thermotolerant Gluconobacter frateurii

A microbial screening indicated that two fungal strains, Beauveria bassiana DSM 1344=ATCC 7159 and Cunninghamella elegans DSM 1908=ATCC 9245, as well as four bacterial strains belonging to the genus Streptomyces were able to hydroxylate 4,5-dianilinophthalimide (DAPH, CGP52411) to 4-(4′-hydroxyanilino)-5-anilinophthalimide. Cunninghamella elegans DSM 1908 turned out to be the most active biocatalyst and was also able to form the dihydroxy derivative, 4,5-bis(4′-hydroxyanilino)phthalimide. The reaction for the monohydroxylated biotransformation product was carried out on a preparative scale, and the culture conditions for the formation of 4-(4′-hydroxy- anilino)-5-anilinophthalimide with this strain were op-timized.     

Fungal metabolism of (-)-deprenyl and pargyline

Deprenyl and pargyline were readily metabolized by Cunninghamella echinulata ATCC 9244 and the products obtained were identified by gas-liquid chromatography and mass spectrometry. Deprenyl was completely metabolized to four products; amphetamine, N-methylamphetamine, 1-phenyl 2-propanone oxime and N-acetylamphetamine. Pargyline metabolism extracts contained substrate and five products; N-propargylbenzylamine, N-hydroxy-N-propargylbenzylamine, N-methylbenzylamine, N-acetylbenzylamine and benzylamine. Substrate concentration influenced the relative amounts of metabolites recovered. C. echinulata is an excellent organism for use as a model of mammalian metabolism.     

Fatty acid lithium salts from Cunninghamella echinulata have cytotoxic and genotoxic effects on HL‐60 human leukemia cells

Polyunsaturated fatty acids, especially gamma linolenic acid (GLA), are potentially useful agents in the treatment of cancer. Cunninghamella echinulata, a fungus species that is able to synthesize GLA, when cultivated under nitrogen‐limited conditions in a medium having glucose as carbon and energy source, accumulated 32–35% of lipids containing 11–18% GLA. The conversion yield of glucose to lipid was around 0.11 g per gram of glucose consumed while the lipid production was 5 g/L. Fatty acid lithium salts (FALS) were prepared from the total Cunninghamella lipids and studied for their effects on HL‐60 human leukemic cells. Cytotoxicity of FALS on HL‐60 leukemic cells was linearly related to the FALS concentration. High FALS concentration (i.e. 15 and 20 μg/mL) induced DNA fragmentation, while concurrent treatment of cells with H₂O₂ (at 100 μM) and FALS resulted in enhanced cytotoxicity of H₂O₂. However, when FALS were employed at low concentrations (i.e. 5 and 10 μg/mL), they demonstrated a protective effect on HL‐60 cells against H₂O₂ genotoxicity, whereas at 20 μg/mL FALS enhanced the ability of H₂O₂ to induce DNA fragmentation. It is concluded that FALS derived from C. echinulata lipids could be an effective preparation against HL‐60 human leukemic cells.     

New Derivatives from Microbial Transformation of ent‐Kaur‐16‐en‐19‐oic Acid by Cunninghamella echinulata

Biotransformation of ent‐kaur‐16‐en‐19‐oic acid using fungus Cunninghamella echinulata resulted in two novel hydroxylated metabolites together with five known compounds. Their structures were elucidated by means of extensive NMR and HR‐ESI‐MS data analysis. The eight compounds were measured for their cytotoxicity against the human breast carcinoma (MCF‐7) and human hepatoblastoma (HepG‐2) cell lines. Seven compounds showed no cytotoxicity to the two cell lines. One compound displayed moderate cytotoxicity against HepG‐2 and MCF‐7 with the IC₅₀ values of 12.6 and 27.1 μM, respectively.     

Fungal hydroxylation of (−)-santonin and its analogues

Santonin (1) was incubated with separate growing cultures of Aspergillus niger ATCC 9142, Mucor plumbeus ATCC 4740, Whetzelinia sclerotiorum ATCC 18687, Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. Three novel metabolites were isolated: 11β,13-dihydroxysantonin (3), 6,7-dehydosantonin (5) and 3,6-dihydroxy-9-keto-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (7). 11β-Hydroxysantonin (2), 14-hydroxysantonin (4) and 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (6) were also isolated. Hydroxylation at C-9 followed by a retro-aldol reaction was postulated to have produced 6 and 7. Through the synthesis and fermentation of the santonin analogues: tetrahydrosantonin (8) and α-desmotroposantonin (12), several new compounds were obtained; the most significant being 9-keto-desmotroposantonin (14), which was indicative of C-9 monohydroxylation.     

Organic nitrogen of tomato waste hydrolysate enhances glucose uptake and lipid accumulation in Cunninghamella echinulata

Aims: To investigate the effect of organic nitrogen on lipogenesis during growth of Cunninghamella echinulata on tomato waste hydrolysate (TWH) media. Methods and Results: Cunninghamella echinulata grown on a TWH medium rapidly took up glucose and produced large amounts of lipids. However, when some quantities of the organic nitrogen were removed from TWH (by acid followed by alkaline precipitation of proteins) the uptake of glucose was dramatically reduced and large quantities of fungal biomass having low lipid content were produced. Nevertheless, when glycerol was used as carbon source instead of glucose, the uptake rate as well as the biomass production and the lipid accumulation processes were unaffected by the TWH organic nitrogen removal. Finally, when the fungus was grown on a glucose supplemented TWH medium that contained no assimilable organic nitrogen (after further precipitation of proteins with methanol), the produced biomass contained non-negligible quantities of lipids, although glucose uptake remained low. Lipid analysis showed that the produced lipids comprised mainly of neutral lipids, which were preferentially consumed during lipid turnover. Lipid production on the original TWH medium having glucose as carbon source was 0·48 g of lipid per gram of dry biomass, corresponding to 8·7 g of lipid per litre of growth medium. The produced lipids contained 11·7%γ-linolenic acid (GLA), hence the GLA yield was more than 1 g l⁻¹. Conclusions: Organic nitrogen compounds found in TWH favour glucose (but not glycerol) uptake and lipid accumulation in C. echinulata. Significance and Impact of the Study: Agro-industrial wastes containing organic nitrogen, such as tomato waste, are produced in vast amounts causing severe environmental problems. These wastes could be used as fermentation feedstock to produce microbial lipids.     

Biotransformation of fluorobiphenyl by Cunninghamella elegans

The fungus Cunninghamella elegans is a useful model of human catabolism of xenobiotics. In this paper, the biotransformation of fluorinated biphenyls by C. elegans was investigated by analysis of the culture supernatants with a variety of analytical techniques. 4-Fluorobiphenyl was principally transformed to 4-fluoro-4′-hydroxybiphenyl, but other mono- and dihydroxylated compounds were detected in organic extracts by gas chromatography–mass spectrometry. Additionally, fluorinated water-soluble products were detected by ¹⁹F NMR and were identified as sulphate and β-glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4′-difluoro- and 2,3,4,5,6-pentafluoro-biphenyl) were catabolised by C. elegans, yielding mono- and dihydroxylated products, but phase II metabolites were detected from 4,4′-difluorobiphenyl only.     

Polysaccharides from mycelium of the fungus <Emphasis Type="Italic">Cunninghamella japonica</Emphasis>

Preliminary data on the polysaccharide composition of mycelium of the fungus Cunninghamella japonica (synonymous with C. echinulata) grown by the method of submerged cultivation were obtained. Mild acidic hydrolysis of mycelium resulted in the formation of glucose, mannose, and galactose; while the treatment with acid under drastic conditions afforded glucosamine as a product of hydrolysis of chitin and chitosan, their total content was about 35%. Several polysaccharide fractions were isolated from mycelium by successive extraction with hot water, 2% aqueous NaOH, and 10% AcOH; their monosaccharide composition was characterized. The yield of chitosan extracted with AcOH was insignificant. Additional purification of the fraction obtained after extraction with alkali afforded polysaccharide which was a linear (1 → 3)-α-D-glucopyranan according to the data of NMR spectroscopy and the chemical methods of structural analysis. The presence of this polysaccharide, as well as a low content of chitosan and polyuronides, distinguishes the studied strain C. japonica from most of the known Mucorales.